Combination of antisense oligomers

Abstract

In the present description, provided is a combination of antisense oligomers, which induce simultaneous skipping of any two or more exons, said exons being consecutive in numerical order, selected from the group consisting of the 45th exon to the 55th exon in the human dystrophin pre-mRNA, pharmaceutically acceptable salts thereof or hydrates of the same.

Description

Antisense oligomer combinations

[0001] The present invention relates to a pharmaceutical composition or a combined drug for treating muscular dystrophy, a method for treating muscular dystrophy, and the like.

[0002] In recent years, exon skipping therapy has attracted attention as a treatment for diseases by skipping exons in genes with disease-causing mutations, thereby producing partially functional proteins. An example of a disease treatable by such exon skipping therapy is Duchenne muscular dystrophy (DMD).

[0003] DMD is the most common genetic progressive muscle disease, occurring in approximately 1 in 3,500 male births. DMD patients exhibit motor function similar to that of healthy individuals during infancy, but begin to show muscle weakness around the age of 4 or 5. DMD patients' muscle weakness then progresses with age, becoming unable to walk by around age 12 and eventually dying from cardiac or respiratory failure in their 20s. For this reason, there is a strong demand for the development of effective therapeutic drugs.

[0004] DMD is known to be caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome and is a large gene consisting of 2.2 million bases of DNA. It is transcribed from DNA into pre-mRNA, which is then spliced ​​to remove introns and combine 79 exons, resulting in a 13,993-base mRNA. This mRNA is translated into 3,685 amino acids to produce the dystrophin protein. Dystrophin protein is involved in maintaining muscle cell membrane stability and is necessary for preventing muscle cell breakdown. Because DMD patients have mutations in the dystrophin gene, functional dystrophin protein is barely expressed in their muscle cells. As a result, DMD patients are unable to maintain muscle cell structure during muscle contraction, resulting in the influx of large amounts of calcium ions into the muscle cells. This results in muscle cell necrosis and fibrosis, gradually making muscle cell regeneration more difficult.

[0005] Becker muscular dystrophy (BMD) is also caused by mutations in the dystrophin gene. Although symptoms of BMD include muscle weakness, they are generally milder than DMD, and the progression of muscle weakness is slower. BMD often develops in adulthood. The difference in clinical symptoms between DMD and BMD is thought to be due to whether the mutation disrupts or maintains the amino acid reading frame during translation of dystrophin mRNA into dystrophin protein (Non-Patent Document 1). In other words, DMD has a mutation that shifts the amino acid reading frame, resulting in the expression of almost no functional dystrophin protein. However, BMD has a mutation that deletes part of an exon, but maintains the amino acid reading frame, resulting in the production of an incomplete but functional dystrophin protein.

[0006] Exon skipping is a promising treatment for DMD. This method involves modifying splicing to restore the amino acid reading frame of dystrophin mRNA, thereby inducing the expression of a partially functional dystrophin protein (Non-Patent Document 2). The amino acid sequence translated from the exon targeted by exon skipping is lost. Therefore, the dystrophin protein expressed by this treatment is shorter than normal, but the amino acid reading frame is maintained, thereby partially retaining its function of stabilizing muscle cells. Therefore, exon skipping is expected to result in DMD exhibiting symptoms similar to milder forms of BMD. Following animal experiments in mice and dogs, exon skipping is currently undergoing clinical trials in human DMD patients.

[0007] Exon skipping can be induced by binding of an antisense nucleic acid targeting either or both of the 5' or 3' splice site regions, or the interior of an exon. An exon is included in mRNA only when both splice sites are recognized by the spliceosome complex. Therefore, exon skipping can be induced by targeting the area around a splice site with an antisense nucleic acid. Furthermore, it is believed that binding of a serine- and arginine-rich SR protein to an exon splicing enhancer (ESE) is required for an exon to be recognized by the splicing machinery, and exon skipping can also be induced by targeting the ESE.

[0008] Because mutations in the dystrophin gene vary among DMD patients, antisense nucleic acids appropriate for the location and type of gene mutation are required. Several reports have been published on antisense nucleic acids that target a single contiguous base sequence in a single exon of the dystrophin gene to induce exon skipping (Patent Documents 1 to 6 and Non-Patent Documents 1 and 2). It has also been reported that mixing two types of antisense nucleic acids targeting the same exon of the dystrophin gene (dual targeting) can enhance skipping activity compared to the use of each antisense nucleic acid alone (Patent Document 7).

[0009] Furthermore, a method called multi-exon skipping, which skips multiple exons (exon groups) rather than skipping a single exon as described above, has attracted attention. This method makes it possible to treat a wide range of dystrophin gene mutations through exon skipping. For example, exons 45 to 55 in the dystrophin gene are known to be hotspots for genetic mutations, and it has been reported that skipping these 11 exons can treat approximately 60% of DMD patients with deletion mutations (Non-Patent Document 3). Furthermore, while congenital deletion of exons 45 to 55 can cause BMD, most patients are asymptomatic or have mild symptoms (Non-Patent Document 4). Based on these findings, drugs capable of inducing exon 45-55 skipping are expected to be promising therapeutic agents for DMD.

[0010] As methods for inducing multi-exon skipping, for example, a method using antisense nucleic acids that target all exons in a region to be subjected to exon skipping (Non-Patent Documents 5, 7, 8, and 10), a method using antisense nucleic acids that target two different exons, one on the 3' side and one on the 5' side of a region to be subjected to exon skipping (Non-Patent Documents 6 and 9 and Patent Documents 8, 9, and 11), and a method using antisense nucleic acids that target only the exon on the 5' side of a region to be subjected to exon skipping (Patent Document 10) have been reported.

[0011] International Publication No. WO 2004 / 048570, International Publication No. WO 2009 / 139630, International Publication No. WO 2010 / 048586, U.S. Patent Publication No. 2010 / 0168212, International Publication No. 2011 / 057350, International Publication No. 2006 / 000057, International Publication No. 2007 / 135105, International Publication No. 2004 / 083446, International Publication No. 2014 / 007620, International Publication No. 2019 / 200185, International Publication No. 2020 / 219820

[0012] Annemieke Aartsma-Rus et al., (2002) Neuromuscular Disorders 12: S71-S77Wilton SD, et al., Molecular Therapy 2007: 15: p. 1288-96Christophe Beroud et al., Human Mutation, 28(2), 2007, 196-202Yusuke Echigoya et al., Molecular Therapy‐Nucleic Acids, 4(2), 2015, e225Yoshitsugu Aoki et al., PNAS, 109(34), 2012, 13763-13768Laura van Vliet et al., BMC Medical Genetics, 9, 105, 2008Joshua Lee et al., PLoS ONE, 13(5), e0197084, 2018Joshua Lee et al., Methods in Molecular Biology, 1828, 141-150, 2018Annemieke Aartsma-Rus et al, Am. J. Hum. Genet. 74(1), 83-92, 2004Yusuke Echigoya et al., Molecular Therapy, 27(11), 1-13, 2019

[0013] Drugs that simultaneously skip multiple exons (exon groups) of a target pre-mRNA have not always been effective. Under the circumstances described above, there has been a demand for a drug that can treat patients with various mutations by simultaneously skipping multiple exons (exon groups) of a target pre-mRNA.

[0014] The present invention provides the following combinations of antisense oligomers or pharmaceutically acceptable salts thereof or hydrates thereof, pharmaceutical compositions, combined drugs, methods for treating muscular dystrophy, etc. (1) A combination of antisense oligomers or pharmaceutically acceptable salts thereof, or hydrates thereof, which simultaneously skip any two or more consecutive exons in numerical order selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA, comprising: (i) a first unit oligomer comprising a base sequence consisting of an 11-base base sequence extending from the 3' end to the 5' end of the 44th intron of the human dystrophin pre-mRNA and a 69-base base sequence extending from the 5' end to the 3' end of the 45th exon, or a base sequence complementary to a part of said base sequence; and a second unit oligomer comprising a base sequence extending from the 52nd base to the 75th base in the 3' end to the 5' end of the 44th intron of the human dystrophin pre-mRNA, or a base sequence complementary to a part of said base sequence. (ii) a second antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate of either thereof, comprising a base sequence complementary to a base sequence consisting of a 33-base base sequence extending from the 3' end to the 5' end of the 54th intron of the human dystrophin pre-mRNA and a 53-base base sequence extending from the 5' end to the 3' end of the 55th exon, or a part of the base sequence. (2) the first unit oligomer comprises a base sequence complementary to 15 to 30 consecutive bases of a base sequence consisting of an 11-base base sequence from the 3' end to the 5' end of the 44th intron of the human dystrophin pre-mRNA and a 69-base base sequence from the 5' end to the 3' end of the 45th exon, and the second unit oligomer comprises a base sequence complementary to 1 to 10 consecutive bases of a base sequence from the 52nd base to the 75th base from the 3' end to the 5' end of the 44th intron of the human dystrophin pre-mRNA,The combination according to (1), wherein the second antisense oligomer comprises a base sequence complementary to 15 to 30 consecutive bases of a base sequence consisting of a 33-base base sequence extending from the 3' end to the 5' end of the 54th intron of the human dystrophin pre-mRNA and a 53-base base sequence extending from the 5' end to the 3' end of the 55th exon. (3) The first unit oligomer comprises a base sequence complementary to: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906; (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906; (c) a base sequence that has 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906 and has a length within ±15% of the length of any one base sequence selected; or (d) a base sequence that is a part of any one base sequence selected from the group consisting of (a), (b), and (c); and / or the second unit oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105; (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, (c) a base sequence that has 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105 and has a length within ±15% of the length of any one selected base sequence, or (d) a base sequence complementary to a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c). (4) The combination according to (1) or (2), wherein the second antisense oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298,(c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298 and having a length within ±15% of the length of said any one base sequence selected, or (d) a base sequence complementary to a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c). (5) The combination according to any one of (1) to (4), wherein the first antisense oligomer comprises a first unit oligomer and the second unit oligomer in this order from the 5' end, wherein the first unit oligomer comprises the base sequence of any one of SEQ ID NOs: 907 to 1602, the second unit oligomer comprises the base sequence of any one of SEQ ID NOs: 106 to 210, and the second antisense oligomer comprises the base sequence of any one of SEQ ID NOs: 4299 to 5090. (6) The combination according to any one of (1) to (5), wherein the first unit oligomer comprises any one base sequence selected from the group consisting of SEQ ID NOs: 1180, 1190, 1201, 1212, 1222, 1224, and 1239. (7) The combination according to any one of (1) to (6), wherein the second unit oligomer comprises any one base sequence selected from the group consisting of SEQ ID NOs: 114, 124, 151, 201, 203, and 205. (8) The first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, and wherein: the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, and the second unit oligomer comprises the base sequence of SEQ ID NO: 151; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, and the second unit oligomer comprises the base sequence of SEQ ID NO: 201; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, and the second unit oligomer comprises the base sequence of SEQ ID NO: 203; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, and the second unit oligomer comprises the base sequence of SEQ ID NO: 205;the first unit oligomer comprises the base sequence of SEQ ID NO: 1239, and the second unit oligomer comprises the base sequence of SEQ ID NO: 114; the first unit oligomer comprises the base sequence of SEQ ID NO: 1224, and the second unit oligomer comprises the base sequence of SEQ ID NO: 124; the first unit oligomer comprises the base sequence of SEQ ID NO: 1180, and the second unit oligomer comprises the base sequence of SEQ ID NO: 151; the first unit oligomer comprises the base sequence of SEQ ID NO: 1190, and the second unit oligomer comprises the base sequence of SEQ ID NO: 151; the first unit oligomer comprises the base sequence of SEQ ID NO: 1212, and the second unit oligomer comprises the base sequence of SEQ ID NO: 151; the first unit oligomer comprises the base sequence of SEQ ID NO: 1222, and the second unit oligomer comprises the base sequence of SEQ ID NO: 151. The combination according to (6) or (7). (9) The combination according to any one of (1) to (8), wherein the second antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 4698, 4702, 4752, 4923, 4926, 4936, and 4977. (10) The first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, and wherein the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 201, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 203, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950;the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 205, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1239, the second unit oligomer comprises the base sequence of SEQ ID NO: 114, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1224, the second unit oligomer comprises the base sequence of SEQ ID NO: 124, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1180, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1190, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1212, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1222, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4698; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4702; orthe first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4752; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4923; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4926; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4936; The combination according to any one of (1) to (9), wherein the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4977, or the first unit oligomer comprises the base sequence of SEQ ID NO: 1180, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4977. (11) The combination according to any one of (5) to (10), wherein the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950 or 4880. (12) The combination according to any one of (1) to (11), further comprising: (iii) a third antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence complementary to a base sequence consisting of a 23-base sequence extending from the 3' end to the 5' end of the 45th exon of the human dystrophin pre-mRNA and a 73-base sequence extending from the 5' end to the 3' end of the 45th intron, or a part of the base sequence.(13) The combination described in (12), wherein the third antisense oligomer comprises a base sequence complementary to 15 to 30 consecutive bases of a base sequence consisting of a 23-base base sequence extending from the 3' end to the 5' end of the 45th exon of the human dystrophin pre-mRNA and a 73-base base sequence extending from the 5' end to the 3' end of the 45th intron. (14) The combination according to (12) or (13), wherein the third antisense oligomer comprises a base sequence complementary to: (a) any one of the base sequences selected from the group consisting of SEQ ID NOs: 1603 to 2554; (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one of the base sequences selected from the group consisting of SEQ ID NOs: 1603 to 2554; (c) a base sequence that has 85% or more sequence identity with any one of the base sequences selected from the group consisting of SEQ ID NOs: 1603 to 2554 and has a length within ±15% of the length of any one of the selected base sequences; or (d) a base sequence complementary to a partial base sequence of any one of the base sequences selected from the group consisting of (a), (b), and (c). (15-1) The third antisense oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554; (b) SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554. A base sequence that hybridizes under stringent conditions to a base sequence complementary to any one of the base sequences selected from the group consisting of:(c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554, and having a length within ±15% of the length of any one selected base sequence; or (d) a base sequence complementary to a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c), (15-2) The combination according to (14). (15-2) The third antisense oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1614 to 1654, 1667 to 1707, 1721 to 1761, 1776 to 1816, 1832 to 1872, 1889 to 1929, 1947 to 1987, 2006 to 2046, 2066 to 2106, 2127 to 2167, 2189 to 2229, 2252 to 2292, 2316 to 2356, 2381 to 2421, 2447 to 2487, and 2514 to 2554; (b) SEQ ID NOs: 1614 to 1654, 1667 to 1707, 1721 to 1761, 1776 to 1816, 1832 to 1872, 1889 to 1929, 1947 to 1987, 2006 to 2046, 2066 to 2106, 2127 to 2167, 2189 to 2229, 2252 to 2292, 2316 to 2356, 2381 to 2421, 2447 to 2487, and 2514 to 2554. A base sequence that hybridizes under stringent conditions to a base sequence complementary to any one of the base sequences selected from the group consisting of:(c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1614-1654, 1667-1707, 1721-1761, 1776-1816, 1832-1872, 1889-1929, 1947-1987, 2006-2046, 2066-2106, 2127-2167, 2189-2229, 2252-2292, 2316-2356, 2381-2421, 2447-2487, and 2514-2554, and having a length within ±15% of the length of any one selected base sequence; or (d) a base sequence complementary to a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c), (16) The combination according to (14). (16) The third antisense oligomer is selected from the group consisting of: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1617 to 1654, 1670 to 1707, 1724 to 1761, 1779 to 1816, 1835 to 1872, 1892 to 1929, 1950 to 1987, 2009 to 2046, 2069 to 2106, 2130 to 2167, 2192 to 2229, 2255 to 2292, 2319 to 2356, 2384 to 2421, 2450 to 2487, and 2517 to 2554; (b) SEQ ID NOs: 1617-1654, 1670-1707, 1724-1761, 1779-1816, 1835-1872, 1892-1929, 1950-1987, 2009-2046, 2069-2106, 2130-2167, 2192-2229, 2255-2292, 2319-2356, 2384-2421, 2450-2487, and 2517-2554. A base sequence that hybridizes under stringent conditions to a base sequence complementary to any one of the base sequences selected from the group consisting of:(c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1617-1654, 1670-1707, 1724-1761, 1779-1816, 1835-1872, 1892-1929, 1950-1987, 2009-2046, 2069-2106, 2130-2167, 2192-2229, 2255-2292, 2319-2356, 2384-2421, 2450-2487, and 2517-2554, and having a length within ±15% of the length of any one selected base sequence; or (d) a base sequence complementary to a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c), The combination according to (14). (17-1) The combination according to any one of (1) to (14), wherein the third antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 3060, 3065, 3077, 3082, 3087, 3090, 3096, 3108, 3119, and 3320. (17-2) The combination according to any one of (1) to (14), wherein the third antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 3077, 3082, 3087, 3090, 3096, 3108, and 3119. (17-3) The combination according to any one of (1) to (14), wherein the third antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 3082, 3087, 3090, 3096, 3108, and 3119. (18) The combination according to any one of (12) to (17), wherein the first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, the first unit oligomer comprising a base sequence of any one of SEQ ID NOs: 907 to 1602, the second unit oligomer comprising a base sequence of any one of SEQ ID NOs: 106 to 210, the second antisense oligomer comprising a base sequence of any one of SEQ ID NOs: 4299 to 5090, and the third antisense oligomer comprising a base sequence of any one of SEQ ID NOs: 2555 to 3506. (19) The combination according to any one of (12) to (17), wherein the first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, andthe first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 201, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 203, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 205, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1239, the second unit oligomer comprises the base sequence of SEQ ID NO: 114, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1224, the second unit oligomer comprises the base sequence of SEQ ID NO: 124, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises the base sequence of SEQ ID NO: 1180, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; orthe first unit oligomer comprises the base sequence of SEQ ID NO: 1190, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1212, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1222, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3060; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3065; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3077; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3087; orthe first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3090; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3096; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3108; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3119; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3320; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4698, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4702, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; orthe first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4752, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4923, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4926, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4936, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4977, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082; or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4977, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3096; The combination according to any one of (1) to (18), wherein the first unit oligomer comprises the base sequence of SEQ ID NO: 1180, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4977, and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3096.(20) The combination according to (18) or (19), wherein the first unit oligomer comprises the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer comprises the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer comprises the nucleotide sequence of SEQ ID NO: 4950 or 4880, and the third antisense oligomer comprises the nucleotide sequence of SEQ ID NO: 3082, 3090, or 3096. (21) The combination according to any of (1) to (20), wherein all of exons 45 to 55 in human dystrophin pre-mRNA are skipped. (22) The combination according to any of (1) to (11), wherein the first to second antisense oligomers are oligonucleotides, or the combination according to any of (12) to (21), wherein the first to third antisense oligomers are oligonucleotides. (23) The combination according to (22), wherein the sugar moiety and / or the phosphate linkage moiety of at least one nucleotide constituting the oligonucleotide is modified. (24) The sugar moiety of at least one nucleotide constituting the oligonucleotide has an —OH group at the 2′-position that is selected from the group consisting of —OR, —R, —R′OR, —SH, —SR, and —NH ２ , -NHR, -NR ２ , -N ３(22) The combination according to (23), wherein R is ribose substituted with any group selected from the group consisting of -CN, -F, -Cl, -Br, and -I (wherein R represents alkyl or aryl, and R' represents alkylene). (25) The combination according to any of (22) to (24), wherein the phosphate bond of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of phosphorothioate bond, phosphorodithioate bond, alkylphosphonate bond, phosphoramidate bond, and boranophosphate bond. (26) The combination according to any of (1) to (11), wherein the first and second antisense oligomers are morpholino oligomers, or the combination according to any of (12) to (21), wherein the first to third antisense oligomers are oligonucleotides. (27) The combination according to (26), wherein the first to third antisense oligomers are phosphorodiamidate morpholino oligomers. (28) The combination according to (26) or (27), wherein the 5'-ends of the first to third antisense oligomers are any of groups represented by the following chemical formulas (1) to (3): (29) (a) A pharmaceutical composition comprising the first and second antisense oligomers according to any one of (1) to (28) or a pharmaceutically acceptable salt thereof, or a hydrate thereof; or (b) A combination pharmaceutical comprising (i) a pharmaceutical composition comprising the first antisense oligomer according to any one of (1) to (28) or a pharmaceutically acceptable salt thereof, or a hydrate thereof, and (ii) a pharmaceutical composition comprising the second antisense oligomer according to any one of (1) to (28) or a pharmaceutically acceptable salt thereof, or a hydrate thereof. (30) (a) a pharmaceutical composition comprising the first to third antisense oligomers according to any one of (12) to (28) or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or (b) a combination pharmaceutical comprising (i) a pharmaceutical composition comprising the first antisense oligomer according to any one of (12) to (28) or a pharmaceutically acceptable salt thereof, or a hydrate thereof, (ii) a pharmaceutical composition comprising the second antisense oligomer according to any one of (12) to (28) or a pharmaceutically acceptable salt thereof, or a hydrate thereof, and (iii) a pharmaceutical composition comprising the third antisense oligomer according to any one of (12) to (28) or a pharmaceutically acceptable salt thereof, or a hydrate thereof. (31) The pharmaceutical composition or combination pharmaceutical according to (29) or (30), wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. (32) The pharmaceutical composition or combination pharmaceutical according to any one of (29) to (31), for the treatment of muscular dystrophy. (33) The pharmaceutical composition or combined pharmaceutical composition according to any one of (29) to (32) for administration to a human patient. (34) A method for treating muscular dystrophy, comprising the step of administering to a patient with muscular dystrophy (i) the first and second antisense oligomers according to any one of (1) to (28) or pharmaceutically acceptable salts thereof, or hydrates thereof, (ii) the first to third antisense oligomers according to any one of (12) to (28) or pharmaceutically acceptable salts thereof, or hydrates thereof, or (iii) the pharmaceutical composition or combined pharmaceutical composition according to any one of (29) to (33). (35) The method for treatment according to (34), wherein the patient with muscular dystrophy has a mutation in the dystrophin gene that is subject to exon 45-55 skipping.(36) The method of treatment according to (34) or (35), wherein the patient is a human.

[0015] The present invention provides a combination of antisense oligomers that simultaneously skip multiple target exons. Another aspect of the present invention provides a pharmaceutical composition or combination for treating patients with muscular dystrophy having various mutations by simultaneously skipping multiple exons of a target pre-mRNA. Another aspect of the present invention makes it possible to simultaneously and highly efficiently skip exons 45 to 55 of human dystrophin pre-mRNA.

[0016] Figure 1 shows the results of RT-PCR analysis of exon 45-55 skipping in mouse dystrophin pre-mRNA in H2K-mdx52 cells (total PMO concentration: 30 μM). Figure 2 shows the results of RT-PCR analysis of exon 45 skipping in mouse dystrophin pre-mRNA in H2K-mdx52 cells (total PMO concentration: 30 μM). Figure 3 shows the results of RT-PCR analysis of exon 45-55 skipping in mouse dystrophin pre-mRNA in H2K-mdx52 cells (total PMO concentration: 30 μM). Figure 4 shows the results of RT-PCR analysis of exon 45-55 skipping in mouse dystrophin pre-mRNA in H2K-mdx52 cells (total PMO concentration: 30 μM). In the figure, 2-2 shows the results of treatment with Mixture 2 + PMO No. 3 (1:1), 2-4 shows the results of treatment with Mixture 2 + PMO No. 3 (1:2), 2-5 shows the results of treatment with PMO No. 3 alone, and 2-7 shows the results of treatment with Mixture 2 alone. NT means no treatment (total PMO concentration: 15 μM). This figure shows the results of RT-PCR analysis of exon 45 skipping of mouse dystrophin pre-mRNA in H2K-mdx52 cells. In the figure, 2-2 shows the results of treatment with Mixture 2 + PMO No. 3 (1:1), 2-4 shows the results of treatment with Mixture 2 + PMO No. 3 (1:2), 2-5 shows the results of treatment with PMO No. 3 alone, and 2-7 shows the results of treatment with Mixture 2 alone. NT means no treatment (not treated) (total PMO concentration: 15 μM). This figure shows the results of RT-PCR analysis of exon 45-55 skipping of mouse dystrophin pre-mRNA in H2K-mdx52 cells. In the figure, 3-2 shows the results of treatment with Mixture 2 + PMO No. 4 (1:1), 3-4 shows the results of treatment with Mixture 2 + PMO No. 4 (1:2), 3-5 shows the results of treatment with PMO No. 4 alone, and 3-7 shows the results of treatment with Mixture 2. NT means no treatment (total PMO concentration: 15 μM). Figure 1 shows the results of RT-PCR analysis of exon 45 skipping of mouse dystrophin pre-mRNA in H2K-mdx52 cells.In the figure, 3-2 shows the results of treatment with Mixture 2 + PMO No. 4 (1:1), 3-4 shows the results of treatment with Mixture 2 + PMO No. 4 (1:2), 3-5 shows the results of treatment with PMO No. 4 alone, and 3-7 shows the results of treatment with Mixture 2; NT means "not treated" (total PMO concentration: 15 μM). This figure shows the results of RT-PCR analysis of exons 45-55 of mouse dystrophin pre-mRNA in H2K-mdx52 cells. In the figure, 2-1 shows the results of treatment with Mixture 2 alone, 2-2 shows the results of treatment with Mixture 2 + PMO No. 3 (1:1), 2-3 shows the results of treatment with Mixture 2 + PMO No. 3 (2:1), and 2-4 shows the results of treatment with Mixture 2 + PMO No. 3 (3:1); NT means "not treated" (total PMO concentration: 15 μM). This figure shows the results of RT-PCR analysis of exon 45 skipping of mouse dystrophin pre-mRNA in H2K-mdx52 cells. In the figure, 2-1 shows the results of treatment with Mixture 2 alone, 2-2 shows the results of treatment with Mixture 2 + PMO No. 3 (1:1), 2-3 shows the results of treatment with Mixture 2 + PMO No. 3 (2:1), and 2-4 shows the results of treatment with Mixture 2 + PMO No. 3 (3:1). NT means no treatment (not treated) (total PMO concentration: 15 μM). This figure shows the results of RT-PCR analysis of exon 45-55 skipping of mouse dystrophin pre-mRNA in H2K-mdx52 cells. In the figure, NC indicates Endo-porter only, Mix2 indicates a mixture containing PMO No. 1 and PMO No. 2 at a final concentration of 25 μM, and Mix2 + hnRNP A1 indicates a mixture containing PMO No. 1 and PMO No. 2 at a final concentration of 18.75 μM and PMO No. 3 at a final concentration of 12.5 μM (total PMO concentration: 50 μM). Figure 1 shows the results of RT-PCR analysis of exon 45 skipping in mouse dystrophin pre-mRNA in H2K-mdx52 cells.In the figure, NC indicates Endo-porter only, Mix2 indicates a mixture containing PMO No. 1 and PMO No. 2 at a final concentration of 25 μM, and Mix2 + hnRNP A1 indicates a mixture containing PMO No. 1 and PMO No. 2 at a final concentration of 18.75 μM and PMO No. 3 at a final concentration of 12.5 μM (total PMO concentration: 50 μM). Figure 1 shows the results of Western blotting analysis of dystrophin protein expression in H2K-mdx52 cells following skipping of exons 45-55 of mouse dystrophin pre-mRNA. In the figure, NC indicates Endo-porter only, Mix2 indicates a mixture containing PMO No. 1 and PMO No. 2 at a final concentration of 25 μM, Mix2 + hnRNP A1 indicates a mixture containing PMO No. 1 and PMO No. 2 at a final concentration of 18.75 μM and PMO No. 3 at a final concentration of 12.5 μM, and NT indicates no treatment (total PMO concentration: 50 μM). Figure 1 shows the results of RT-PCR analysis of exon 45-55 multiexon skipping in normal human myoblasts. Figure 2 shows the results of RT-PCR analysis of exon 45 skipping in normal human myoblasts. Figure 3 shows the results of RT-PCR analysis of exon 45-55 multiexon skipping in myoblasts derived from a DMD patient with an exon 48-50 deletion. Figure 1 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 48-50 deletion by RT-PCR. Figure 2 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 48-50 deletion by RT-PCR. Figure 3 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 48-50 deletion by RT-PCR. Figure 4 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 48-50 deletion by Western blotting. Figure 5 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 46-51 deletion by RT-PCR.Figure 1 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 46-51 deletion by RT-PCR. Figure 2 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 46-51 deletion by RT-PCR. Figure 3 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 46-51 deletion by RT-PCR. Figure 4 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 46-51 deletion by Western blotting. Figure 5 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 6 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 1 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 2 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 3 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 4 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 5 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 6 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 1 shows the results of examining exon 45-55 multi-exon skipping by RT-PCR in myoblasts derived from a DMD patient with exon 51 deletion. Figure 2 shows the results of examining exon 45 skipping by RT-PCR in myoblasts derived from a DMD patient with exon 51 deletion. Figure 3 shows the results of examining exon 45-55 multi-exon skipping by RT-PCR in myoblasts derived from a DMD patient with exon 51 deletion.Figure 1 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 2 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 3 shows the results of examining exon 45 skipping in myoblasts derived from a DMD patient with exon 51 deletion by RT-PCR. Figure 4 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 51 deletion by Western blotting. Figure 5 shows the results of examining exon 45-55 multi-exon skipping in myoblasts derived from a DMD patient with exon 51 deletion by Western blotting.

[0017] The present invention will be described in detail below. The following embodiments are merely examples for explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be embodied in various forms without departing from the gist of the present invention.

[0018] 1. Combination of Antisense Oligomers The present invention provides a combination of antisense oligomers or pharmaceutically acceptable salts thereof, or hydrates of these, which simultaneously skip any two or more consecutive exons in numerical order selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA, comprising: (i) a first unit oligomer comprising a base sequence consisting of an 11-base base sequence extending from the 3' to the 5' end of the 44th intron of the human dystrophin pre-mRNA and a 69-base base sequence extending from the 5' to the 3' end of the 45th exon, or a base sequence complementary to a portion of said base sequence; and a second unit oligomer comprising a base sequence extending from the 52nd to the 75th base in the 3' to the 5' end of the 44th intron of the human dystrophin pre-mRNA, or a base sequence complementary to a portion of said base sequence. (ii) a second antisense oligomer, or a pharmaceutically acceptable salt or hydrate of the second antisense oligomer, comprising a base sequence consisting of a 33-base sequence extending from the 3' end to the 5' end of the 54th intron of the human dystrophin pre-mRNA and a 53-base sequence extending from the 5' end to the 3' end of the 55th exon of the human dystrophin pre-mRNA, or a base sequence complementary to a portion of the base sequence. Hereinafter, the above combination will also be referred to as the "combination of the present invention."

[0019] As used herein, "combination" refers to a combination product, a combined pharmaceutical, a combined agent, etc. In one embodiment, each antisense oligomer in the combination of the present invention is contained in a single pharmaceutical composition and administered simultaneously. In another embodiment, each antisense oligomer in the combination of the present invention is contained in multiple pharmaceutical compositions and administered separately (simultaneously or sequentially). As used herein, "simultaneously" administering multiple pharmaceutical compositions means administering multiple pharmaceutical compositions at the same time. As used herein, "sequentially" administering multiple pharmaceutical compositions means administering them at different times. Specifically, one pharmaceutical composition can be administered before or after another pharmaceutical composition, and the administration interval in this case may be, but is not limited to, for example, several minutes, several hours, or several days.

[0020] Hereinafter, the first antisense oligomer or a pharmaceutically acceptable salt thereof or a hydrate thereof, the second antisense oligomer or a pharmaceutically acceptable salt thereof or a hydrate thereof (and optionally the third antisense oligomer described herein or a pharmaceutically acceptable salt thereof or a hydrate thereof) are collectively referred to as the "antisense oligomer of the present invention." The antisense oligomer of the present invention may also refer to the antisense oligomer, a pharmaceutically acceptable salt thereof, or a hydrate thereof. Furthermore, the above (i) first antisense oligomer, a pharmaceutically acceptable salt thereof, or a hydrate thereof is also referred to as the "first antisense oligomer of the present invention," and (ii) the second antisense oligomer, a pharmaceutically acceptable salt thereof, or a hydrate thereof is also referred to as the "second antisense oligomer of the present invention."

[0021] As used herein, the term "gene" includes not only genomic genes but also cDNA, pre-mRNA, and mRNA, and preferably refers to pre-mRNA. As used herein, "pre-mRNA" refers to an RNA molecule containing exons and introns transcribed from a target gene on the genome, and is a precursor to mRNA.

[0022] Human dystrophin pre-mRNA is an RNA molecule containing exons and introns transcribed from the human dystrophin gene on the genome, and is a precursor to mRNA. Those skilled in the art can obtain information on the nucleotide sequence of human dystrophin pre-mRNA by analogy with the genomic sequence of the human dystrophin gene (GenBank Accession No. NG_012232.1).

[0023] In the human genome, the human dystrophin gene is located at locus Xp21.2. The human dystrophin gene is approximately 3.0 Mbp in size, making it the largest known human gene. However, the coding region of the human dystrophin gene is only approximately 14 kb, and this coding region is distributed within the dystrophin gene as 79 exons (Roberts, R.G., et al., Genomics, 16: 536-538 (1993)). The pre-mRNA, which is a transcript of the human dystrophin gene, is spliced ​​to generate a mature mRNA of approximately 14 kb. The nucleotide sequence of the wild-type human dystrophin gene mature mRNA is known (GenBank Accession No. NM_004006).

[0024] The first antisense oligomer of the present invention comprises a first unit oligomer and a second unit oligomer, or consists of a first unit oligomer and a second unit oligomer.

[0025] The first unit oligomer targets a base sequence of 11 bases extending from the 3' end to the 5' end of the 44th intron of human dystrophin pre-mRNA and a base sequence of 69 bases extending from the 5' end to the 3' end of the 45th exon. As used herein, the term "target" means that the base sequence of interest is a base sequence complementary to the base sequence of a target region or a part of the base sequence of the target sequence.

[0026] The target sequence of the first unit oligomer can be expressed as a range of -11 bases to +69 bases, where the boundary between the 3' end of intron 44 and the 5' end of exon 45 is taken as base point 0, the base sequence region on the 5' side (upstream) of the base point on the dystrophin gene is indicated by "-" (minus), and the base sequence region on the 3' side (downstream) of the base point is indicated by "+". In this case, the region expressed in the range of -11 bases to -1 base belongs to intron 44, and the region expressed in the range of +1 base to +69 bases belongs to exon 45.

[0027] The first unit oligomer contains a base sequence complementary to a base sequence of 11 bases extending from the 3' end to the 5' end of the 44th intron of human dystrophin pre-mRNA and a base sequence of 69 bases extending from the 5' end to the 3' end of the 45th exon, or a part of the base sequence thereof.

[0028] The second unit oligomer targets the base sequence from the 52nd base to the 75th base in the 3'-5' direction of the 44th intron of human dystrophin pre-mRNA.

[0029] The target sequence of the second unit oligomer can be expressed as a range of -75 bases to -52 bases, where the boundary between the 3' end of intron 44 and the 5' end of exon 45 is taken as base point 0, the base sequence region on the 5' side (upstream) of the base point on the dystrophin gene is indicated by "-" (minus), and the base sequence region on the 3' side (downstream) of the base point is indicated by "+". In this case, the region expressed in the range of -75 bases to -52 bases belongs to intron 44.

[0030] The second unit oligomer contains a base sequence complementary to the base sequence from the 52nd base to the 75th base in the direction from the 3' end to the 5' end of the 44th intron of human dystrophin pre-mRNA, or a part of that base sequence.

[0031] The second antisense oligomer of the present invention targets a base sequence consisting of a 33-base sequence extending from the 3' end to the 5' end of the 54th intron of human dystrophin pre-mRNA and a 53-base sequence extending from the 5' end to the 3' end of the 55th exon.

[0032] The target sequence of the second antisense oligomer can be expressed as a range of -33 bases to +53 bases, where the boundary between the 3' end of intron 54 and the 5' end of exon 55 is taken as base point 0, the base sequence region on the 5' side (upstream) of the base point on the dystrophin gene is indicated by "-" (minus), and the base sequence region on the 3' side (downstream) of the base point is indicated by "+". In this case, the region expressed in the range of -33 bases to -1 base belongs to intron 54, and the region expressed in the range of +1 base to +53 bases belongs to exon 55.

[0033] The antisense oligomer 2 contains a base sequence complementary to a 33-base sequence extending from the 3' end to the 5' end of the 54th intron of human dystrophin pre-mRNA and a 53-base sequence extending from the 5' end to the 3' end of the 55th exon, or a part of the base sequence thereof.

[0034] The combination of the present invention may further comprise, in addition to the first and second antisense oligomers of the present invention, a third antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence consisting of a 23-base sequence extending from the 3' to the 5' end of the 45th exon of human dystrophin pre-mRNA and a 73-base sequence extending from the 5' to the 3' end of the 45th intron, or a base sequence complementary to a portion of that base sequence. Hereinafter, the third antisense oligomer, a pharmaceutically acceptable salt thereof, or a hydrate thereof will also be referred to as the "third antisense oligomer of the present invention."

[0035] The third antisense oligomer of the present invention targets a base sequence consisting of a 23-base sequence extending from the 3' end to the 5' end of the 45th exon of human dystrophin pre-mRNA and a 73-base sequence extending from the 5' end to the 3' end of the 45th intron.

[0036] The target sequence of the third antisense oligomer can be expressed as a range of -23 bases to +73 bases, where the boundary between the 3' end of exon 45 and the 5' end of intron 46 is taken as base point 0, the base sequence region on the 5' side (upstream) of the base point on the dystrophin gene is indicated by "-" (minus), and the base sequence region on the 3' side (downstream) of the base point is indicated by "+". In this case, the region expressed in the range of -23 bases to -1 base belongs to exon 45, and the region expressed in the range of +1 base to +73 bases belongs to intron 46.

[0037] The third antisense oligomer comprises a base sequence complementary to a base sequence consisting of a 23-base sequence extending from the 3' end to the 5' end of the 45th exon of human dystrophin pre-mRNA and a 73-base sequence extending from the 5' end to the 3' end of the 45th intron, or a part of the base sequence.

[0038] Specific examples of the sequences surrounding the target sequence of the first unit oligomer, second unit oligomer, second antisense oligomer, and third antisense oligomer contained in the first antisense oligomer of the present invention include those listed in Table 1 below.

[0039]

[0040] Specific examples of target sequences for the first unit oligomer, second unit oligomer, second antisense oligomer, and third antisense oligomer contained in the first antisense oligomer of the present invention include those listed in Table 2 below.

[0041] As used herein, thymine "T" and uracil "U" are interchangeable, and the exon skipping activity of the antisense oligomer of the present invention is not essentially affected whether "T" or "U" is used. Therefore, the base sequences shown herein include cases where "T" is replaced with "U," and are represented by the same SEQ ID NO. In the tables below, even base sequences in pre-mRNA may be represented as "T" instead of "U," but those skilled in the art can understand the RNA sequence by appropriately substituting "T" for "U."

[0042] In this specification, the target base sequence is written as "Ha_b-c".

[0043] "Ha" represents the a-th exon of the human dystrophin gene, "b" represents the 5'-terminal base of the target base sequence, and "c" represents the 3'-terminal base of the target base sequence. When "b" and "c" are positive integers, "b" and "c" respectively represent the base number from the 3'-terminal end when the 5'-terminal base of the a-th exon is the first base. On the other hand, when "b" and "c" are negative integers, "b" and "c" respectively represent the base number from the 5'-terminal end when the 3'-terminal base of the (a-1)-th intron is the -1 base.

[0044] For example, "H55_(-75)-(-52)" means a base sequence in which the 5' end of the target base sequence is the 75th base from the 3' end of the 54th intron toward the 5' end, and the 3' end of the target base sequence is the 52nd base from the 3' end of the 54th intron toward the 5' end.

[0045] Furthermore, the sequences surrounding the target region of the antisense oligomer of the present invention or its target sequence include not only wild-type sequences (e.g., the nucleotide sequences shown in SEQ ID NOs: 5021 to 5027) but also mutant sequences of human dystrophin pre-mRNA. Such mutant sequences have, for example, any one of the nucleotide sequences selected from the group consisting of the following nucleotide sequences (B0) and (B1) to (B16). (B0) A base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027; (B1) A base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 15% of the length of any one selected base sequence. (B2) A base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 14% of the length of any one selected base sequence. (B3) A base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 13% of the length of any one selected base sequence. (B4) A base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 12% of the length of any one base sequence selected. (B5) A base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 11% of the length of any one base sequence selected. (B6) A base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 10% of the length of any one base sequence selected. (B7) A base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 9% of the length of any one base sequence selected.(B8) A base sequence having 92% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 8% of the length of any one base sequence selected. (B9) A base sequence having 93% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 7% of the length of any one base sequence selected. (B10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 6% of the length of any one base sequence selected. (B11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 5% of the length of any one base sequence selected. (B12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 4% of the length of any one base sequence selected. (B13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 3% of the length of any one base sequence selected. (B14) A base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 2% of the length of any one base sequence selected. (B15) A base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ± 1% of the length of any one base sequence selected. (B16) A base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027, and having a length within ±0.5% of the length of any one selected base sequence.

[0046] As used herein, the term "a base sequence that hybridizes under stringent conditions" refers to a base sequence obtained by colony hybridization, plaque hybridization, Southern hybridization, or the like, using as a probe all or part of a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027. Hybridization methods that can be used include those described in, for example, "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press, 2001" and "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997."

[0047] As used herein, the term "complementary base sequence" is not limited to a base sequence that forms a Watson-Crick pair with a target base sequence, but also includes a base sequence that forms a wobble base pair. Here, Watson-Crick pair refers to base pairs in which a hydrogen bond is formed between adenine-thymine, adenine-uracil, and guanine-cytosine, and wobble base pair refers to base pairs in which a hydrogen bond is formed between guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine. Furthermore, a "complementary base sequence" does not necessarily have to be 100% complementary to the target base sequence. For example, the "complementary base sequence" may contain one, two, three, four, or five non-complementary bases relative to the target base sequence, or may be a base sequence that is one, two, three, four, or five bases shorter than the target base sequence.

[0048] As used herein, "stringent conditions" may refer to low stringent conditions, moderate stringent conditions, or high stringent conditions. "Low stringent conditions" refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringent conditions" refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C, or 5x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formamide, and 42°C. "Highly stringent conditions" refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C, or 0.2x SSC, 0.1% SDS, and 65°C. Under these conditions, it is expected that increasing the temperature will result in more efficient production of base sequences with higher sequence identity. However, factors that affect hybridization stringency include temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and those skilled in the art can achieve similar stringency by appropriately selecting these factors. When using a commercially available hybridization kit, for example, the AlkPhos Direct Labeling and Detection System (GE Healthcare) can be used. In this case, incubation with the labeled probe is performed overnight according to the protocol provided with the kit, and then the membrane is washed with a primary wash buffer containing 0.1% (w / v) SDS at 55°C, after which hybridization can be detected. Alternatively, when preparing a probe based on all or part of a base sequence complementary to any one of the base sequences selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, if the probe is labeled with digoxigenin (DIG) using a commercially available reagent (e.g., PCR Labeling Mix (Roche Diagnostics)), hybridization can be detected using a DIG Nucleic Acid Detection Kit (Roche Diagnostics), etc.

[0049] The identity of nucleotide sequences can be determined using the BLAST (Basic Local Alignment Search Tool) algorithm by Carlin and Altschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993). Programs based on the BLAST algorithm, such as BLASTN and BLASTX, have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing nucleotide sequences using BLASTN, the parameters are set to, for example, score = 100 and wordlength = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

[0050] The antisense oligomer of the present invention comprises a base sequence complementary to the base sequence of a target region of the present invention or a portion thereof. "A portion thereof" refers to a region excluding the entire length of the target region, i.e., a partial region of the target region. The length of the partial region may be 10 to 60 bases, 10 to 55 bases, 10 to 50 bases, 10 to 45 bases, 10 to 40 bases, 10 to 35 bases, 10 to 30 bases, 10 to 25 bases, 15 to 60 bases, 15 to 55 bases, 15 to 50 bases, 15 to 45 bases, 15 to 40 bases, 15 to 35 bases, 15 to 30 bases, 15 to 25 bases, 16 to 60 bases, 16 to 55 bases, 16 to 50 bases, 16 to 45 bases, 16 to 40 bases, 16 to 35 bases, 16 to 30 bases, 16 to 25 bases, 17 to 60 base length, 17 to 55 base length, 17 to 50 base length, 17 to 45 base length, 17 to 40 base length, 17 to 35 base length, 17 to 30 base length, 17 to 25 base length, 18 to 60 base length, 18 to 55 base length, 18 to 50 base length, 18 to 45 base length, 18 to 40 base length, 18 to 35 base length, 18 to 30 base length, 18 to 25 base length, 19 to 60 base length, 19 to 55 base length, 19 to 50 base length, 19 to 45 base length, 19 to 40 base length, 19 to 35 base length, 19 to 30 base length, 19 to 25 base length, 20 to 60 base length, 20 to 55 base length, 20 to 50 base length, 20 to 45 base length, 20 to 40 base length, 20 to 35 base length, 20 to 30 base length, 20 to 25 base length, 15 to 30 base length, 15 to 29 base length, 15 to 28 base length, 15 to 27 base length, 15 to 26 base length, 15 to 25 base length, 15 to 24 base length, 15 to 23 base length, 15 to 22 base length, 15 to 21 base length, 15 to 20 base length, 15 to 19 base length, 15 to 18 base length, 16 to 30 base length, 16 to 29 base length, 16 to 28 base length, 16 to 27 base length, 16 to 26 base length, 16 to 25 base length, 16 to 24 base length, 16 to 23 base length, 16 to 22 base length, 16 to 21 base length, 16 to 20 base length, 16 to 19 base length, 16 to 18 base length, 17 to 30 base length, 17 to 29 base length, 17 to 28 base length, 17 to 27 base length, 17 to 26 base length, 17 to 25 base length, 17 to 24 base length, 17 to 23 base length, 17 to 22 base length, 17 to 21 base length, 17 to 20 base length, 17 to 19 base length, 17 to 18 base length, 18 to 30 base length, 18 to 29 base length, 18 to 28 base length, 18 to 27 base length,18-26 base length, 18-25 base length, 18-24 base length, 18-23 base length, 18-22 base length, 18-21 base length, 18-20 base length, 18-19 base length, 19-30 base length, 19-29 base length, 19-28 base length, 19-27 base length, 19-26 base length, 19-25 base length, 19-24 base length, 19-23 base length, 19-22 base length, 19-21 base length, 19-20 base length, 20-30 base length, 20-29 base length, 20-28 base length, 20-27 base length, 20-26 base length, 20-25 base length, 20-24 base length, 20-23 base length, 20-22 base length Base length, 20-21 base length, 5-25 base length, 5-24 base length, 5-23 base length, 5-22 base length, 5-21 base length, 5-20 base length, 5-19 base length, 5-18 base length, 5-17 base length, 5-16 base length, 5-15 base length, 5-14 base length, 5-13 base length, 5-12 base length, 7-25 base length, 7-24 base length, 7-23 base length, 7-22 base length, 7-21 base length, 7-20 base length, 7-19 base length, 7-18 base length, 7-17 base length, 7-16 base length, 7-15 base length, 7-14 base length, 7-13 base length, 7-12 base length, 9-25 base length, 9-24 base length, 9-23 bases, 9-22 bases, 9-21 bases, 9-20 bases, 9-19 bases, 9-18 bases, 9-17 bases, 9-16 bases, 9-15 bases, 9-14 bases, 9-13 bases, 9-12 bases, 10-25 bases, 10-24 bases, 10-23 bases, 10-22 bases, 10-21 bases, 10-20 bases, 10-19 bases, 10-18 bases, 10-17 bases, 10-16 bases, 10-15 bases, 10-14 bases, 10-13 bases, 10-12 bases, 60 bases, 59 bases, 58 bases, 57 bases, 56 Base length, 55 base length, 54 base length, 53 base length, 52 base length, 51 base length, 50 base length, 49 base length, 48 base length, 47 base length, 46 base length, 45 base length, 44 base length, 43 base length, 42 base length, 41 base length, 40 base length, 39 base length, 38 base length, 37 base length, 36 base length, 35 base length, 34 base length, 33 base length, 32 base length, 31 base length, 30 base length, 29 base length, 28 base length, 27 base length, 26 base length, 25 base length, 24 base length, 23 base length, 22 base length, 21 base length, 20 base length, 19 base length, 18 base length, 17 base length, 16 base length, 15 base length,The length may be, but is not limited to, 14 bases, 13 bases, 12 bases, 11 bases, 10 bases, 9 bases, 8 bases, 7 bases, 6 bases, or 5 bases, and 1, 2, or 3 bases may be added to or subtracted from the above lengths.

[0051] The antisense oligomer of the present invention has the activity of simultaneously skipping any two or more consecutive exons in numerical order selected from the group consisting of exons 45 to 55 in human dystrophin pre-mRNA. Herein, such skipping of two or more consecutive exons in numerical order from a target pre-mRNA is referred to as "multi-exon skipping" or "multi-skipping," and this activity is referred to as "multi-exon skipping activity" or "multi-skipping activity."

[0052] As used herein, "simultaneous skipping" of two or more consecutive exons in numerical order refers not only to the case where the timing of removal of each exon from pre-mRNA is completely identical, but also to the case where each exon is removed sequentially during the period from pre-mRNA to mature mRNA. In other words, "simultaneous skipping" of two or more consecutive exons in numerical order refers to the removal of two or more consecutive exons from pre-mRNA.

[0053] As used herein, "two or more consecutive exons in numerical order" refers to multiple exons (the total number of exons is referred to as "Texon") contained in the target pre-mRNA whose exon numbers increase by one. The exon number refers to a number in which the exon located most 5' in the pre-mRNA is defined as the first exon, and the exons located sequentially from the 5' side to the 3' side are assigned numbers such as second, third, and so on. When skipping two or more consecutive exons in a gene, the exon numbers a1, ..., aj can be expressed as a sequence {aj}. The general term aj of the sequence {aj} is expressed by the following formula: [Formula 1] aj=m+(j-1) where m is any natural number satisfying 1≦m≦(Texon-1), and j is a natural number satisfying 2≦(m+j)≦Texon+1. For example, when the target pre-mRNA is human dystrophin pre-mRNA, Texon is 79. In one embodiment, j is any natural number selected from 1 to 11. In another embodiment, j is 11, j is 10, j is 9, j is 8, j is 7, j is 6, j is 5, j is 4, j is 3, j is 2, and j is 1.

[0054] Here, "any two or more consecutive exons in numerical order selected from the group consisting of the 45th exon to the 55th exon" refers to a plurality of exons whose exon numbers increase by one among the 11 exons contained in the pre-mRNA, from the 45th exon to the 55th exon. The exon number refers to a number assigned to the exons located sequentially from the 5' side to the 3' side of the 79 exons contained in the human dystrophin pre-mRNA, with the exon located most 5'-side of the pre-mRNA defined as the first exon, and the exons located sequentially from the 5' side to the 3' side being assigned numbers such as the second, third, ..., 79th. Note that introns are numbered the same as the exon located on their 5' side. In other words, the 45th intron has the 45th exon on its 5' side and the 46th exon on its 3' side. Furthermore, in this specification, an "nth" exon or intron refers to the nth exon or intron in the pre-mRNA, counting from the 5' end to the 3' end.

[0055] Table 3 shows combinations of exons contained in any two or more consecutive exons in numerical order selected from the group consisting of the 45th exon to the 55th exon.

[0056]

[0057] Among the exon combinations listed in Table 3, skipping patterns expected to exhibit a higher therapeutic effect on DMD include, for example, combinations 1, 2, 3, 4, 6, 8, 10, 18, 20, 21, 23, 25, 27, 28, 30, 32, 34, 36, 38, 40, 41, 43, 45, 46, 50, 52, and 55. Multi-exon skipping using these combinations is expected to exhibit a therapeutic effect on a greater number of DMD patients. In one embodiment, the combination of the present invention skips all of exons 45 to 55 in human dystrophin pre-mRNA.

[0058] Furthermore, any two or more consecutive exons selected from the group consisting of the 45th exon to the 55th exon may include multiple consecutive exon groups, and may be, but are not limited to, (Example 1) exons 45-46 (first exon group) and exons 48-53 (second exon group), or (Example 2) exons 46-47 (first exon group), exons 49-50 (second exon group), and exons 52-54 (third exon group).

[0059] In the present invention, "skipping activity" (i.e., multi-skipping activity) refers to, taking human dystrophin pre-mRNA as an example, the activity of generating human dystrophin mRNA in which any two or more consecutive exons selected from the group consisting of exons 45 to 55 in human dystrophin pre-mRNA are deleted in numerical order. In other words, this activity means that when the antisense oligomer of the present invention binds to a target site in human dystrophin pre-mRNA, the 5'-terminal nucleotide of the exon immediately downstream of the exon to be deleted is linked to the 3'-terminal nucleotide of the exon immediately upstream of the exon to be deleted when the pre-mRNA is spliced, resulting in the formation of a mature mRNA in which no codon frameshift has occurred (i.e., a mature mRNA in which exons have been deleted without frameshifting).

[0060] The antisense oligomers of the present invention exhibit multiskipping activity under physiological conditions. The term "under physiological conditions" refers to conditions controlled to pH, salt composition, and temperature similar to those in vivo. Examples include conditions at 25 to 40°C, preferably 37°C, pH 5 to 8, preferably pH 7.4, and a sodium chloride concentration of 150 mM.

[0061] Whether or not multi-skipping has occurred can be confirmed by introducing the combination of the present invention into dystrophin-expressing cells (e.g., human rhabdomyosarcoma cells), RT-PCR-amplifying the region surrounding exons 45 to 55 of the mRNA of the human dystrophin gene from the total RNA of the dystrophin-expressing cells, and then performing nested PCR or sequence analysis on the PCR amplification product. Multi-skipping efficiency can be determined by recovering human dystrophin gene mRNA from test cells, measuring the polynucleotide amount "A" of a band in which any two or more consecutive exons from exons 45 to 55 have been skipped in numerical order, the polynucleotide amount "B" of a band in which any one exon from exons 45 to 55 has been skipped, and the polynucleotide amount "C" of a band in which no skipping has occurred, and then calculating the skipping efficiency according to the following formula based on the measured values ​​of "A," "B," and "C." Skipping efficiency (%) = A / (A+B+C)×100

[0062] For example, the multi-skipping efficiency of exons 45 to 55 can be calculated according to the above formula based on the measured values ​​of "A," "B," and "C," by measuring the polynucleotide amount "A" of a band in which exons 45 to 55 have been multi-skipped using a forward primer for exon 44 and a reverse primer for exon 56, measuring the polynucleotide amount "B" of a band in which exon 45 has been single-skipped using a forward primer for exon 44 and a reverse primer for exon 46, and measuring the polynucleotide amount "C" of a band in which no skipping has occurred using a forward primer for exon 44 and a reverse primer for exon 46.

[0063] The number of exons deleted in human dystrophin mRNA by the antisense oligomer of the present invention is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. This is called a deletion pattern, but various deletion patterns may be present in the results obtained from a single skipping experiment or skipping treatment. For example, when the antisense oligomer of the present invention is introduced into cells expressing human dystrophin pre-mRNA and the resulting mRNA is recovered, a mixture of mRNAs in which 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 exons are deleted will be obtained.

[0064] In one embodiment, the "skipping activity" can be defined as (C1) to (C10) below.

[0065] (C1) Any two consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the two consecutive exons in numerical order may be the 45th and 46th exons, the 46th and 47th exons, the 47th and 48th exons, the 48th and 49th exons, the 49th and 50th exons, the 50th and 51st exons, the 51st and 52nd exons, the 52nd and 53rd exons, the 53rd and 54th exons, or the 54th and 55th exons.

[0066] (C2) Any three consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the three consecutive exons in numerical order may be the 45th to 47th exons, the 46th to 48th exons, the 47th to 49th exons, the 48th to 50th exons, the 49th to 51st exons, the 50th to 52nd exons, the 51st to 53rd exons, the 52nd to 54th exons, or the 53rd to 55th exons.

[0067] (C3) Any four consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA in numerical order are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the four consecutive exons in numerical order may be the 45th to 48th exons, the 46th to 49th exons, the 47th to 50th exons, the 48th to 51st exons, the 49th to 52nd exons, the 50th to 53rd exons, the 51st to 54th exons, or the 52nd to 55th exons.

[0068] (C4) Any five consecutive exons selected from the group consisting of exons 45 to 55 in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the five consecutive exons in number order may be exons 45 to 49, exons 46 to 50, exons 47 to 51, exons 48 to 52, exons 49 to 53, exons 50 to 54, or exons 51 to 55.

[0069] (C5) Any six consecutive exons selected from the group consisting of exons 45 to 55 in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the six consecutive exons in numbered order may be exons 45 to 50, exons 46 to 51, exons 47 to 52, exons 48 to 53, exons 49 to 54, or exons 50 to 55.

[0070] (C6) Any seven consecutive exons selected from the group consisting of exons 45 to 55 in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the seven consecutive exons in number order may be exons 45 to 51, exons 46 to 52, exons 47 to 53, exons 48 to 54, or exons 49 to 55.

[0071] (C7) Any eight consecutive exons selected from the group consisting of exons 45 to 55 in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the eight consecutive exons in number order may be exons 45 to 52, exons 46 to 53, exons 47 to 54, or exons 48 to 55.

[0072] (C8) Any nine consecutive exons selected from the group consisting of exons 45 to 55 in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the nine consecutive exons in number order may be exons 45 to 53, exons 46 to 54, or exons 47 to 55.

[0073] (C9) Any 10 consecutive exons selected from the group consisting of exons 45 to 55 in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the 10 consecutive exons in number order may be exons 45 to 54 or exons 46 to 55.

[0074] (C10) Eleven consecutive exons selected from the group consisting of the 45th to 55th exons in human dystrophin pre-mRNA are skipped with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Here, the 11 consecutive exons are the 45th to 55th exons.

[0075] The antisense oligomers of the present invention may be 10 to 60 bases long, 10 to 55 bases long, 10 to 50 bases long, 10 to 45 bases long, 10 to 40 bases long, 10 to 35 bases long, 10 to 30 bases long, 10 to 25 bases long, 15 to 60 bases long, 15 to 55 bases long, 15 to 50 bases long, 15 to 45 bases long, 15 to 40 bases long, 15 to 35 bases long, 15 to 30 bases long, 15 to 25 bases long, 16 to 60 bases long, 16 to 55 bases long, 16 to 50 bases long, 16 to 45 bases long, 16 to 40 bases long, 16 to 35 bases long, 16 to 30 bases long, 16 to 25 bases long, 17 to 60 bases long, 17 to 55 bases long , 17-50 base length, 17-45 base length, 17-40 base length, 17-35 base length, 17-30 base length, 17-25 base length, 18-60 base length, 18-55 base length, 18-50 base length, 18-45 base length, 18-40 base length, 18-35 base length, 18-30 base length, 18-25 base length, 19-60 base length, 19-55 base length, 19-50 base length, 19-45 base length, 19-40 base length, 19-35 base length, 19-30 base length, 19-25 base length, 20-60 base length, 20-55 base length, 20-50 base length, 20-45 base length, 20-40 base length, 20-35 Base length, 20-30 base length, 20-25 base length, 15-30 base length, 15-29 base length, 15-28 base length, 15-27 base length, 15-26 base length, 15-25 base length, 15-24 base length, 15-23 base length, 15-22 base length, 15-21 base length, 15-20 base length, 15-19 base length, 15-18 base length, 16-30 base length, 16-29 base length, 16-28 base length, 16-27 base length, 16-26 base length, 16-25 base length, 16-24 base length, 16-23 base length, 16-22 base length, 16-21 base length, 16-20 base length, 16-19 base length, 16 up to 18 bases long, 17-30 bases long, 17-29 bases long, 17-28 bases long, 17-27 bases long, 17-26 bases long, 17-25 bases long, 17-24 bases long, 17-23 bases long, 17-22 bases long, 17-21 bases long, 17-20 bases long, 17-19 bases long, 17-18 bases long, 18-30 bases long, 18-29 bases long, 18-28 bases long, 18-27 bases long, 18-26 bases long, 18-25 bases long, 18-24 bases long, 18-23 bases long, 18-22 bases long, 18-21 bases long, 18-20 bases long, 18-19 bases long, 19-30 bases long, 19-29 bases long,19-28 base length, 19-27 base length, 19-26 base length, 19-25 base length, 19-24 base length, 19-23 base length, 19-22 base length, 19-21 base length, 19-20 base length, 20-30 base length, 20-29 base length, 20-28 base length, 20-27 base length, 20-26 base length, 20-25 base length, 20-24 base length, 20-23 base length, 20-22 base length, 20-21 base length, 60 base length, 59 base length, 58 base length, 57 base length, 56 base length, 55 base length, 54 base length, 53 base length, 52 base length, 51 base length, 50 base length, 49 base length, 48 base length, 47 base length, 46 base length, 45 base length, The length may be, but is not limited to, 44 ​​bases, 43 bases, 42 bases, 41 bases, 40 bases, 39 bases, 38 bases, 37 bases, 36 bases, 35 bases, 34 bases, 33 bases, 32 bases, 31 bases, 30 bases, 29 bases, 28 bases, 27 bases, 26 bases, 25 bases, 24 bases, 23 bases, 22 bases, 21 bases, 20 bases, 19 bases, 18 bases, 17 bases, 16 bases, 15 bases, 14 bases, 13 bases, 12 bases, 11 bases, or 10 bases, and 1, 2, or 3 bases may be added to or subtracted from the above lengths.

[0076] The first antisense oligomer of the present invention is a linked antisense oligomer having a configuration in which a plurality of unit oligomers are linked to each other, or a pharmaceutically acceptable salt thereof, or a hydrate of either (hereinafter also referred to as the "linked antisense oligomer of the present invention"). A unit oligomer refers to each oligomer that constitutes the linked antisense oligomer of the present invention. In other words, a unit oligomer refers to a portion (unit) containing a nucleotide sequence that hybridizes with a target nucleotide sequence having a consecutive nucleotide sequence when the linked antisense oligomer of the present invention binds to the target nucleotide sequence on human dystrophin pre-mRNA.

[0077] The unit oligomers may be linked via a linker that does not contribute to hybridization, or may be linked directly without a linker. When the unit oligomers are linked directly to each other, the 3' end of the unit located on the 5' end side and the 5' end of the unit located on the 3' end side form a phosphate bond or any of the following groups: (Wherein, X is —OH, —CH ２ R １ , —O—CH ２ R １ , -S-CH ２ R １ , -NR ２ R ３ or F; R １ represents H or alkyl; R ２ and R ３ are the same or different and represent H, alkyl, cycloalkyl, or aryl; Y １ is O, S, CH ２ , or NR １ represents; Y ２ is O, S, or NR １ Z represents O or S.

[0078] The first unit oligomer constituting the linked antisense oligomer of the present invention may comprise a base sequence consisting of an 11-base sequence extending from the 3' to the 5' end of the 44th intron of human dystrophin pre-mRNA and a 69-base sequence extending from the 5' to the 3' end of the 45th exon, or a base sequence complementary to a portion of that base sequence. The second unit oligomer constituting the linked antisense oligomer of the present invention may comprise a base sequence complementary to the 52nd to 75th bases extending from the 3' to the 5' end of the 44th intron of human dystrophin pre-mRNA, or a portion of that base sequence.

[0079] Furthermore, with regard to the target sequence of a unit oligomer, "a part thereof" means a continuous partial region excluding the entire length of the target sequence. The length of the partial region is 5 to 30 bases long, such as 5 to 29 bases long, 5 to 28 bases long, 5 to 27 bases long, 5 to 26 bases long, 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 bases long, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 bases long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 30 bases long, 7 to 29 bases long, 7 to 28 bases long, 7 to 27 bases long, 7 to 26 bases long, and 7 to 25 bases long. , 7-24 base length, 7-23 base length, 7-22 base length, 7-21 base length, 7-20 base length, 7-19 base length, 7-18 base length, 7-17 base length, 7-16 base length, 7-15 base length, 7-14 base length, 7-13 base length, 7-12 base length, 9-30 base length, 9-29 base length, 9-28 base length, 9-27 base length, 9-26 base length, 9-25 base length, 9-24 base length, 9-23 base length, 9-22 base length, 9-21 base length, 9-20 base length, 9-19 base length, 9-18 base length, 9-19 base length, 9-18 base length, 9-19 base length, 9-21 base length, 9-22 base length, 9-23 base length, 9-22 base length, 9-21 base length, 9-20 base length, 9-19 base length, 9-18 base length, 9-19 ... 7 base length, 9-16 base length, 9-15 base length, 9-14 base length, 9-13 base length, 9-12 base length, 10-30 base length, 10-29 base length, 10-28 base length, 10-27 base length, 10-26 base length, 10-25 base length, 10-24 base length, 10-23 base length, 10-22 base length, 10-21 base length, 10-20 base length, 10-19 base length, 10-18 base length, 10-17 base length, 10-16 base length, 10-15 base length, 10-14 base length, 10-13 base length, 10-1 The length may be 2 bases, 30 bases, 29 bases, 28 bases, 27 bases, 26 bases, 25 bases, 24 bases, 23 bases, 22 bases, 21 bases, 20 bases, 19 bases, 18 bases, 17 bases, 16 bases, 15 bases, 14 bases, 13 bases, 12 bases, 11 bases, 10 bases, 9 bases, 8 bases, 7 bases, 6 bases, or 5 bases, but is not limited to these, and 1, 2, or 3 bases may be added to or subtracted from the above lengths.

[0080] The size of each unit oligomer is 5 to 30 bases long, and includes 5 to 29 bases long, 5 to 28 bases long, 5 to 27 bases long, 5 to 26 bases long, 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 bases long, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 bases long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 30 bases long, 7 to 29 bases long, 7 to 28 bases long, 7 to 27 bases long, 7 to 26 ... 25 base length, 7-24 base length, 7-23 base length, 7-22 base length, 7-21 base length, 7-20 base length, 7-19 base length, 7-18 base length, 7-17 base length, 7-16 base length, 7-15 base length, 7-14 base length, 7-13 base length, 7-12 base length, 9-30 base length, 9-29 base length, 9-28 base length, 9-27 base length, 9-26 base length, 9-25 base length, 9-24 base length, 9-23 base length, 9-22 base length, 9-21 base length, 9-20 base length, 9-19 base length, 9-18 base length , 9-17 base length, 9-16 base length, 9-15 base length, 9-14 base length, 9-13 base length, 9-12 base length, 10-30 base length, 10-29 base length, 10-28 base length, 10-27 base length, 10-26 base length, 10-25 base length, 10-24 base length, 10-23 base length, 10-22 base length, 10-21 base length, 10-20 base length, 10-19 base length, 10-18 base length, 10-17 base length, 10-16 base length, 10-15 base length, 10-14 base length, 10-13 base length, 10 The length may be, but is not limited to, up to 12, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 bases, and 1, 2, or 3 bases may be added to or subtracted from the above lengths. The size of each unit oligomer may be the same or different.

[0081] In the first antisense oligomer, the order of the first unit oligomer and the second unit oligomer is not limited. The first antisense oligomer may contain the first unit oligomer and the second unit oligomer in this order from the 5' end, or may contain the second unit oligomer and the first unit oligomer in this order from the 5' end.

[0082] In one embodiment, the first unit oligomer comprises or consists of a base sequence complementary to 15 to 30 consecutive bases of a base sequence consisting of an 11-base base sequence extending from the 3' to 5' direction of the 44th intron of human dystrophin pre-mRNA and a 69-base base sequence extending from the 5' to 3' direction of the 45th exon. In one embodiment, the second unit oligomer comprises or consists of a base sequence complementary to 1 to 10 consecutive bases of a base sequence extending from the 52nd to 75th bases extending from the 3' to 5' direction of the 44th intron of human dystrophin pre-mRNA. In one embodiment, the second antisense oligomer comprises or consists of a base sequence complementary to 15 to 30 consecutive bases of a base sequence consisting of a 33-base base sequence extending from the 3' to 5' direction of the 54th intron of human dystrophin pre-mRNA and a 53-base base sequence extending from the 5' to 3' direction of the 55th exon. In one embodiment, the third antisense oligomer comprises or consists of a base sequence complementary to 15 to 30 consecutive bases of a base sequence consisting of a 23-base base sequence extending from the 3' end to the 5' end of the 45th exon of the human dystrophin pre-mRNA and a 73-base base sequence extending from the 5' end to the 3' end of the 45th intron.

[0083] Examples of target sequences of the first unit oligomer and their complementary sequences (antisense sequences) are shown in Table 4 below.

[0084] In one embodiment, the first unit oligomer comprises a base sequence complementary to: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906; (b) a base sequence that hybridizes under stringent conditions to a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906; (c) a base sequence that has 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906 and has a length within ±15% of the length of any one selected base sequence; or (d) a base sequence that is a portion of any one base sequence selected from the group consisting of (a), (b), and (c).

[0085] Here, the base sequence of (c) is a mutant of the base sequence of (a), and the mutant is: (c-1) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±15% of the length of said any one base sequence selected; (c-2) a base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±14% of the length of said any one base sequence selected; (c-3) a base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±13% of the length of said any one base sequence selected; (c-4) A base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±12% of the length of any one selected base sequence; (c-5) A base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±11% of the length of any one selected base sequence; (c-6) A base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±10% of the length of any one selected base sequence; (c-7) A base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±9% of the length of any one selected base sequence; (c-8) A base sequence having a sequence identity of 92% or more with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and a length within ±8% of the length of any one selected base sequence; (c-9) A base sequence having a sequence identity of 93% or more with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and a length within ±7% of the length of any one selected base sequence;(c-10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±6% of the length of any one base sequence selected; (c-11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±5% of the length of any one base sequence selected; (c-12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±4% of the length of any one base sequence selected; (c-13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and having a length within ±3% of the length of any one base sequence selected; (c-14) A base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and a length within ±2% of the length of any one selected base sequence; (c-15) A base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and a length within ±1% of the length of any one selected base sequence; and (c-16) A base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, and a length within ±0.5% of the length of any one selected base sequence.

[0086] In one embodiment, the first unit oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602; or (b) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602 and having a length within ±15% of the length of any one selected base sequence, or consists of the base sequence of (a) or (b).

[0087] Here, the base sequence of (b) is a mutant of the base sequence of (a), and the mutant is: (b-1) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and having a length within ±15% of the length of said any one base sequence selected; (b-2) a base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and having a length within ±14% of the length of said any one base sequence selected; (b-3) a base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and having a length within ±13% of the length of said any one base sequence selected; (b-4) A base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and a length within ±12% of the length of any one selected base sequence; (b-5) A base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and a length within ±11% of the length of any one selected base sequence; (b-6) A base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and a length within ±10% of the length of any one selected base sequence; (b-7) A base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and a length within ±9% of the length of any one selected base sequence; (b-8) A base sequence having 92% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and having a length within ±8% of the length of any one selected base sequence; (b-9) A base sequence having 93% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and having a length within ±7% of the length of any one selected base sequence;(b-10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and a length within ±6% of the length of any one selected base sequence; (b-11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and a length within ±5% of the length of any one selected base sequence; (b-12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and a length within ±4% of the length of any one selected base sequence; (b-13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and a length within ±3% of the length of any one selected base sequence; (b-14) A base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and having a length within ±2% of the length of any one selected base sequence; (b-15) A base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and having a length within ±1% of the length of any one selected base sequence; and (b-16) A base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, and having a length within ±0.5% of the length of any one selected base sequence.

[0088] In one embodiment, the first unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602.

[0089] In one embodiment, the first unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 1180, 1190, 1201, 1212, 1222, 1224, and 1239.

[0090] Examples of target sequences of second unit oligomers and their complementary sequences (antisense sequences) are shown in Table 5 below.

[0091] In one embodiment, the second unit oligomer comprises a base sequence complementary to: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105; (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105; (c) a base sequence that has 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105 and has a length within ±15% of the length of any one selected base sequence; or (d) a base sequence that is a portion of any one base sequence selected from the group consisting of (a), (b), and (c).

[0092] Here, the base sequence of (c) is a mutant of the base sequence of (a), and the mutant is: (c-1) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and having a length within ±15% of the length of said selected base sequence; (c-2) a base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and having a length within ±14% of the length of said selected base sequence; (c-3) a base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and having a length within ±13% of the length of said selected base sequence; (c-4) a base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and having a length within ±12% of the length of said selected base sequence; (c-5) a base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±11% of the length of any one selected base sequence; (c-6) a base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±10% of the length of any one selected base sequence; (c-7) a base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±9% of the length of any one selected base sequence; (c-8) a base sequence having 92% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±8% of the length of any one selected base sequence; (c-9) a base sequence having a sequence identity of 93% or more with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and having a length within ±7% of the length of any one selected base sequence;(c-10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±6% of the length of any one selected base sequence; (c-11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±5% of the length of any one selected base sequence; (c-12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±4% of the length of any one selected base sequence; (c-13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±3% of the length of any one selected base sequence; (c-14) a base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±2% of the length of any one selected base sequence; (c-15) a base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±1% of the length of any one selected base sequence; and (c-16) a base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, and a length within ±0.5% of the length of any one selected base sequence.

[0093] In one embodiment, the second unit oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210; or (b) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210 and having a length within ±15% of the length of any one selected base sequence, or consists of the base sequence of (a) or (b).

[0094] Here, the base sequence of (b) is a mutant of the base sequence of (a), and the mutant is: (b-1) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and having a length within ±15% of the length of said any one base sequence selected; (b-2) a base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and having a length within ±14% of the length of said any one base sequence selected; (b-3) a base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and having a length within ±13% of the length of said any one base sequence selected; (b-4) a base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±12% of the length of any one selected base sequence; (b-5) a base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±11% of the length of any one selected base sequence; (b-6) a base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±10% of the length of any one selected base sequence; (b-7) a base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±9% of the length of any one selected base sequence; (b-8) A base sequence having 92% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±8% of the length of any one selected base sequence; (b-9) A base sequence having 93% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±7% of the length of any one selected base sequence;(b-10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±6% of the length of any one selected base sequence; (b-11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±5% of the length of any one selected base sequence; (b-12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±4% of the length of any one selected base sequence; (b-13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and a length within ±3% of the length of any one selected base sequence; (b-14) a base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and having a length within ±2% of the length of any one selected base sequence; (b-15) a base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and having a length within ±1% of the length of any one selected base sequence; and (b-16) a base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and having a length within ±0.5% of the length of any one selected base sequence.

[0095] In one embodiment, the second unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210.

[0096] In one embodiment, the second unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 114, 124, 151, 201, 203, and 205.

[0097] In one embodiment, the first unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602, the second unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and the first antisense oligomer comprises the first unit oligomer and the second unit oligomer in this order from the 5' end.

[0098] In one embodiment, the first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, and wherein: the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 201; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 203; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 205; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1239, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 114; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1224, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 124; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1180, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1190, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1212, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151;The first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1222, and the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151.

[0099] Examples of target sequences for the second antisense oligomer of the present invention and their complementary sequences (antisense sequences) are shown in Table 6 below.

[0100] In one embodiment, the second antisense oligomer of the present invention comprises a base sequence complementary to: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298; (b) a base sequence that hybridizes under stringent conditions to a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298; (c) a base sequence that has 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298 and has a length within ±15% of the length of any one selected base sequence; or (d) a base sequence complementary to a portion of any one base sequence selected from the group consisting of (a), (b), and (c).

[0101] Here, the base sequence of (c) is a mutant of the base sequence of (a), and the mutant is: (c-1) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±15% of the length of said any one base sequence selected; (c-2) a base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±14% of the length of said any one base sequence selected; (c-3) a base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±13% of the length of said any one base sequence selected; (c-4) A base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±12% of the length of any one base sequence selected; (c-5) A base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±11% of the length of any one base sequence selected; (c-6) A base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±10% of the length of any one base sequence selected; (c-7) A base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±9% of the length of any one base sequence selected; (c-8) A base sequence having 92% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±8% of the length of any one selected base sequence; (c-9) A base sequence having 93% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±7% of the length of any one selected base sequence;(c-10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±6% of the length of any one base sequence selected; (c-11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±5% of the length of any one base sequence selected; (c-12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±4% of the length of any one base sequence selected; (c-13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±3% of the length of any one base sequence selected; (c-14) A base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±2% of the length of any one selected base sequence; (c-15) A base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±1% of the length of any one selected base sequence; and (c-16) A base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, and having a length within ±0.5% of the length of any one selected base sequence.

[0102] In one embodiment, the second antisense oligomer of the present invention comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090; or (b) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090 and having a length within ±15% of the length of any one selected base sequence, or consists of the base sequence of (a) or (b).

[0103] Here, the base sequence of (b) is a mutant of the base sequence of (a), and the mutant is: (b-1) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and having a length within ±15% of the length of said any one base sequence selected; (b-2) a base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and having a length within ±14% of the length of said any one base sequence selected; (b-3) a base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and having a length within ±13% of the length of said any one base sequence selected; (b-4) A base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±12% of the length of any one base sequence selected; (b-5) A base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±11% of the length of any one base sequence selected; (b-6) A base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±10% of the length of any one base sequence selected; (b-7) A base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±9% of the length of any one base sequence selected; (b-8) A base sequence having 92% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and having a length within ±8% of the length of any one selected base sequence; (b-9) A base sequence having 93% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and having a length within ±7% of the length of any one selected base sequence;(b-10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±6% of the length of any one selected base sequence; (b-11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±5% of the length of any one selected base sequence; (b-12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±4% of the length of any one selected base sequence; (b-13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±3% of the length of any one selected base sequence; (b-14) A base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±2% of the length of any one selected base sequence; (b-15) A base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±1% of the length of any one selected base sequence; and (b-16) A base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090, and a length within ±0.5% of the length of any one selected base sequence.

[0104] In one embodiment, the second antisense oligomer of the present invention comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 4299 to 5090.

[0105] In one embodiment, the second antisense oligomer comprises or consists of a base sequence selected from the group consisting of SEQ ID NOs: 4698, 4702, 4752, 4923, 4926, 4936, 4950, and 4977.

[0106] Examples of target sequences for the third antisense oligomer of the present invention and their complementary sequences (antisense sequences) are shown in Table 7 below.

[0107] In one embodiment, the third antisense oligomer of the present invention comprises a base sequence complementary to: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554; (b) a base sequence that hybridizes under stringent conditions to a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554; (c) a base sequence that has 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554 and has a length within ±15% of the length of any one selected base sequence; or (d) a base sequence complementary to a portion of any one base sequence selected from the group consisting of (a), (b), and (c).

[0108] In one embodiment, the third antisense oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1611-1654, 1664-1707, 1718-1761, 1773-1816, 1829-1872, 1886-1929, 1944-1987, 2003-2046, 2063-2106, 2124-2167, 2186-2229, 2249-2292, 2313-2356, 2378-2421, 2444-2487, and 2511-2554; (b) SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554. A base sequence that hybridizes under stringent conditions to a base sequence complementary to any one of the base sequences selected from the group consisting of: (c) SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554. A base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554, and having a length within ± 15% of the length of any one selected base sequence, or (d) a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c). It contains a base sequence complementary to or consists of said base sequence.In one embodiment, the third antisense oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1614-1654, 1667-1707, 1721-1761, 1776-1816, 1832-1872, 1889-1929, 1947-1987, 2006-2046, 2066-2106, 2127-2167, 2189-2229, 2252-2292, 2316-2356, 2381-2421, 2447-2487, and 2514-2554; (b) SEQ ID NOs: 1614 to 1654, 1667 to 1707, 1721 to 1761, 1776 to 1816, 1832 to 1872, 1889 to 1929, 1947 to 1987, 2006 to 2046, 2066 to 2106, 2127 to 2167, 2189 to 2229, 2252 to 2292, 2316 to 2356, 2381 to 2421, 2447 to 2487, and 2514 to 2554. A base sequence that hybridizes under stringent conditions to a base sequence complementary to any one of the base sequences selected from the group consisting of: (c) SEQ ID NOs: 1614-1654, 1667-1707, 1721-1761, 1776-1816, 1832-1872, 1889-1929, 1947-1987, 2006-2046, 2066-2106, 2127-2167, 2189-2229, 2252-2292, 2316-2356, 2381-2421, 2447-2487, and 2514-2554. A base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1614-1654, 1667-1707, 1721-1761, 1776-1816, 1832-1872, 1889-1929, 1947-1987, 2006-2046, 2066-2106, 2127-2167, 2189-2229, 2252-2292, 2316-2356, 2381-2421, 2447-2487, and 2514-2554, and having a length within ±15% of the length of any one selected base sequence, or (d) a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c). It contains a base sequence complementary to or consists of said base sequence.In one embodiment, the third antisense oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1617-1654, 1670-1707, 1724-1761, 1779-1816, 1835-1872, 1892-1929, 1950-1987, 2009-2046, 2069-2106, 2130-2167, 2192-2229, 2255-2292, 2319-2356, 2384-2421, 2450-2487, and 2517-2554; (b) SEQ ID NOs: 1617-1654, 1670-1707, 1724-1761, 1779-1816, 1835-1872, 1892-1929, 1950-1987, 2009-2046, 2069-2106, 2130-2167, 2192-2229, 2255-2292, 2319-2356, 2384-2421, 2450-2487, and 2517-2554. A base sequence that hybridizes under stringent conditions to a base sequence complementary to any one of the base sequences selected from the group consisting of: (c) SEQ ID NOs: 1617-1654, 1670-1707, 1724-1761, 1779-1816, 1835-1872, 1892-1929, 1950-1987, 2009-2046, 2069-2106, 2130-2167, 2192-2229, 2255-2292, 2319-2356, 2384-2421, 2450-2487, and 2517-2554. A base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1617-1654, 1670-1707, 1724-1761, 1779-1816, 1835-1872, 1892-1929, 1950-1987, 2009-2046, 2069-2106, 2130-2167, 2192-2229, 2255-2292, 2319-2356, 2384-2421, 2450-2487, and 2517-2554, and having a length within ±15% of the length of any one selected base sequence, or (d) a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c). It contains a base sequence complementary to or consists of said base sequence.

[0109] Here, the base sequence of (c) is a mutant of the base sequence of (a), and the mutant is: (c-1) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and having a length within ±15% of the length of said any one base sequence selected; (c-2) a base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and having a length within ±14% of the length of said any one base sequence selected; (c-3) a base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and having a length within ±13% of the length of said any one base sequence selected; (c-4) A base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±12% of the length of any one selected base sequence; (c-5) A base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±11% of the length of any one selected base sequence; (c-6) A base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±10% of the length of any one selected base sequence; (c-7) A base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±9% of the length of any one selected base sequence; (c-8) A base sequence having 92% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±8% of the length of any one selected base sequence; (c-9) A base sequence having 93% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±7% of the length of any one selected base sequence;(c-10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±6% of the length of any one base sequence selected; (c-11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±5% of the length of any one base sequence selected; (c-12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±4% of the length of any one base sequence selected; (c-13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±3% of the length of any one base sequence selected; (c-14) A base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±2% of the length of any one selected base sequence; (c-15) A base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±1% of the length of any one selected base sequence; and (c-16) A base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554, and a length within ±0.5% of the length of any one selected base sequence.

[0110] In one embodiment, the third antisense oligomer comprises: (a) any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506; or (b) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506 and having a length within ±15% of the length of any one selected base sequence, or consists of the base sequence of (a) or (b).

[0111] Here, the base sequence of (b) is a mutant of the base sequence of (a), and the mutant is: (b-1) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and having a length within ±15% of the length of said any one base sequence selected; (b-2) a base sequence having 86% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and having a length within ±14% of the length of said any one base sequence selected; (b-3) a base sequence having 87% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and having a length within ±13% of the length of said any one base sequence selected; (b-4) A base sequence having 88% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±12% of the length of any one base sequence selected; (b-5) A base sequence having 89% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±11% of the length of any one base sequence selected; (b-6) A base sequence having 90% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±10% of the length of any one base sequence selected; (b-7) A base sequence having 91% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±9% of the length of any one base sequence selected; (b-8) A base sequence having a sequence identity of 92% or more with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±8% of the length of any one selected base sequence; (b-9) A base sequence having a sequence identity of 93% or more with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±7% of the length of any one selected base sequence;(b-10) A base sequence having 94% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±6% of the length of any one base sequence selected; (b-11) A base sequence having 95% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±5% of the length of any one base sequence selected; (b-12) A base sequence having 96% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±4% of the length of any one base sequence selected; (b-13) A base sequence having 97% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and a length within ±3% of the length of any one base sequence selected; (b-14) A base sequence having 98% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and having a length within ±2% of the length of any one selected base sequence; (b-15) A base sequence having 99% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and having a length within ±1% of the length of any one selected base sequence; and (b-16) A base sequence having 99.5% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506, and having a length within ±0.5% of the length of any one selected base sequence.

[0112] In one embodiment, the third antisense oligomer of the present invention comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506.

[0113] In one embodiment, the third antisense oligomer comprises or consists of a base sequence selected from the group consisting of SEQ ID NOs: 3060, 3065, 3077, 3082, 3087, 3090, 3096, 3108, 3119, and 3320. In one embodiment, the third antisense oligomer comprises or consists of a base sequence selected from the group consisting of SEQ ID NOs: 3077, 3082, 3087, 3090, 3096, 3108, and 3119. In one embodiment, the third antisense oligomer comprises or consists of a base sequence selected from the group consisting of SEQ ID NOs: 3082, 3087, 3090, 3096, 3108, and 3119.

[0114] The combination of the first unit oligomer and second unit oligomer constituting the first antisense oligomer of the present invention, and the second antisense oligomer of the present invention (and optionally the third antisense oligomer of the present invention) is not limited, and any combination can be used.

[0115] In one embodiment, the first antisense oligomer comprises a first unit oligomer and the second unit oligomer in this order from the 5' end, and wherein: the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 201, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 203, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 205, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1239, the second unit oligomer comprises the base sequence of SEQ ID NO: 114, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1224, the second unit oligomer comprises the base sequence of SEQ ID NO: 124, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1180, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, or the first unit oligomer comprises the base sequence of SEQ ID NO: 1190, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950,the first unit oligomer comprises the base sequence of SEQ ID NO: 1212, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1222, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4698; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4702; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4752; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4923; the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4926, or the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4936,the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4977, or the first unit oligomer comprises the base sequence of SEQ ID NO: 1180, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4977.

[0116] In one embodiment, the first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, wherein the first unit oligomer comprises the base sequence of any of SEQ ID NOs: 907 to 1602, the second unit oligomer comprises the base sequence of any of SEQ ID NOs: 106 to 210, and the second antisense oligomer comprises the base sequence of any of SEQ ID NOs: 4299 to 5090. In one embodiment, the first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, wherein the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950 or 4880 (preferably 4950).

[0117] In one embodiment, the first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, and the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082, the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 201, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 203, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 205, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082,the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1239, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 114, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1224, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 124, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1180, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082, or the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1190, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082, the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1212, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082,the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1222, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082, or the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3060, the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3065; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3077; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3087,the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3090; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3096; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3108; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3119; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4950, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3320,the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4698, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4702, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4752, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4923, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4926, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082,the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4936, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4977, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3082; the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4977, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3096, or the first unit oligomer comprises or consists of the base sequence of SEQ ID NO: 1180, the second unit oligomer comprises or consists of the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 4977, and the third antisense oligomer comprises or consists of the base sequence of SEQ ID NO: 3096.

[0118] In one embodiment, the first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, wherein the first unit oligomer comprises a base sequence of any of SEQ ID NOs: 907 to 1602, the second unit oligomer comprises a base sequence of any of SEQ ID NOs: 106 to 210, the second antisense oligomer comprises a base sequence of any of SEQ ID NOs: 4299 to 5090, and the third antisense oligomer comprises a base sequence of any of SEQ ID NOs: 2555 to 3506. In one embodiment, the first antisense oligomer comprises a first unit oligomer and a second unit oligomer in this order from the 5' end, wherein the first unit oligomer comprises the base sequence of SEQ ID NO: 1201, the second unit oligomer comprises the base sequence of SEQ ID NO: 151, the second antisense oligomer comprises the base sequence of SEQ ID NO: 4950 or 4880 (preferably 4950), and the third antisense oligomer comprises the base sequence of SEQ ID NO: 3082, 3090, or 3096.

[0119] The antisense oligomer of the present invention (including the linked antisense oligomer of the present invention) may be an oligonucleotide, a morpholino oligomer, or a peptide nucleic acid (PNA) oligomer (hereinafter, these will also be referred to as the "antisense oligonucleotide of the present invention," the "antisense morpholino oligomer of the present invention," or the "antisense peptide nucleic acid oligomer of the present invention," respectively).

[0120] The antisense oligonucleotide of the present invention is an antisense oligomer having nucleotides as its constituent units, and such nucleotides may be any of ribonucleotides, deoxyribonucleotides, or modified nucleotides.

[0121] A modified nucleotide refers to a ribonucleotide or deoxyribonucleotide in which all or part of the nucleic acid base, sugar moiety, and phosphate linkage moiety that constitute the ribonucleotide or deoxyribonucleotide have been modified.

[0122] Examples of nucleic acid bases include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (e.g., 5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 1-methyluracil, 1-methylhydroxy ... Examples of amino acids include xanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, and xanthine.

[0123] Modifications of the sugar moiety include, for example, modification of the 2'-position of ribose and modifications of other parts of the sugar. Modifications of the 2'-position of ribose include, for example, replacing the -OH group at the 2'-position of ribose with -OR, -R, -R'OR, -SH, -SR, -NH ２ , -NHR, -NR ２ , -N ３, -CN, -F, -Cl, -Br, or -I. Here, R represents alkyl or aryl. R' represents alkylene. Modifications of other sugar moieties include, but are not limited to, substitution of O at the 4'-position of ribose or deoxyribose with S, and bridging of the 2' and 4'-positions of the sugar, such as LNA (Locked Nucleic Acid) or ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids).

[0124] Modifications of the phosphate linkage moiety include, for example, substitution of a phosphodiester bond with a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, or a boranophosphate bond (see, for example, Enya et al.: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (see, for example, Republished Patent Publication No. 2006 / 129594 and Republished Patent Publication No. 2006 / 038608).

[0125] In this specification, the alkyl is preferably a linear or branched alkyl having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, and isohexyl. The alkyl may be substituted, and examples of such substituents include halogen, alkoxy, cyano, and nitro, and the alkyl may be substituted with 1 to 3 of these.

[0126] In this specification, the cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, and cyclododecyl.

[0127] In this specification, halogen includes fluorine, chlorine, bromine, and iodine.

[0128] In this specification, examples of alkoxy include straight-chain or branched-chain alkoxy having 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy, etc. In particular, alkoxy having 1 to 3 carbon atoms is preferred.

[0129] In this specification, the aryl is preferably an aryl having 6 to 10 carbon atoms. Specific examples include phenyl, α-naphthyl, and β-naphthyl. Phenyl is particularly preferred. The aryl may be substituted, and examples of such substituents include alkyl, halogen, alkoxy, cyano, and nitro, and the aryl may be substituted with 1 to 3 of these.

[0130] In this specification, the alkylene is preferably a linear or branched alkylene having 1 to 6 carbon atoms, such as methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene.

[0131] As used herein, acyl includes straight-chain or branched-chain alkanoyl or aroyl. Examples of alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl, and hexanoyl. Examples of aroyl include benzoyl, toluoyl, and naphthoyl. Such aroyl may be substituted at any substitutable position, and may be substituted with alkyl.

[0132] The antisense oligonucleotide of the present invention is preferably an antisense oligomer of the present invention having a constituent unit represented by the following general formula, in which the —OH group at the 2'-position of ribose is substituted with methoxy and the phosphate linkage is a phosphorothioate linkage: (In the formula, Base represents a nucleic acid base.)

[0133] The antisense oligonucleotides of the present invention can be easily synthesized using various automated synthesizers (e.g., AKTA oligopilot plus 10 / 100 (GE Healthcare)), or can be produced by outsourcing to a third party (e.g., Promega or Takara).

[0134] The antisense morpholino oligomer of the present invention is an antisense oligomer having a group represented by the following general formula as a constituent unit. (In the formula, Base has the same meaning as defined above; W represents a group represented by any of the following formulas: (Wherein, X is —CH ２ R １ , —O—CH ２ R １ , -S-CH ２ R １ , -NR ２ R ３ or F; R １ represents H or alkyl; R ２ and R ３ are the same or different and represent H, alkyl, cycloalkyl, or aryl; Y １ is O, S, CH ２ , or NR １ represents Y ２ is O, S, or NR １ Z represents O or S.

[0135] Examples of morpholino monomer compounds used to synthesize the antisense morpholino oligomers of the present invention include, but are not limited to, morpholino monomer compound (A), morpholino monomer compound (C), morpholino monomer compound (T), and morpholino monomer compound (G) shown in Table 8.

[0136]

[0137] In the present invention, the morpholino oligomer is preferably an oligomer having a group represented by the following formula as a constituent unit (phosphorodiamidate morpholino oligomer (hereinafter referred to as "PMO")). (In the formula, Base, R ２ , R ３ has the same meaning as above.)

[0138] Morpholino oligomers can be produced, for example, according to the methods described in WO 1991 / 009033 or WO 2009 / 064471. In particular, PMOs can be produced according to the methods described in WO 2009 / 064471 or WO 2013 / 100190.

[0139] The antisense peptide nucleic acid oligomer of the present invention is an antisense oligomer having a group represented by the following general formula as a constituent unit: (wherein, Base has the same meaning as defined above.) Peptide nucleic acid oligomers can be produced, for example, according to the following literature: 1) PE Nielsen, M. Egholm, RH Berg, O. Buchardt, Science, 254, 1497 (1991)2) M. Egholm, O. Buchardt, PE Nielsen, RH Berg, JACS, 114, 1895 (1992)3) KL Dueholm, M. Egholm, C. Behrens, L. Christensen, HF Hansen, T. Vulpius, KH Petersen, RH Berg, PE Nielsen, O. Buchardt, J. Org. Chem., 59, 5767 (1994) 4) L. Christensen, R. Fitzpatrick, B. Gildea, KH Petersen, HF Hansen, T. Koch, M. Egholm, O. Buchardt, PE Nielsen, J. Coull, RH Berg, J. Pept. Sci., 1, 175 (1995)5) T. Koch, HF Hansen, P. Andersen, T. Larsen, HG Batz, K. Otteson, H. Orum, J. Pept. Res., 49, 80 (1997)

[0140] The antisense oligomer of the present invention (including the linked antisense oligomer of the present invention) may be in the form of a pharmaceutically acceptable salt, a hydrate, or a hydrate of a pharmaceutically acceptable salt thereof.

[0141] Examples of pharmaceutically acceptable salts of the antisense oligomers of the present invention include alkali metal salts such as sodium salt, potassium salt, and lithium salt, and alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; ammonium salt; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, pipette salt, and the like. Examples of suitable salts include organic amine salts such as tetramethylammonium salts and tris(hydroxymethyl)aminomethane salts; hydrohalide salts such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate and phosphate; lower alkane sulfonate salts such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; arylsulfonate salts such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate and aspartate. These salts can be prepared by known methods. Alternatively, the antisense oligomer of the present invention may be in the form of a hydrate.

[0142] The third antisense oligomer of the present invention can function as a suppressor antisense oligomer. In the present invention, a suppressor antisense oligomer refers to an antisense oligomer that suppresses single exon skipping (hereinafter referred to as "single skipping"). By suppressing single skipping, the suppressor antisense oligomer can enhance the effect of multi-exon skipping by the antisense oligomer. Therefore, a combination of the present invention containing a third antisense oligomer can have a higher multi-exon skipping effect than one that does not contain the third antisense oligomer.

[0143] Specifically, the third antisense oligomer of the present invention can suppress the single skipping of any one exon selected from the group consisting of exons 45 to 55 of human dystrophin pre-mRNA. More specifically, the third antisense oligomer of the present invention can suppress the single skipping of exon 45 of human dystrophin pre-mRNA.

[0144] The third antisense oligomer of the present invention can suppress single-skipping by targeting, for example, a splicing silencer sequence, a branch site sequence, or a splice site sequence on human dystrophin pre-mRNA and thereby inhibiting splicing. The third antisense oligomer of the present invention can reduce the efficiency of single-skipping of a target exon compared to a control.

[0145] In one embodiment, the third antisense oligomer of the present invention targets the recognition sequence of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which is a splicing silencer sequence. A splicing silencer sequence is a base sequence element on pre-mRNA that functions to suppress exon recognition. The target sequence of the third antisense oligomer is as described herein.

[0146] Whether or not the suppressor antisense oligomer enhances the effect of multi-exon skipping can be confirmed by preparing (i) an experimental system for multi-exon skipping using only the antisense oligomer of the present invention, and (ii) an experimental system for multi-exon skipping using the antisense oligomer of the present invention and a suppressor antisense oligomer, while maintaining the same other conditions, and observing the difference between the multi-exon skipping effect obtained in experimental system (ii) and the multi-exon skipping effect obtained in experimental system (i).

[0147] [Method for Producing PMO] The antisense oligomer of the present invention may be a PMO. One embodiment of the PMO is, for example, a compound represented by the following general formula (I) (hereinafter referred to as PMO(I)): (In the formula, each Base, R ２ , R ３ has the same meaning as above; and n is any integer in the range of 1 to 99, preferably any integer in the range of 18 to 28.

[0148] PMO(I) can be produced according to known methods (see, for example, WO 2009 / 064471 or WO 2013 / 100190).

[0149] Furthermore, the 5'-end of the antisense oligomer of the present invention may be any of the groups represented by the following chemical formulae (1) to (3), preferably (3) -OH. Hereinafter, the groups represented by (1), (2), and (3) above will be referred to as "group (1)," "group (2)," and "group (3)," respectively.

[0150] The antisense oligomer of the present invention may be conjugated with a functional peptide (e.g., a membrane-permeable peptide for improving the efficiency of delivery to target cells) or an antibody fragment (e.g., the Fab of an antibody against a muscle cell-specific receptor such as the transferrin receptor) for the purpose of improving efficacy (see WO 2008 / 036127, WO 2009 / 005793, WO 2012 / 150960, WO 2016 / 187425, WO 2018 / 118662, WO 2011 / 013700, WO 2018 / 118599, WO 2018 / 118627, JP 2022-47613, JD Ramsey, NH Flynn, Pharmacology & Therapeutics 154, 78-86 (2015), MK Tsoumpra et al., EBioMedicine, 45, 630-645 (2019), WO 2020 / 028832, WO 2021 / 142307, WO 2021 / 142313, WO 2022 / 020107, WO 2022 / 020108). The binding site is not particularly limited, but preferably the 5' or 3' end of the antisense oligomer is bound to the amino or carboxyl terminus of the functional peptide or antibody fragment. In another embodiment, the antisense oligomer of the present invention and the functional peptide or antibody fragment may form a complex via a linker. The linker is not particularly limited, but preferably the 5' or 3' end of the antisense oligomer is bound to one end of the linker, and the amino or carboxyl terminus of the functional peptide or antibody fragment is bound to the other end of the linker. In addition, additional amino acids may be present between the functional peptide or antibody fragment and the linker.

[0151] Pharmaceutical Use In one embodiment, the present invention provides a pharmaceutical composition (hereinafter referred to as the "pharmaceutical composition of the present invention") comprising a first antisense oligomer and a second antisense oligomer of the present invention (including pharmaceutically acceptable salts or hydrates thereof). The pharmaceutical composition of the present invention may further comprise a third antisense oligomer of the present invention (including pharmaceutically acceptable salts or hydrates thereof) and / or a pharmaceutically acceptable carrier.

[0152] In one embodiment, the present invention provides a pharmaceutical combination comprising a pharmaceutical composition comprising a first antisense oligomer of the present invention and a pharmaceutical composition comprising a second antisense oligomer of the present invention (hereinafter also referred to as the "pharmaceutical combination of the present invention"). The pharmaceutical combination of the present invention may further comprise a third antisense oligomer and / or a pharmaceutically acceptable carrier.

[0153] The pharmaceutical composition of the present invention includes any combination of the antisense oligomers of the present invention. The combined pharmaceutical of the present invention also includes any combination of the antisense oligomers of the present invention. Details of the combination of antisense oligomers are as described herein.

[0154] In one embodiment, the antisense oligomers in the combination of the present invention are contained in a single pharmaceutical composition and administered simultaneously. In another embodiment, the antisense oligomers in the combination of the present invention are contained in multiple pharmaceutical compositions (combined pharmaceutical compositions of the present invention) and administered separately (simultaneously or sequentially). As used herein, "simultaneously" administering multiple pharmaceutical compositions means administering multiple pharmaceutical compositions at the same time. As used herein, "sequentially" administering multiple pharmaceutical compositions means administering them at different times. Specifically, one pharmaceutical composition can be administered before or after another pharmaceutical composition, and the administration interval in this case may be, but is not limited to, several minutes, several hours, or several days.

[0155] The pharmaceutical composition of the present invention and the pharmaceutical combination of the present invention can be used, respectively, for the treatment of, for example, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, oncopharyngeal muscular dystrophy, cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), Alport syndrome, etc. Furthermore, the pharmaceutical combination of the present invention and the pharmaceutical composition of the present invention can be administered to human patients, particularly human patients with muscular dystrophy. Patients to whom the pharmaceutical combination of the present invention or the pharmaceutical composition of the present invention can be administered may be human patients with mutations in the dystrophin gene that are subject to skipping of two or more exons selected from the group consisting of exons 45 to 55. As used herein, the mutations that are the subject of exon skipping are not limited, but include, for example, deletions in exons of the dystrophin gene (e.g., exon 46 deletion, exon 46-47 deletion, exon 46-48 deletion, exon 46-50 deletion, exon 46-51 deletion, exon 46-52 deletion, exon 46-53 deletion, exon 46-55 deletion, exon 47-50 deletion, exon 48 deletion, exon 49 deletion, exon 50 deletion, exon 51 deletion, exon 52 deletion, exon 53 deletion, exon 54 deletion, exon 55 deletion, exon 56 deletion, exon 57 deletion, exon 58 deletion, exon 59 deletion, exon 60 deletion, exon 61 deletion, exon 62 deletion, exon 63 deletion, exon 64 deletion, exon 65 deletion, exon 66 deletion, exon 67 deletion, exon 68 deletion, exon 69 deletion, exon 70 deletion, exon 71 deletion, exon 72 deletion, exon 73 deletion, exon 74 deletion, exon 75 deletion, exon 76 deletion, exon 77 deletion, exon 78 deletion, exon 79 deletion, exon 80 deletion, exon 81 deletion, exon 82 deletion, exon 83 deletion, exon 84 deletion, exon 85 deletion, exon 86 deletion, exon 87 deletion, exon 88 deletion, exon 89 deletion, exon 90 deletion, exon 91 deletion, exon 92 deletion, exon 93 deletion, exon 94 deletion, exon 95 deletion, exon 96 deletion, exon 97 deletion, exon 98 deletion, exon 99 deletion, exon 100 deletion, exon 101 deletion Examples of such deletions include patients with exon 47-52 deletion, exon 48-50 deletion, exon 48-52 deletion, exon 48-54 deletion, exon 49-50 deletion, exon 49-52 deletion, exon 49-54 deletion, exon 50 deletion, exon 50-52 deletion, exon 51 deletion, exon 51-53 deletion, exon 52 deletion, exon 53 deletion, or exon 53-54 deletion.

[0156] In one aspect of the present invention, there is provided a method for treating muscular dystrophy, comprising the step of administering the antisense oligomer combination of the present invention to a patient with muscular dystrophy. In another aspect of the present invention, there is provided a method for treating muscular dystrophy, comprising the step of administering the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention to a patient with muscular dystrophy. The treatment method may involve skipping any two or more consecutive exons in numerical order selected from the group consisting of exon 45 to exon 55 of human dystrophin pre-mRNA. In the treatment method, the patient with muscular dystrophy may be a patient with a mutation in the dystrophin gene that is subject to exon 45-55 skipping. The patient may be human, or may be a human patient with a mutation in the dystrophin gene that is subject to exon 45-55 skipping.

[0157] The present invention further provides use of the antisense oligomer combination of the present invention, or the pharmaceutical composition of the present invention, or the pharmaceutical combination of the present invention in the manufacture of a medicament for treating muscular dystrophy.

[0158] The present invention further provides an antisense oligomer combination of the present invention, a pharmaceutical composition of the present invention, or a combination drug of the present invention for use in treating muscular dystrophy. The treatment may involve skipping any two or more consecutive exons in numerical order selected from the group consisting of exon 45 to exon 55 of human dystrophin pre-mRNA. In the treatment, the muscular dystrophy patient may be a patient with a mutation in the dystrophin gene that is subject to exon 45-55 skipping. The patient may be human, or may be a human patient with a mutation in the dystrophin gene that is subject to exon 45-55 skipping.

[0159] The administration form of the antisense oligomer combination of the present invention, the pharmaceutical composition of the present invention, or the combination drug of the present invention is not particularly limited as long as it is a pharmaceutically acceptable administration form and can be selected depending on the treatment method, but from the viewpoint of ease of delivery to muscle tissue, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, oral administration, intratissue administration, transdermal administration, etc. are preferred. In addition, the dosage form that the composition of the present invention can take is not particularly limited, and examples thereof include various injections, oral agents, infusions, inhalants, ointments, lotions, etc.

[0160] When the antisense oligomer of the present invention is administered to a patient with muscular dystrophy, the composition of the present invention preferably contains a carrier that promotes delivery of the oligomer to muscle tissue. Such a carrier is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include cationic carriers such as cationic liposomes and cationic polymers, or carriers that utilize viral envelopes. Examples of cationic liposomes include liposomes formed from 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids as essential components (hereinafter referred to as "Liposome A"), Oligofectamine (registered trademark) (Invitrogen), Lipofectin (registered trademark) (Invitrogen), Lipofectamine (registered trademark) (Invitrogen), Lipofectamine 2000 (registered trademark) (Invitrogen), DMRIE-C (registered trademark) (Invitrogen), GeneSilencer (registered trademark) (Gene Therapy Systems), TransMessenger (registered trademark) (QIAGEN), TransIT TKO (registered trademark) (Mirus), and Nucleofector II (Lonza). Among these, Liposome A is preferred. Examples of cationic polymers include JetSI (registered trademark) (manufactured by Qbiogene) and Jet-PEI (registered trademark) (polyethyleneimine, manufactured by Qbiogene). Examples of carriers utilizing viral envelopes include GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Kaisha). Alternatively, the pharmaceutical device described in Japanese Patent No. 2924179 and the cationic carriers described in Republished Patent Publication Nos. 2006 / 129594 and 2008 / 096690 can also be used.

[0161] The concentration of the antisense oligomer of the present invention contained in the pharmaceutical composition of the present invention and / or the combination drug of the present invention varies depending on the type of carrier, etc., but is suitably in the range of 0.1 nM to 100 μM, preferably in the range of 1 nM to 10 μM, and more preferably in the range of 10 nM to 1 μM. Furthermore, the weight ratio of the antisense oligomer of the present invention to the carrier contained in the composition of the present invention (carrier / antisense oligomer of the present invention) varies depending on the properties of the oligomer, the type of the carrier, etc., but is suitably in the range of 0.1 to 100, preferably in the range of 1 to 50, and more preferably in the range of 10 to 20.

[0162] In one embodiment, each antisense oligomer in the combination of the present invention is contained in a single pharmaceutical composition and administered simultaneously. In another embodiment, each antisense oligomer in the combination of the present invention is contained in multiple pharmaceutical compositions (combination pharmaceuticals of the present invention) and administered separately (simultaneously or sequentially). When each antisense oligomer in the combination of the present invention is contained in one or more pharmaceutical compositions, the concentration of each antisense oligomer is as follows:

[0163] The pharmaceutical composition of the present invention and / or the combined pharmaceutical of the present invention may be in the form of an aqueous solution. In this case, the pharmaceutical composition of the present invention and / or the combined pharmaceutical of the present invention may contain each antisense oligomer of the present invention at a concentration of 2.5 to 500 mg / mL, 5 to 450 mg / mL, 10 to 400 mg / mL, 15 to 350 mg / mL, 20 to 300 mg / mL, 20 to 250 mg / mL, 20 to 200 mg / mL, 20 to 150 mg / mL, 20 to 100 mg / mL, 20 to 50 mg / mL, 20 to 40 mg / mL, 20 to 30 mg / mL, 23 to 27 mg / mL, 24 to 26 mg / mL, or 25 mg / mL. Alternatively, the pharmaceutical composition of the present invention and / or the combination drug of the present invention may contain each antisense oligomer of the present invention at a concentration of 10 to 100 mg / mL, 15 to 95 mg / mL, 20 to 80 mg / mL, 25 to 75 mg / mL, 30 to 70 mg / mL, 35 to 65 mg / mL, 40 to 60 mg / mL, 45 to 55 mg / mL, 47 to 53 mg / mL, 48 to 52 mg / mL, 49 to 51 mg / mL, or 50 mg / mL.

[0164] The pharmaceutical composition of the present invention and / or the combined pharmaceutical of the present invention may be in a dry form. In this case, to prepare the pharmaceutical composition of the present invention and / or the combined pharmaceutical of the present invention in the form of an aqueous solution, for example, 125 mg or 250 mg of each antisense oligomer of the present invention in a dry form may be mixed with 0.5 mL to 100 mL of water (corresponding to a concentration of each antisense oligomer of the present invention of 1.25 mg / mL to 250 mg / mL or 2.5 mg / mL to 500 mg / mL), preferably 1 mL to 50 mL of water (corresponding to a concentration of each antisense oligomer of the present invention of 2.5 mg / mL to 125 mg / mL or 5 mg / mL to 250 mg / mL), and more preferably 5 mL to 10 mL of water (corresponding to a concentration of each antisense oligomer of the present invention of 12.5 mg / mL to 25 mg / mL or 25 mg / mL to 50 mg / mL).

[0165] When each antisense oligomer in the combination of the present invention is contained in one or more pharmaceutical compositions, the total concentration of the antisense oligomers is as follows: When the pharmaceutical composition of the present invention and / or the combined drug of the present invention is in the form of an aqueous solution, the pharmaceutical composition of the present invention and / or the combined drug of the present invention may contain the antisense oligomer of the present invention at a concentration of 2.5 to 500 mg / mL, 5 to 450 mg / mL, 10 to 400 mg / mL, 15 to 350 mg / mL, 20 to 300 mg / mL, 20 to 250 mg / mL, 20 to 200 mg / mL, 20 to 150 mg / mL, 20 to 100 mg / mL, 20 to 50 mg / mL, 20 to 40 mg / mL, 20 to 30 mg / mL, 23 to 27 mg / mL, 24 to 26 mg / mL, or 25 mg / mL, or at a concentration of 5 to 1000 mg / mL, 10 to 900 mg / mL, 20 to 800 mg / mL, 30 to 700 mg / mL, 40 to 600 mg / mL, or at a concentration of 2.5 to 500 mg / mL, 5 to 450 mg / mL, 10 to 400 mg / mL, 15 to 350 mg / mL, 20 to 300 mg / mL, 20 to 250 mg / mL, 20 to 200 mg / mL, 20 to 150 mg / mL, 20 to 100 mg / mL, 20 to 50 mg / mL, 20 to 40 mg / mL, 20 to 30 mg / mL, 23 to 27 mg / mL, 24 to 26 mg / mL, or 25 mg / mL. mL, 40-500 mg / mL, 40-400 mg / mL, 40-300 mg / mL, 40-200 mg / mL, 40-100 mg / mL, 40-80 mg / mL, 40-60 mg / mL, 46-54 mg / mL, 48-52 mg / mL, or 50 mg / mL, or 7.5-1500 mg / mL, 15-1350 mg / mL, 30-1200 mg / mL The composition may contain a total concentration of 45-1150 mg / mL, 60-900 mg / mL, 60-750 mg / mL, 60-600 mg / mL, 60-450 mg / mL, 60-300 mg / mL, 60-150 mg / mL, 60-120 mg / mL, 60-90 mg / mL, 69-81 mg / mL, 72-78 mg / mL, or 75 mg / mL.Alternatively, the pharmaceutical composition of the present invention and / or the combination drug of the present invention may contain the antisense oligomer of the present invention at a concentration of 10 to 100 mg / mL, 15 to 95 mg / mL, 20 to 80 mg / mL, 25 to 75 mg / mL, 30 to 70 mg / mL, 35 to 65 mg / mL, 40 to 60 mg / mL, 45 to 55 mg / mL, 47 to 53 mg / mL, 48 to 52 mg / mL, 49 to 51 mg / mL, or 50 mg / mL, or at a concentration of 20 to 200 mg / mL, 30 to 190 mg / mL, 40 to 160 mg / mL, 50 to 150 mg / mL, 60 to 140 mg / mL, 70 to 80 mg / mL, 80 to 90 mg / mL, 90 to 100 mg / mL, 10 to 120 mg / mL, 11 to 140 mg / mL, 15 to 160 mg / mL, 17 to 180 mg / mL, 19 to 210 mg / mL, 22 to 230 mg / mL, 24 to 250 mg / mL, 26 to 270 mg / mL, 27 to 280 mg / mL, 28 to 300 mg / mL, 31 to 320 mg / mL, 33 to 340 mg / mL, 35 to 360 mg / mL, 37 to 380 mg / mL, 39 to 400 mg / mL, 41 to 420 mg / mL, 42 to 430 mg / mL, 43 to 440 mg / mL, 44 to 450 mg / mL, 45 to 55 mg / mL, 47 to 53 mg / It may contain a total concentration of 130 mg / mL, 80-120 mg / mL, 90-110 mg / mL, 94-106 mg / mL, 96-104 mg / mL, 98-102 mg / mL, or 100 mg / mL, or 30-300 mg / mL, 45-285 mg / mL, 60-240 mg / mL, 75-225 mg / mL, 90-210 mg / mL, 105-195 mg / mL, 120-180 mg / mL, 130-165 mg / mL, 141-159 mg / mL, 144-156 mg / mL, 147-153 mg / mL, or 150 mg / mL.

[0166] When the pharmaceutical composition of the present invention and / or the combined drug of the present invention is in a dry form, to prepare the pharmaceutical composition of the present invention and / or the combined drug of the present invention in the form of an aqueous solution, for example, a total of 125 mg or 250 mg of the antisense oligomer of the present invention in a dry form is mixed with 0.5 mL to 100 mL of water (corresponding to a total concentration of the antisense oligomer of the present invention of 1.25 mg / mL to 250 mg / mL or 2.5 mg / mL to 500 mg / mL), preferably with 1 mL to 50 mL of water (corresponding to a total concentration of the antisense oligomer of the present invention of 2.5 mg / mL to 125 mg / mL or corresponds to a total concentration of the antisense oligomer of the present invention of 5 mg / mL to 250 mg / mL), more preferably mixed with 5 mL to 10 mL of water (corresponding to a total concentration of the antisense oligomer of the present invention of 12.5 mg / mL to 25 mg / mL or 25 mg / mL to 50 mg / mL), or, for example, a total of 250 mg or 500 mg of the antisense oligomer of the present invention in dry form is mixed with 0.5 mL to 100 mL of water (corresponding to a total concentration of the antisense oligomer of the present invention of 2.5 mg / mL to 500 mg / mL or 5 mg / mL to 1000 mg / mL). -total concentration), preferably mixed with 1 mL to 50 mL of water (corresponding to a total concentration of the antisense oligomer of the present invention of 5 mg / mL to 250 mg / mL or 10 mg / mL to 500 mg / mL), more preferably mixed with 5 mL to 10 mL of water (corresponding to a total concentration of the antisense oligomer of the present invention of 25 mg / mL to 50 mg / mL or 50 mg / mL to 100 mg / mL), or for example, by mixing a total of 375 mg or 750 mg of the antisense oligomer of the present invention in dry form with 0.5 mL to 100 mL of water ( The antisense oligomers of the present invention may be used in a concentration range of 3.75 mg / mL to 750 mg / mL or 7.5 mg / mL to 1500 mg / mL, preferably mixed with 1 mL to 50 mL of water (corresponding to a total antisense oligomer concentration of 7.5 mg / mL to 375 mg / mL or 15 mg / mL to 750 mg / mL), more preferably mixed with 5 mL to 10 mL of water (corresponding to a total antisense oligomer concentration of 37.5 mg / mL to 75 mg / mL or 75 mg / mL to 150 mg / mL).

[0167] In addition to the antisense oligomer of the present invention and the above-mentioned carrier, the pharmaceutical composition and / or combination drug of the present invention may optionally contain pharmaceutically acceptable additives. Examples of such additives include emulsifiers (e.g., fatty acids having 6 to 22 carbon atoms or pharmaceutically acceptable salts thereof, albumin, and dextran), stabilizers (e.g., cholesterol, phosphatidic acid, mannitol, and sorbitol), isotonicity agents (e.g., sodium chloride, glucose, maltose, lactose, sucrose, and trehalose), and pH adjusters (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, and triethanolamine). These additives may be used alone or in combination. The content of the additive in the composition of the present invention is suitably 90% by weight or less, preferably 70% by weight or less, and more preferably 50% by weight or less.

[0168] The pharmaceutical composition and / or combination drug of the present invention can be prepared by adding the antisense oligomer of the present invention to a dispersion of a carrier and stirring appropriately. The additives can be added at any appropriate step, either before or after the addition of the antisense oligomer of the present invention. The aqueous solvent that can be used when adding the antisense oligomer of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution. In such cases, the conditions, such as pH and temperature, can be appropriately selected by those skilled in the art.

[0169] The pharmaceutical composition of the present invention and / or the combination drug of the present invention can be, for example, a liquid formulation or a lyophilized formulation thereof. Such a lyophilized formulation can be prepared by lyophilizing the composition of the present invention in liquid form using standard methods. For example, after appropriate sterilization of the composition of the present invention in liquid form, a predetermined amount can be dispensed into vials, pre-frozen for approximately 2 hours at a temperature ranging from approximately −40°C to −20°C, primary dried under reduced pressure at a temperature ranging from approximately 0°C to 10°C, and then secondary dried under reduced pressure at a temperature ranging from approximately 15°C to 25°C, thereby achieving lyophilization. The interior of the vial can then generally be purged with nitrogen gas and the vial can be sealed to obtain a lyophilized formulation of the composition of the present invention.

[0170] The lyophilized formulation of the pharmaceutical composition of the present invention and / or the combination drug of the present invention can generally be reconstituted and used by adding any appropriate solution (reconstitution liquid). Examples of such reconstitution liquid include water for injection, physiological saline, and other general infusion solutions. The volume of this reconstitution liquid varies depending on the application, etc., and is not particularly limited, but is suitably 0.5 to 2 times the volume of the liquid before lyophilization, or 500 mL or less.

[0171] The dosage of the pharmaceutical composition of the present invention and / or the combination drug of the present invention is preferably adjusted taking into consideration the type of antisense oligomer of the present invention contained therein, the dosage form, the patient's condition such as age and body weight, the route of administration, and the nature and severity of the disease. For adults, the amount of the antisense oligomer of the present invention per administration is 0.1 mg to 1 g per kg of body weight, preferably 1 mg to 100 mg per kg of body weight, more preferably 1 mg to 90 mg per kg of body weight, and even more preferably 1 mg to 80 mg per kg of body weight. The administration frequency may be once every 1 to 3 days, once a week, or once every 2 to 3 weeks. This value may vary depending on the type of disease, administration form, and target molecule being targeted. Therefore, a lower dose or administration frequency may be sufficient in some cases, while a higher dose or administration frequency may be required in other cases.

[0172] Another embodiment of the pharmaceutical composition and / or combination drug of the present invention is a pharmaceutical composition comprising a vector capable of expressing the antisense oligomer of the present invention and the above-described carrier. Such an expression vector may be capable of expressing multiple antisense oligomers of the present invention. Similar to the composition of the present invention containing the antisense oligomer of the present invention, pharmaceutically acceptable additives may be added to the composition. The concentration of the expression vector contained in the composition varies depending on the type of carrier, etc., but is suitably in the range of 0.1 nM to 100 μM, preferably in the range of 1 nM to 10 μM, and more preferably in the range of 10 nM to 1 μM. The weight ratio of the expression vector to the carrier contained in the composition (carrier / expression vector) varies depending on the properties of the expression vector, the type of carrier, etc., but is suitably in the range of 0.1 to 100, preferably in the range of 1 to 50, and more preferably in the range of 10 to 20. The content of the carrier contained in the composition is the same as in the composition of the present invention containing the antisense oligomer of the present invention, and the preparation method, etc., are also the same as in the composition of the present invention.

[0173] The present invention will be explained in more detail below with reference to examples and test examples, but the present invention is not limited to the scope shown in the examples.

[0174] [Example 1: Production of antisense oligomers] The antisense oligomers shown in Table 9 (PMO Nos. 1 to 5 (SEQ ID NOs: 5098 to 5102)) were synthesized according to the method described in Example 1 of International Publication WO2013 / 100190. The theoretical molecular weight of each antisense oligomer and the actual measured value by ESI-TOF-MS are also shown. The 5'-end of the PMO is the following group (1). The synthesized PMO was dissolved in water for injection (Otsuka Pharmaceutical Factory Co., Ltd.).

[0175]

[0176]

[0177] The target base sequences of the antisense oligomers of the present invention are described as "Ma1_b1-c1," "Ma2_b2-c2_Ma3_b3-c3," etc.

[0178] "Ma1" represents the a-th exon of the mouse dystrophin gene, "b1" represents the 5'-terminal base of the target base sequence, and "c1" represents the 3'-terminal base of the target base sequence. When "b1" and "c1" are positive integers, "b1" and "c1" respectively represent the base number from the 3'-terminal end when the 5'-terminal base of the a-th exon is the first base. On the other hand, when "b1" and "c1" are negative integers, "b1" and "c1" respectively represent the base number from the 5'-terminal end when the 3'-terminal base of the (a-1)-th intron is the -1st base.

[0179] For example, "M55_(-4)-24" means a base sequence in which the 5' end of the target base sequence is the fourth base from the 3' end of the 54th intron toward the 5' end, and the 3' end of the target base sequence is the 24th base from the 5' end of the 55th exon toward the 3' end.

[0180] The first part of "Ma2_b2-c2_Ma3_b3-c3", "Ma2_b2-c2", refers to the target base sequence of the 3'-end unit oligomer that constitutes the antisense oligomer, and the second part, "Ma3_b3-c3", refers to the target base sequence of the 5'-end unit oligomer that constitutes the antisense oligomer.

[0181] If "Ma2" and "Ma3" are the same, the "_Ma3" portion can be omitted.

[0182] For example, "M45_(-66)-(-61)_19-40" or "M45_(-66)-(-61)_M45_19-40" means a base sequence in which the target base sequence of the 3'-terminal unit oligomer constituting the antisense oligomer is "M45_(-66)-(-61)" and the target base sequence of the 5'-terminal unit oligomer is "M45_19-40."

[0183] [Example 2: Test of multi-exon skipping activity of antisense oligomers] <Test Example 1> Assay of exon 45-55 multi-exon skipping in cultured cells derived from a model mouse (1): Induction of multi-exon skipping (total concentration added: 30 μM) Procedure

[0184] H2K-mdx52 cells (immortalized myoblasts established from a cross between mdx52 mice, a Duchenne muscular dystrophy model, and H-2kb-tsA58 transgenic mice) were cultured at 1 × 10 in a 0.4% gelatin-coated 48-well plate (AGC Technoglass). ４ The cells were seeded at 1 / well and cultured in a growth medium (High glucose Dulbecco's Modified Eagle Medium (DMEM) (containing GlutaMax) (Thermo Fisher Scientific) supplemented with 20% FBS (Sigma-Aldrich), 2% chicken embryo extract (US Biologicals, the same applies hereinafter), 2% L-glutamine (Sigma-Aldrich, the same applies hereinafter), 1% penicillin / streptomycin (Sigma-Aldrich, the same applies hereinafter), and 20 U / mL Recombinant Murine IFN-γ (PeproTech). Scientific) in 0.5 mL at 37°C and 5% CO ２ The cells were cultured for 3 days under these conditions. After 48 hours, the growth medium was replaced with a differentiation medium (DMEM supplemented with 5% horse serum (Thermo Fisher Scientific), 2% L-glutamine, and 1% penicillin / streptomycin). After culturing for 3 days, the cells were transfected with 30 μM PMO using 6 μM Endo-Porter (Gene Tools; the same applies below). The PMOs used are listed in Table 10 below.

[0185]

[0186] After culturing for an additional 3 days, the cells were washed once with PBS (Takara Bio Inc.), and total RNA was extracted using an RNeasy Mini Kit (Qiagen). 350 μL of Buffer RLT (Qiagen) containing 1% 2-mercaptoethanol (Nacalai Tesque) was added to the cells, left at room temperature for several minutes to lyse the cells, and then collected in a QIAshredder homogenizer (Qiagen). The homogenate was centrifuged at 15,000 rpm for 2 minutes. Total RNA was extracted according to the protocol provided with the RNeasy Mini Kit (Qiagen). The concentration of the extracted total RNA was measured using a NanoDrop One C (Thermo Fisher Scientific). One-step RT-PCR was performed on 400 ng of extracted total RNA using the QIAGEN OneStep RT-PCR Kit (Qiagen). The reaction solution was prepared according to the protocol attached to the kit. The thermal cycler used was a Veriti 96 Well Thermal Cycler (Thermo Fisher Scientific). The RT-PCR program used was as follows:

[0187] 50°C, 30 minutes: reverse transcription reaction 95°C, 15 minutes: polymerase activation, reverse transcriptase inactivation, cDNA heat denaturation [94°C, 10 seconds; 57°C, 30 seconds; 72°C, 1 minute] x 33 cycles: PCR amplification 72°C, 10 minutes: final extension reaction

[0188] The nucleotide sequences of the forward and reverse primers used in RT-PCR are as follows: Forward primer: 5'-cagttgaaaaatggcgacac-3' (SEQ ID NO: 5103) Reverse primer 1: 5'-ttagctgctgctcatctcca-3' (SEQ ID NO: 5104) Reverse primer 2: 5'-ttccagggatctcaggattt-3' (SEQ ID NO: 5105)

[0189] The combination of the forward primer and reverse primer 1 can detect a transcript without skipping (429 bp) or a transcript with exon 45 skipping (253 bp), while the combination of the forward primer and reverse primer 2 can detect a transcript with multi-exon skipping of exons 45 to 55 (218 bp).

[0190] The PCR reaction products were analyzed using MultiNA (Shimadzu Corporation). The amount of polyoligonucleotide "A" in the band in which exons 45 to 55 were skipped, the amount of polynucleotide "B" in the band in which exon 45 was skipped, and the amount of polyoligonucleotide "C" in the band in which no skipping occurred were measured. Based on the measured values ​​of "A," "B," and "C," the skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping were calculated according to the following formulas: Skipping efficiency (%) of exon 45 to 55 skipping = A / (A + B + C) × 100 Skipping efficiency (%) of exon 45 skipping = B / (A + B + C) × 100

[0191] The results are shown in Figures 1 and 2. Compared with the mixture of PMO No. 1 and PMO No. 2 (15 μM each, Mixture 2) alone, the mixture containing PMO No. 3 targeting hnRNP A1 (10 μM each, Mixture 2 + PMO No. 3) increased the skipping efficiency of exons 45-55 (Figure 1), but decreased the skipping efficiency of exon 45 (Figure 2).

[0192] Test Example 2: Assay of exon 45-55 multiexon skipping in cultured cells derived from a mouse model (2): Induction of multiexon skipping (total concentration added: 15 μM, varying the mixing ratio)

[0193] Procedure: H2K-mdx52 cells were plated in a 0.4% gelatin-coated 24-well plate at 5 x 10 ４ Seeded in 1 / well in 1 mL of growth medium at 37°C and 5% CO ２After culturing for 48 hours under these conditions, the proliferation medium was changed to differentiation medium. After culturing for 3 days, 15 μM PMO was transfected using 6 μM Endo-Porter. In addition to the PMOs used in Test Example 1, the PMOs listed in Table 11 below were also used.

[0194] After culturing for another 3 days, the cells were collected in the same manner as in Test Example 1, total RNA was extracted, and one-step RT-PCR was performed. The PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0195] (Results) The results are shown in Figures 3 to 8. Compared with the mixture of PMO No. 1 and PMO No. 2 (Mixture 2) alone, the mixture containing PMO No. 3 targeting hnRNP A1 (Mixture 2 + hnRNP A1) showed increased exon 45-55 skipping efficiency (Figure 3), but decreased exon 45 skipping efficiency (Figure 4). The mixture containing PMO No. 4 targeting hnRNP A1 (Mixture 2 + PMO No. 4) showed exon 45-55 skipping (Figure 5), but decreased exon 45 skipping efficiency (Figure 6).

[0196] When we investigated the effect of varying the ratio of Mixture 2 to PMO No. 3 targeting hnRNP A1, we found that a 3:1 mixture of the two compounds resulted in the highest exon 45-55 skipping efficiency (Figure 7), while the exon 45 skipping efficiency was the lowest (Figure 8).

[0197] Test Example 3: Assay of exon 45-55 multiexon skipping in cultured cells derived from a mouse model (3): Induction of multiexon skipping (total concentration added: 50 μM)

[0198] Procedure: H2K-mdx52 cells were plated in a 0.4% gelatin-coated 12-well plate at 6.7 × 10 ４ Seeded per well in 2 mL of growth medium at 37°C and 5% CO ２The cells were cultured for one day under these conditions. After two days of culture, the medium was changed from proliferation medium to differentiation medium. After three days of culture, 50 μM PMO was transfected using 6 μM Endo-Porter. After an additional three days of culture, the cells were collected as in Test Example 1, total RNA was extracted, and one-step RT-PCR was performed. The PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0199] (Results) The results are shown in Figures 9 and 10. Compared with the mixture of PMO No. 1 and PMO No. 2 (Mix 2) alone, the mixture containing PMO No. 3 targeting hnRNP A1 (Mix 2 + hnRNP A1) increased the skipping efficiency of exons 45-55 (Figure 9), but decreased the skipping efficiency of exon 45 (Figure 10). Note that Mix 2 is synonymous with Mixture 2 in Test Examples 1 and 2.

[0200] <Test Example 4> Assay of exon 45-55 multi-exon skipping in cultured cells derived from a model mouse (4): Restoration of dystrophin protein expression by multi-exon skipping

[0201] Procedure: H2K-mdx52 cells were plated in a 0.4% gelatin-coated 12-well plate at 6.7 × 10 ４ Seeded per well in 2 mL of growth medium at 37°C and 5% CO ２The cells were cultured for one day under these conditions. After two days of culture, the medium was changed from proliferation medium to differentiation medium. After three days of culture, 50 μM PMO was transfected using 6 μM Endo-Porter. After another three days of culture, the medium was changed to differentiation medium, and after another day of culture, the cells were collected using cell lysis buffer Pierce RIPA Buffer (Thermo Fisher Scientific) supplemented with the protease inhibitor cocktail cComplete Mini (Roche Diagnostics). The cells were disrupted using an ultrasonic disrupter Bioruptor UCD-250 (manufactured by Sonic Bio) (output: H, 30 seconds, 3 times), and centrifuged in a refrigerated centrifuge (TOMY MX-305, rotor: AR015-24, manufactured by Tomy Seiko Co., Ltd.) (15,000 rpm, 4°C, 15 minutes). The supernatant was used as a cell lysate. Using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), absorbance at 562 nm was measured with a plate reader Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments), and the protein concentration in the cell lysate was determined using data analysis software Gen5 version 2.09.2 (BioTek Instruments). The cell lysate (30 μg protein) was electrophoresed (150 V, 75 minutes) on a 15-well polyacrylamide gel, NuPAGE 3 to 8%, Tris-Acetate, 1.5 mm, Mini Protein Gel (Thermo Fisher Scientific). HiMark Pre-Stained Protein Standard (Thermo Fisher Scientific) was used as a molecular weight marker.

[0202] After electrophoresis, the sample was transferred to an Immobilon-P Transfer membrane (Merck Millipore) by semi-dry blotting (4 mA / cm ２Western blotting was performed using an anti-dystrophin antibody (NCL-Dys1, Leica Biosystems Newcastle) diluted 1:100 as the primary antibody and a goat anti-mouse IgG (H+L)-Horseradish Peroxidase complex (Bio-Rad Laboratories) diluted 2,500:1 as the secondary antibody. After the antibody reaction, the gel was induced to emit light using an ECL Prime Western Blotting Detection System (Cytiva), and the luminescence was detected and images were taken using a chemiluminescence / gel imaging device, ChemiDoc Touch MP Imaging System (Bio-Rad Laboratories).

[0203] (Results) The results are shown in Figure 11. Dystrophin protein was not expressed in the negative control or in the mixture of PMO No. 1 and PMO No. 2 (Mix 2), but expression of dystrophin protein corresponding to exon 45-55 skipping was confirmed in the mixture containing PMO No. 3 targeting hnRNP A1 (Mix 2 + hnRNP A1) (Figure 11). Note that Mix 2 is synonymous with Mixture 2 in Test Examples 1 and 2.

[0204] [Example 3: Production of antisense oligomers (2)] Antisense oligomers (PMO Nos. 6 to 33) shown in Table 12 were synthesized in the same manner as in Example 1. The theoretical molecular weight of each antisense oligomer and the measured value by ESI-TOF-MS are also shown. The 5'-end of the PMO is group (1), as in Example 1. The synthesized PMO was dissolved in water for injection (Otsuka Pharmaceutical Factory).

[0205]

[0206] The target base sequences of the antisense oligomers of the present invention are described as "Ha1_b1-c1," "Ha2_b2-c2_Ha3_b3-c3," etc.

[0207] "Ha1" represents the a-th exon of the human dystrophin gene, "b1" represents the 5'-terminal base of the target base sequence, and "c1" represents the 3'-terminal base of the target base sequence. When "b1" and "c1" are positive integers, "b1" and "c1" respectively represent the base number from the 3'-terminal end when the 5'-terminal base of the a-th exon is the first base. On the other hand, when "b1" and "c1" are negative integers, "b1" and "c1" respectively represent the base number from the 5'-terminal end when the 3'-terminal base of the (a-1)-th intron is the -1st base.

[0208] For example, "H55_(-18)-10" means a base sequence in which the 5' end of the target base sequence is the 18th base from the 3' end of the 54th intron toward the 5' end, and the 3' end of the target base sequence is the 10th base from the 5' end of the 55th exon toward the 3' end.

[0209] The first part of "Ha2_b2-c2_Ha3_b3-c3", "Ha2_b2-c2", refers to the target base sequence of the 3'-end unit oligomer that constitutes the antisense oligomer, and the second part, "Ha3_b3-c3", refers to the target base sequence of the 5'-end unit oligomer that constitutes the antisense oligomer.

[0210] If "Ha2" and "Ha3" are the same, the "_Ha3" portion can be omitted.

[0211] For example, "H45_(-66)-(-61)_19-40" or "H45_(-66)-(-61)_H45_19-40" means a base sequence in which the target base sequence of the 3'-terminal unit oligomer constituting the antisense oligomer is "H45_(-66)-(-61)" and the target base sequence of the 5'-terminal unit oligomer is "H45_19-40."

[0212] [Example 4: Test of multi-exon skipping activity of antisense oligomers (2)] <Test Example 1> Assay of exon 45-55 multi-exon skipping in normal human myoblasts (1): Induction of multi-exon skipping

[0213] Normal human myoblasts (manufactured by LONZA) were directly immunofluorescently stained with a PE-labeled anti-human CD82 antibody (manufactured by BioLegend, hereinafter the same), and then sorted using a Cell Sorter SH800S (manufactured by Sony, hereinafter the same) to obtain CD82-positive normal human myoblasts. 5 × 10 normal human myoblasts were placed in a 96-well collagen I-coated microplate (manufactured by AGC Technoglass) coated with Corning® Matrigel basement membrane matrix (manufactured by Corning, hereinafter the same). ４ The cells were seeded at 1 / well and grown in a normal human myoblast growth medium (DMEM supplemented with 20% fetal bovine serum (FBS) (manufactured by Corning, the same applies hereinafter), 0.1% hBFGF (manufactured by Sigma-Aldrich), and 1% penicillin / streptomycin (P / S) (manufactured by Sigma-Aldrich, the same applies hereinafter) containing high glucose, GlutaMAX ＴＭ Supplement, pyruvate (Thermo Fisher Scientific, same below) 0.1 mL, 37°C, 5% CO ２ The day after seeding, the medium was changed from the proliferation medium to 0.2 mL of normal human myoblast differentiation medium (DMEM supplemented with 2% horse serum (manufactured by Thermo Fisher Scientific), 1% ITS liquid medium supplement (100x) (manufactured by Sigma-Aldrich), and P / S, high glucose, GlutaMAX ＴＭ The medium was replaced with 100mg of ...

[0214]

[0215] The PMOs used and their concentrations in the medium are shown in Table 14 below.

[0216] After an additional 3 days of culture, the medium was replaced with 0.25 mL of normal human myoblast differentiation medium. Seven days after PMO addition, the cells were washed once with PBS (Takara Bio), and total RNA was extracted using an RNeasy Micro Kit (Qiagen). 75 μL of Buffer RLT (Qiagen) containing 1% 2-mercaptoethanol (Nacalai Tesque) was added to the cells, and the mixture was left at room temperature for several minutes to lyse the cells. Total RNA was extracted according to the protocol provided with the RNeasy Micro Kit (Qiagen). The concentration of the extracted total RNA was measured using a NanoDrop One C (Thermo Fisher Scientific). One-Step RT-PCR was performed on 100 ng of extracted total RNA using the QIAGEN OneStep RT-PCR Kit (Qiagen). The reaction solution was prepared according to the protocol attached to the kit. The thermal cycler used was a Veriti 96 Well Thermal Cycler (Thermo Fisher Scientific). The RT-PCR program used was as follows: 50°C, 30 minutes: reverse transcription; 95°C, 15 minutes: polymerase activation, reverse transcriptase inactivation, cDNA thermal denaturation; [94°C, 30 seconds; 57°C, 30 seconds; 72°C, 1 minute] x 33 cycles: PCR amplification; 72°C, 10 minutes: final extension reaction

[0217] The base sequences of the forward and reverse primers used in RT-PCR are shown in Table 15 below.

[0218] The combination of forward primer 1 and reverse primer 1 can detect a transcript (301 bp) in which multi-exon skipping of exons 45 to 55 has occurred. The combination of forward primer 2 and reverse primer 2 can detect a transcript (245 bp) in the region of exons 37 to 38 that is not affected by skipping.

[0219] The PCR reaction products were analyzed using MultiNA (Shimadzu Corporation). The amount of polyoligonucleotide "A" in the band where exons 45 to 55 were skipped and the amount of polyoligonucleotide "B" in the band where skipping did not occur were measured. Based on the measured values ​​of "A" and "B," the skipping efficiency of exons 45 to 55 skipping was calculated according to the following formula: Skipping efficiency of exons 45 to 55 skipping (%) = A / B × 100

[0220] For exon 45 skipping, one-step RT-PCR was performed in the same manner as for the detection of exon 45-55 skipping, using the primers in Table 16 below.

[0221] The combination of the forward primer and the reverse primer makes it possible to detect a transcript in which exon 45 skipping has occurred (268 bp) and a transcript in which skipping has not occurred (444 bp).

[0222] The PCR reaction products were analyzed using MultiNA (Shimadzu Corporation). The amount of polynucleotide "A" in the band where exon 45 was skipped and the amount of polynucleotide "B" in the band where skipping did not occur were measured. Based on the measured values ​​of "A" and "B," the skipping efficiency of exon 45 skipping was calculated according to the following formula: Exon 45 skipping efficiency (%) = A / (A + B) × 100

[0223] Results The results are shown in Figures 12 and 13. In normal human cultured cells, PMO No. 6 alone (condition 8) and mixtures containing PMO No. 6 (conditions 2 to 6) were confirmed to induce exon 45-55 skipping (Figure 12). On the other hand, mixtures containing PMO No. 6 and PMO No. 7 targeting hnRNP A1 (conditions 2 to 4, 6) reduced the skipping efficiency of exon 45 skipping and suppressed single skipping (Figure 13).

[0224] Test Example 2: Assay of exon 45-55 multiexon skipping in myoblasts derived from exon 48-50 deleted DMD patients (1): Induction of multiexon skipping Procedure: Myoblasts derived from exon 48-50 deleted DMD patients obtained from the NCNP Biobank were directly immunofluorescently stained with a PE-labeled anti-human CD82 antibody and an APC-labeled anti-human CD56 antibody (Miltenyi Biotec, the same applies hereinafter), and then sorted using a Cell Sorter SH800S to obtain CD56- and CD82-positive myoblasts derived from exon 48-50 deleted DMD patients. Exon 48-50 deleted DMD patient-derived myoblasts (CD56-positive, CD82-positive) were plated at 5 x 10 cells per well onto a Corning BioCoat Collagen I 48-well clear microplate coated with Corning® Matrigel basement membrane matrix. ４ The cells were seeded at 1 / well and cultured in 0.25 mL of growth medium for DMD patient-derived myoblasts (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12) (Thermo Fisher Scientific, same below) supplemented with 20% fetal bovine serum (FBS) and 1% P / S) at 37°C and 5% CO ２ The cells were cultured for 1 day under the conditions of 0.5 mL. The day after seeding, the growth medium was replaced with 0.5 mL of differentiation medium for DMD patient-derived myoblasts (DMEM / F12 supplemented with 2% horse serum, 1% ITS liquid medium supplement (100x), and 1% P / S). After 6 days of culture in the differentiation medium, the cells were transfected with 6 μM of PMO using Endo-Porter. The same PMO as in Test Example 1 was used, and its concentration in the medium is as shown in Table 17 below.

[0225]

[0226] After culturing for an additional 3 days, the medium was replaced with 0.5 mL of differentiation medium. Seven days after the addition of PMO, total RNA was extracted from the cells in the same manner as in Test Example 1 of Example 2, and one-step RT-PCR was performed using 200 ng of total RNA in the same manner as in Test Example 1. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0227] Results The results are shown in Figures 14 and 15. Exon 45-55 skipping was confirmed in myoblasts derived from a DMD patient with an exon 48-50 deletion using a mixture containing PMO No. 6 and PMO No. 8 (condition 4). Furthermore, exon 45-55 skipping was also confirmed to be induced in mixtures containing PMO No. 7, which targets hnRNP A1 (conditions 2 and 3) (Figure 14). However, the skipping efficiency of exon 45 skipping was reduced, and single skipping was suppressed (Figure 15).

[0228] <Test Example 3> Assay of exon 45-55 multi-exon skipping in myoblasts derived from DMD patients with exon 48-50 deletion (2): Induction of multi-exon skipping

[0229] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a DMD patient with exon 48-50 deletion, prepared in the same manner as in Test Example 2, were plated at 2 x 10 in a Corning BioCoat Collagen I 48-well clear microplate coated with Corning® Matrigel basement membrane matrix. ４ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２ The cells were cultured for 3 days under the conditions. Three days after seeding, the medium was replaced with 0.5 mL of differentiation medium for DMD patient-derived myoblasts. After culturing in the differentiation medium for 8 days, the cells were transfected with 6 μM of PMO using Endo-Porter. The same PMOs as in Test Examples 1 and 2 were used, and their concentrations in the medium are shown in Table 18 below.

[0230]

[0231] After culturing for an additional 3 days, the medium was replaced with 0.5 mL of differentiation medium. Six days after the addition of PMO, total RNA was extracted in the same manner as in Test Example 2, and 200 ng of total RNA was used to perform one-step RT-PCR in the same manner as in Test Examples 1 and 2. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0232] Results The results are shown in Figures 16 and 17. Compared with PMO No. 6 alone (Condition 4), the skipping efficiency of exons 45-55 was improved in the mixtures containing PMO No. 6 plus PMO No. 8 or PMO No. 7 and PMO No. 8 (Conditions 2 and 3) (Figure 16). On the other hand, the skipping efficiency of exon 45 was reduced in the mixture containing PMO No. 7 targeting hnRNP A1 (Condition 2), and single-strand skipping was suppressed (Figure 17).

[0233] <Test Example 4> Assay of exon 45-55 multi-exon skipping in myoblasts derived from DMD patients with exon 48-50 deletion (3): Restoration of dystrophin protein expression by multi-exon skipping

[0234] Myoblasts (CD56-positive, CD82-positive) derived from a DMD patient with exon 48-50 deletion were plated at 1.0 x 10 cells per well in a 24-well collagen I-coated microplate (AGC Technoglass Co., Ltd.) coated with Corning® Matrigel basement membrane matrix. ５ 1 / well in 1 mL of DMD patient-derived myoblast growth medium, 37°C, 5% CO ２The cells were cultured for one day under these conditions. The day after seeding, the medium was changed from a proliferation medium for DMD patient-derived myoblasts to a differentiation medium for DMD patient-derived myoblasts. After six days of culture, the cells were transfected with 6 μM Endo-Porter. After an additional three days of culture, the medium was changed to a differentiation medium, and after seven days of culture from the addition of PMO, a cell lysate was prepared in the same manner as in Test Example 4 of Example 2, and the protein concentration in the cell lysate was determined. Western blotting was performed to detect dystrophin protein in the same manner as in Test Example 4 of Example 2, except that 30 μg of protein was used in the cell lysate, the electrophoresis time was 120 minutes, and the anti-dystrophin antibody (NCL-DYS1) was diluted 250-fold. The electrophoresed samples are shown in Table 19 below. As positive controls for dystrophin expression, a lysate of mouse C2C12 cells cultured for muscle differentiation for 12 days (normal dystrophin control) and a lysate of skeletal muscle from a transgenic mouse lacking exons 45-55 (dystrophin expression control lacking exons 45-55) were used.

[0235]

[0236] (Results) The results are shown in Figure 18. In the negative control (Condition 3), no dystrophin protein was expressed, but expression of dystrophin protein corresponding to exon 45-55 skipping was confirmed in the mixture of PMO No. 6 and PMO No. 8 (Condition 5) and the mixture of PMO Nos. 6 to 8 (Conditions 4 and 6) (Figure 18: arrowheads).

[0237] <Test Example 5> Assay of exon 45-55 multi-exon skipping in myoblasts derived from DMD patients with exon 46-51 deletion (1): Induction of multi-exon skipping

[0238] Procedure Myoblasts derived from DMD patients with exon 46-51 deletion obtained from the NCNP Biobank were sorted in the same manner as in Test Example 2. The resulting myoblasts (CD56-positive, CD82-positive) were then plated at 5 x 10 in a Corning BioCoat Collagen I 48-well clear microplate coated with Corning® Matrigel basement membrane matrix.４ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２ The cells were cultured for 1 day under the conditions. The day after seeding, the medium was replaced with 0.3 mL of differentiation medium for DMD patient-derived myoblasts. After culturing in the differentiation medium for 6 days, the cells were transfected with 6 μM of PMO using Endo-Porter. The same PMOs as in Test Examples 1 to 4 were used, and their concentrations in the medium are shown in Table 20 below.

[0239]

[0240] After an additional 3 days of culture, the medium was replaced with 0.3 mL of differentiation medium. Five days after the addition of PMO, total RNA was extracted in the same manner as in Test Examples 2-3, and 100 ng of total RNA was used to perform one-step RT-PCR in the same manner as in Test Examples 1-3. The resulting PCR reaction products were analyzed to determine the skipping efficiency of exon 45-55 skipping. Furthermore, one-step RT-PCR was performed in the same manner as in Test Examples 1-3, except that the primers listed in Table 21 below were used. The skipping efficiency of exon 45 skipping was determined by analyzing the PCR reaction products. Using a combination of forward and reverse primers, a transcript in which exon 45 skipping had occurred (162 bp) and a transcript in which skipping had not occurred (338 bp) could be detected.

[0241]

[0242] Results The results are shown in Figures 19 and 20. In myoblasts derived from a DMD patient with exon 46-51 deletion, it was confirmed that exon 45-55 skipping was induced by PMO No. 6 alone (condition 3) and a mixture containing PMO Nos. 6-8 (condition 2) (Figure 19). Furthermore, the mixture containing PMO Nos. 6-8 (condition 2) showed a lower skipping efficiency of exon 45 skipping than PMO No. 6 alone (condition 3), and single skipping was suppressed (Figure 20).

[0243] <Test Example 6> Assay of exon 45-55 multi-exon skipping in myoblasts derived from DMD patients with exon 46-51 deletion (2): Induction of multi-exon skipping

[0244] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a DMD patient with exon 46-51 deletion, prepared in the same manner as in Test Example 5, were plated at 6.3 x 10 cells / well onto a Corning BioCoat Collagen I 48-well clear microplate coated with Corning® Matrigel basement membrane matrix. ３ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２ The cells were cultured for 4 days under the conditions. Four days after seeding, the medium was replaced with 0.3 mL of differentiation medium for DMD patient-derived myoblasts. After 7 days of culture in the differentiation medium, the cells were transfected with 6 μM of PMO using Endo-Porter. The same PMOs as in Test Examples 1 to 5 were used, and their concentrations in the medium are shown in Table 22 below.

[0245]

[0246] After culturing for an additional 3 days, the medium was replaced with 0.3 mL of differentiation medium. Seven days after the addition of PMO, total RNA was extracted in the same manner as in Test Examples 2 to 3 and 5. One-step RT-PCR was performed in the same manner as in Test Example 5, except that 80 ng of total RNA was used. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0247] Results The results are shown in Figures 21 and 22. In myoblasts derived from a DMD patient with an exon 46-51 deletion, it was confirmed that exon 45-55 skipping was induced by PMO No. 6 alone (condition 6) and mixtures containing PMO No. 6 (conditions 2, 3, and 5) (Figure 21). Furthermore, the mixture containing PMO No. 6 and PMO No. 7 (condition 5) showed a lower skipping efficiency of exon 45 skipping than the mixture containing PMO No. 6 and PMO No. 8 (condition 3), indicating that single skipping was suppressed (Figure 22).

[0248] <Test Example 7> Assay of exon 45-55 multi-exon skipping in myoblasts derived from DMD patients with exon 46-51 deletion (4): Restoration of dystrophin protein expression by multi-exon skipping

[0249] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a DMD patient with exon 46-51 deletion were plated at 8.0 × 10 cells per well in a 24-well collagen I-coated microplate coated with Corning® Matrigel basement membrane matrix. ４ The cells were seeded in 1000 x 1000 cells / well and incubated at 37°C in 5% CO in 1 mL of growth medium for DMD patient-derived myoblasts. ２ The cells were cultured for one day under these conditions. The day after seeding, the medium was changed from the DMD patient-derived myoblast growth medium to 1 mL of DMD patient-derived myoblast differentiation medium. After 4 days of culture, the medium was changed, and after 7 days of culture, PMO was transfected using 6 μM Endo-Porter. After an additional 3 days of culture, the medium was changed to differentiation medium, and after 7 days of culture from the addition of PMO, a cell lysate was prepared in the same manner as in Test Example 4 of Example 2 and Test Example 4 of this Example. Western blotting was performed to detect dystrophin protein in the same manner as in Test Example 4 of Example 2 and Test Example 4 of this Example, except that 24 μg of the cell lysate was used in protein amount. The samples electrophoresed are shown in Table 23 below.

[0250]

[0251] (Results) The results are shown in Figure 23. In the negative control (Condition 3), no dystrophin protein was expressed, but expression of dystrophin protein corresponding to exon 45-55 skipping was confirmed in the mixture of PMO No. 6 and PMO No. 8 (Condition 5) and the mixture of PMO Nos. 6-8 (Condition 4) (Figure 23: arrowheads).

[0252] Test Example 8: Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (1): Study of the first antisense oligomer (1)

[0253] Procedure: Myoblasts derived from exon 51-deleted DMD patients obtained from the NCNP Biobank were sorted in the same manner as in Examples 3 and 6. The resulting myoblasts (CD56-positive, CD82-positive) were then plated at 2.5 x 10 cells per well onto a 24-well collagen I-coated microplate (AGC Technoglass) coated with Corning® Matrigel basement membrane matrix. ４ The cells were seeded in 0.5 mL of growth medium for DMD patient-derived myoblasts and incubated at 37°C in 5% CO ２ The cells were cultured for 3 days under the conditions of (1) to (3). Three days after seeding, the medium was replaced with 0.5 mL of differentiation medium for DMD patient-derived myoblasts. After 4 days of culture in differentiation medium for DMD patient-derived myoblasts, the cells were transfected with PMO using 6 μM Endo-Porter. In addition to the PMOs used in Test Examples 1 to 7, the PMOs listed in Table 24 below were also used.

[0254]

[0255] PMOs were added to the medium at the concentrations shown in Table 25 below.

[0256]

[0257] After culturing for an additional 3 days, the medium was replaced with 0.5 mL of differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, total RNA was extracted from the cells in the same manner as in Test Examples 2, 3, 5, and 6. One-step RT-PCR was performed using 200 ng of total RNA in the same manner as in Test Examples 1 to 3. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0258] Results The results are shown in Figures 24 and 25. It was confirmed that exon 45-55 skipping was induced by a mixture containing PMO No. 6 and PMO No. 8 (condition 2) in myoblasts derived from a DMD patient with exon 51 deletion (Figure 24). It was also confirmed that exon 45-55 skipping was induced by a mixture containing PMO No. 6, PMO No. 8, and PMO No. 7 targeting hnRNP A1 (condition 3) (Figure 24). Furthermore, it was confirmed that exon 45-55 skipping was induced under conditions 4, 6, and 8, in which PMO No. 9-11 were added to PMO No. 8 as the first antisense oligomer. Furthermore, PMO No. 10 targeting hnRNP A1 was also induced. It was confirmed that exon 45-55 skipping was also induced under conditions 5, 7, and 9, in which 7 was also added (Figure 24). However, the skipping efficiency of exon 45 skipping was reduced, and single skipping was suppressed (Figure 25).

[0259] <Test Example 9> Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (2): Study of the first antisense oligomer (2)

[0260] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a patient with DMD with exon 51 deletion, prepared in the same manner as in Test Example 8, were plated at 5 x 10 in a 48-well clear microplate coated with Corning® Matrigel basement membrane matrix (Corning BioCoat Collagen I). ４ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２ The cells were cultured for 1 day under the conditions of 0.25 mL. The day after seeding, the medium was replaced with 0.25 mL of differentiation medium for DMD patient-derived myoblasts. After 7 days of culture in differentiation medium for DMD patient-derived myoblasts, the cells were transfected with PMO using 6 μM Endo-Porter. In addition to the PMOs used in Test Examples 1 to 7, the PMOs listed in Table 26 below were used.

[0261]

[0262] PMOs were added to the medium at the concentrations shown in Table 27 below.

[0263] After culturing for an additional 3 days, the medium was replaced with 0.3 mL of differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, total RNA was extracted from the cells in the same manner as in Test Examples 2, 3, 5, 6, and 8. One-step RT-PCR was performed using 200 ng of total RNA in the same manner as in Test Examples 1 to 3 and 8. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0264] Results The results are shown in Figures 26 and 27. Exon 45-55 skipping was also confirmed to be induced under conditions 4, 6, 8, and 10, in which PMO No. 12-17 was added together with PMO No. 8 as the first antisense oligomer. Furthermore, under conditions 5, 7, 9, and 11, in which PMO No. 7 targeting hnRNP A1 was also added, exon 45-55 skipping was confirmed to be induced (Figure 26). However, the skipping efficiency of exon 45 skipping was reduced, and single skipping was suppressed (Figure 27).

[0265] <Test Example 10> Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (3): Study of the third antisense oligomer (1)

[0266] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a patient with DMD with exon 51 deletion, prepared in the same manner as in Test Examples 8 and 9, were placed in a 48-well clear microplate coated with Corning® Matrigel basement membrane matrix (Corning BioCoat Collagen I) at 5 x 10 ４ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２ The cells were cultured for 1 day under the conditions of (1). The day after seeding, the medium was replaced with 0.25 mL of differentiation medium for DMD patient-derived myoblasts. After 7 days of culture in differentiation medium for DMD patient-derived myoblasts, the cells were transfected with PMO using 6 μM Endo-Porter. In addition to the PMOs used in Test Examples 1 to 7, the PMOs listed in Table 28 below were used.

[0267]

[0268] PMOs were added to the medium at the concentrations shown in Table 29 below.

[0269]

[0270] After culturing for an additional 3 days, the medium was replaced with 0.3 mL of differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, total RNA was extracted from the cells in the same manner as in Test Examples 2, 3, 5, 6, 8, and 9. One-step RT-PCR was performed using 200 ng of total RNA in the same manner as in Test Examples 1 to 3, 8, and 9. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0271] Results The results are shown in Figures 28 and 29. When PMO No. 6 and PMO No. 8 were combined with PMO No. 20-23 as a third antisense oligomer (conditions 6-9), the induction of exon 45-55 skipping was confirmed to be comparable to that observed when PMO No. 7 was added (condition 5) (Figure 28). Furthermore, exon 45 skipping was reduced, indicating a tendency for single skipping to be suppressed (Figure 29). Meanwhile, the mixtures containing PMO No. 18 and 19 (conditions 3 and 4) did not decrease exon 45 skipping efficiency compared to the mixture containing PMO No. 7 (condition 5), but they induced greater exon 45-55 skipping than the negative control (condition 1).

[0272] <Test Example 11> Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (4): Study of the third antisense oligomer (2)

[0273] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a patient with DMD with exon 51 deletion, prepared in the same manner as in Test Examples 8 to 10, were plated at 5 x 10 in a Corning BioCoat Collagen I 48-well clear microplate coated with Corning® Matrigel basement membrane matrix. ４ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２The cells were cultured for one day under the conditions. The day after seeding, the medium was replaced with 0.25 mL of differentiation medium for DMD patient-derived myoblasts. After culturing for three days in differentiation medium for DMD patient-derived myoblasts, the cells were transfected with PMO using 6 μM Endo-Porter. In addition to the PMOs used in Test Examples 1 to 7, the PMOs listed in Table 30 below were used.

[0274]

[0275] PMOs were added to the medium at the concentrations shown in Table 31 below.

[0276] After culturing for an additional 3 days, the medium was replaced with 0.3 mL of differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, total RNA was extracted from the cells in the same manner as in Test Examples 2, 3, 5, 6, and 8 to 10. One-step RT-PCR was performed using 200 ng of total RNA in the same manner as in Test Examples 1 to 3 and 8 to 10. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0277] Results The results are shown in Figures 30 and 31. When PMO No. 24-26 were added as a third antisense oligomer to PMO No. 6 and PMO No. 8 (conditions 2, 5, and 8), the efficiency of exon 45-55 skipping was reduced when PMO No. 24 and 26 were added (conditions 2 and 8) compared to when PMO No. 7 was added (condition 4), whereas the addition of PMO No. 25 (condition 5) induced a similar level of exon 45-55 skipping (Figure 30). Furthermore, the addition of PMO No. 25 (condition 5) reduced the efficiency of exon 45 skipping, suppressing single skipping (Figure 31).

[0278] <Test Example 12> Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (5): Study of the second antisense oligomer (1)

[0279] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a patient with DMD with exon 51 deletion, prepared in the same manner as in Test Examples 8 to 11, were plated at 5 x 10 in a 48-well clear microplate coated with Corning® Matrigel basement membrane matrix (Corning BioCoat Collagen I). ４ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２ The cells were cultured for 1 day under the conditions of (1). The day after seeding, the medium was replaced with 0.25 mL of differentiation medium for DMD patient-derived myoblasts. After 7 days of culture in differentiation medium for DMD patient-derived myoblasts, the cells were transfected with PMO using 6 μM Endo-Porter.

[0280] In addition to the PMOs used in Test Examples 1 to 7, the PMOs listed in Table 32 below were used.

[0281] PMOs were added to the medium at the concentrations shown in Table 33 below.

[0282] After culturing for an additional 3 days, the medium was replaced with 0.3 mL of differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, total RNA was extracted from the cells in the same manner as in Test Examples 2, 3, 5, 6, and 8 to 11. One-step RT-PCR was performed using 200 ng of total RNA in the same manner as in Test Examples 1 to 3 and 8 to 11. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0283] The results are shown in Figures 32 and 33. When PMO No. 27-33 was added together with PMO No. 6 as the second antisense oligomer (conditions 2-7, 9), exon 45-55 skipping was induced to a similar extent as when PMO No. 8 was added (condition 8) (Figure 32). Changing the second antisense oligomer did not reduce the efficiency of exon 45 skipping, and single skipping was not suppressed (Figure 33).

[0284] <Test Example 13> Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (6): Study of the second antisense oligomer (2)

[0285] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a patient with DMD with exon 51 deletion, prepared in the same manner as in Test Examples 8 to 12, were plated at 5 x 10 in a 48-well clear microplate coated with Corning® Matrigel basement membrane matrix (Corning BioCoat Collagen I). ４ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２ The cells were cultured for 1 day under the conditions of (1). The day after seeding, the medium was replaced with 0.25 mL of differentiation medium for DMD patient-derived myoblasts. After culturing for 3 days in differentiation medium for DMD patient-derived myoblasts, the cells were transfected with 6 μM PMO using Endo-Porter. The PMO used in Test Example 12 was added to the medium at the concentrations shown in Table 34 below.

[0286]

[0287] After culturing for an additional 3 days, the medium was replaced with 0.3 mL of differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, total RNA was extracted from the cells in the same manner as in Test Examples 2, 3, 5, 6, and 8 to 12. One-step RT-PCR was performed using 200 ng of total RNA in the same manner as in Test Examples 1 to 3 and 8 to 12. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0288] The results are shown in Figures 34 and 35. When PMO Nos. 27-29 and 32 were added together with PMO No. 6 and PMO No. 7 (conditions 2-5), exon 45-55 skipping was induced to a similar extent as when PMO No. 8 was added (condition 6) (Figure 34). Except for when PMO No. 32 was added (condition 5), there was almost no induction of exon 45 skipping, and single skipping was suppressed (Figure 35).

[0289] <Test Example 14> Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (7): Study of the second antisense oligomer (3)

[0290] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a patient with DMD with exon 51 deletion, prepared in the same manner as in Test Examples 8 to 13, were plated at 5 x 10 in a 48-well clear microplate coated with Corning® Matrigel basement membrane matrix (Corning BioCoat Collagen I). ４ The cells were seeded in 0.25 mL of DMD patient-derived myoblast growth medium at 37°C and 5% CO ２ The cells were cultured for 1 day under the conditions of (1). The day after seeding, the medium was replaced with 0.25 mL of differentiation medium for DMD patient-derived myoblasts. After 7 days of culture in differentiation medium for DMD patient-derived myoblasts, the cells were transfected with 6 μM of PMO using Endo-Porter. The PMO used in Test Example 12 was added at the medium concentrations shown in Table 35 below.

[0291]

[0292] After culturing for an additional 3 days, the medium was replaced with 0.3 mL of differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, total RNA was extracted from the cells in the same manner as in Test Examples 2, 3, 5, 6, and 8 to 13. One-step RT-PCR was performed using 200 ng of total RNA in the same manner as in Test Examples 1 to 3 and 8 to 13. The resulting PCR reaction products were analyzed to determine the skipping efficiencies of exon 45-55 skipping and exon 45 skipping.

[0293] The results are shown in Figures 36 and 37. When PMO No. 30, 31, or 33 was added as a second antisense oligomer along with PMO No. 6 and PMO No. 7 (conditions 3, 4, and 6), exon 45-55 skipping was observed to a similar extent as when PMO No. 8 was added (condition 5) (Figure 36). Furthermore, the skipping efficiency of exon 45 skipping was reduced in all cases compared to when only PMO No. 6 or PMO No. 8 was added (condition 2), and single skipping was suppressed (Figure 37).

[0294] <Test Example 15> Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (8): Restoration of dystrophin protein expression by multi-exon skipping

[0295] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a patient with DMD with exon 51 deletion, prepared in the same manner as in Test Examples 8 to 14, were placed in a 12-well collagen I-coated microplate (manufactured by AGC Technoglass Co., Ltd.) coated with Corning® Matrigel basement membrane matrix at a density of 2.0 × 10 ５ 1 / well in 1 mL of DMD patient-derived myoblast growth medium, 37°C, 5% CO ２ The cells were cultured for one day under these conditions. The day after seeding, the medium was changed from the proliferation medium for DMD patient-derived myoblasts to the differentiation medium for DMD patient-derived myoblasts. After 3 days of culture in the differentiation medium, the cells were transfected with 6 μM Endo-Porter PMO. After another 3 days of culture, the medium was changed to the differentiation medium. After 7 and 11 days of culture from the addition of PMO, Western blotting was performed as in Test Example 2 of Example 2, and Test Examples 4 and 7 of this Example to detect dystrophin protein. The electrophoresed samples are shown in Table 36 below. As positive controls for dystrophin expression, a lysate of mouse C2C12 cells cultured for muscle differentiation for 12 days (normal dystrophin control) and a lysate of skeletal muscle from a transgenic mouse with exon 45-55 deletion (dystrophin expression control with exon 45-55 deletion) were used.

[0296]

[0297] (Results) The results are shown in Figure 38. In the negative controls (conditions 3 and 6), no band was observed at the same position (arrowhead in Figure 38) as the band in the positive control (condition 2) for exon 45-55 deleted dystrophin, but in the samples transfected with the PMO cocktail (conditions 4, 5, 7, and 8), expression of dystrophin protein corresponding to exon 45-55 skipping was confirmed (arrowhead in Figure 38).

[0298] <Test Example 16> Assay of exon 45-55 multi-exon skipping in myoblasts derived from exon 51-deleted DMD patients (9): Restoration of dystrophin protein expression by multi-exon skipping (2)

[0299] Procedure: Myoblasts (CD56-positive, CD82-positive) derived from a patient with DMD with exon 51 deletion, prepared in the same manner as in Test Examples 8 to 15, were placed in a 12-well collagen I-coated microplate (manufactured by AGC Technoglass Co., Ltd.) coated with Corning® Matrigel basement membrane matrix at a density of 2.0 × 10 ５ 1 / well in 1 mL of DMD patient-derived myoblast growth medium, 37°C, 5% CO ２ The cells were cultured for one day under the conditions. The day after seeding, the medium was changed from the proliferation medium for DMD patient-derived myoblasts to the differentiation medium for DMD patient-derived myoblasts. After culturing in the differentiation medium for 7 days, the cells were transfected with PMO using 6 μM Endo-Porter. The PMOs used are shown in Table 37 below.

[0300]

[0301] After culturing for an additional 3 days, the medium was replaced with a differentiation medium. After culturing for 7 days from the addition of PMO, Western blotting was performed in the same manner as in Test Example 2 of Example 2 and Test Examples 4, 7, and 15 of this Example to detect dystrophin protein. The electrophoresed samples are shown in Table 38 below.

[0302]

[0303] (Results) The results are shown in Figure 39. In the negative control (condition 3), no band was observed at the same position as the band in the positive control (condition 2) for exon 45-55 deleted dystrophin, but in the samples transfected with the PMO cocktail (conditions 4, 5, and 6), expression of dystrophin protein corresponding to exon 45-55 skipping was confirmed (Figure 39: arrowheads).

Claims

Claim 1 An antisense oligomer that simultaneously skips any two or more consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA, or a pharmaceutically acceptable salt thereof, or a combination of their hydrates, wherein (i) a first unit oligomer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 11 nucleotides in the 3'-terminal to 5'-terminal direction of the 44th intron of the human dystrophin pre-mRNA and 69 nucleotides in the 5'-terminal to 3'-terminal direction of the 45th exon, or a partial nucleotide sequence thereof, and a second unit oligomer comprising a nucleotide sequence complementary to a nucleotide sequence from the 52nd nucleotide to the 75th nucleotide in the 3'-terminal to 5'-terminal direction of the 44th intron of the human dystrophin pre-mRNA, or a partial nucleotide sequence thereof is included, the first antisense oligomer or a pharmaceutically acceptable salt thereof, or their hydrates, and (ii) a second antisense oligomer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 33 nucleotides in the 3'-terminal to 5'-terminal direction of the 54th intron of the human dystrophin pre-mRNA and 53 nucleotides in the 5'-terminal to 3'-terminal direction of the 55th exon, or a partial nucleotide sequence thereof, or a pharmaceutically acceptable salt thereof, or their hydrates is included, the combination. Claim 2 The first unit oligomer comprises a nucleotide sequence complementary to 15 to 30 consecutive nucleotides of a nucleotide sequence consisting of 11 nucleotides in the 3'-terminal to 5'-terminal direction of the 44th intron of the human dystrophin pre-mRNA and 69 nucleotides in the 5'-terminal to 3'-terminal direction of the 45th exon, The second unit oligomer comprises a nucleotide sequence complementary to 1 to 10 consecutive nucleotides of a nucleotide sequence from the 52nd nucleotide to the 75th nucleotide in the 3'-terminal to 5'-terminal direction of the 44th intron of the human dystrophin pre-mRNA, The second antisense oligomer contains a base sequence complementary to 15 to 30 consecutive bases of a base sequence consisting of a 33-base base sequence from the 3'-end to the 5'-end of the 54th intron of the human dystrophin pre-mRNA and a 53-base base sequence from the 5'-end to the 3'-end of the 55th exon. The combination according to claim 1.

3. The first unit oligomer is (a) any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906, (c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 211 to 906 and having a length within ±15% of the length of any one base sequence selected, or (d) a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c) and contains a base sequence complementary thereto, and / or The second unit oligomer is (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105, (c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 105 and having a length within ±15% of the length of any one base sequence selected, or (d) a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c) and contains a base sequence complementary thereto. The combination according to claim 1 or 2.

4. The second antisense oligomer is (a) any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298, (c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 3507 to 4298 and having a length within ±15% of the length of any one base sequence selected, or (d) A base sequence of a part of any one base sequence selected from the group consisting of the above (a), (b), and (c) comprising a base sequence complementary to The combination according to claim 1 or 2.

5. The first antisense oligomer includes the first unit oligomer and the second unit oligomer in this order from the 5'-end, the first unit oligomer includes any one base sequence selected from SEQ ID NOs: 907 to 1602, the second unit oligomer includes any one base sequence selected from SEQ ID NOs: 106 to 210, and the second antisense oligomer includes any one base sequence selected from SEQ ID NOs: 4299 to 5090. The combination according to claim 1 or 2.

6. The combination according to claim 1 or 2, wherein the first unit oligomer includes any one base sequence selected from the group consisting of SEQ ID NOs: 1180, 1190, 1201, 1212, 1222, 1224, and 1239.

7. The combination according to claim 1 or 2, wherein the second unit oligomer includes any one base sequence selected from the group consisting of SEQ ID NOs: 114, 124, 151, 201, 203, and 205.

8. The first antisense oligomer includes the first unit oligomer and the second unit oligomer in this order from the 5'-end, and the first unit oligomer includes the base sequence of SEQ ID NO: 1201, and the second unit oligomer includes the base sequence of SEQ ID NO: 151, or the first unit oligomer includes the base sequence of SEQ ID NO: 1201, and the second unit oligomer includes the base sequence of SEQ ID NO: 201, or the first unit oligomer includes the base sequence of SEQ ID NO: 1201, and the second unit oligomer includes the base sequence of SEQ ID NO: 203, or the first unit oligomer includes the base sequence of SEQ ID NO: 1201, and the second unit oligomer includes the base sequence of SEQ ID NO: 205, or the first unit oligomer includes the base sequence of SEQ ID NO: 1239, and the second unit oligomer includes the base sequence of SEQ ID NO: 114, or the first unit oligomer includes the base sequence of SEQ ID NO: 1224, and the second unit oligomer includes the base sequence of SEQ ID NO: 124, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1180, and does the second unit oligomer contain the nucleotide sequence of SEQ ID NO: 151, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1190, and does the second unit oligomer contain the nucleotide sequence of SEQ ID NO: 151, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1212, and does the second unit oligomer contain the nucleotide sequence of SEQ ID NO: 151, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1222, and the second unit oligomer contains the nucleotide sequence of SEQ ID NO:

151. The combination according to claim 6.

9. The combination according to claim 1 or 2, wherein the second antisense oligomer contains a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4698, 4702, 4752, 4923, 4926, 4936, 4950, and 4977.

10. The first antisense oligomer contains the first unit oligomer and the second unit oligomer in this order from the 5'-end, and the first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, or the first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 201, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, or the first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 203, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, or the first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 205, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, or the first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1239, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 114, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1224, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 124, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1180, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1190, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1212, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1222, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4950, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4698, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4702, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4752, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4923, The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4926, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4936, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and does the second antisense oligomer contain the nucleotide sequence of SEQ ID NO: 4977, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1180, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4977. The combination according to claim 1 or 2.

11. The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, and the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950. The combination according to claim 5.

12. Furthermore, (iii) a third antisense oligomer containing a nucleotide sequence complementary to a nucleotide sequence consisting of 23 nucleotides in the 3'-terminal to 5'-terminal direction of the 45th exon of the human dystrophin pre-mRNA and 73 nucleotides in the 5'-terminal to 3'-terminal direction of the 45th intron or a partial nucleotide sequence thereof, or a pharmaceutically acceptable salt thereof or a hydrate thereof. The combination according to claim 1.

13. The third antisense oligomer contains a nucleotide sequence complementary to 15 to 30 consecutive nucleotides of a nucleotide sequence consisting of 23 nucleotides in the 3'-terminal to 5'-terminal direction of the 45th exon of the human dystrophin pre-mRNA and 73 nucleotides in the 5'-terminal to 3'-terminal direction of the 45th intron. The combination according to claim 12.

14. The third antisense oligomer is (a) any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554 (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554; (c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1603 to 2554 and having a length within ±15% compared to the length of any one base sequence so selected, or (d) a partial base sequence of any one base sequence selected from the group consisting of (a), (b), and (c) above containing a base sequence complementary thereto, The combination according to claim 12 or 13.

15. The third antisense oligomer is (a) any one base sequence selected from the group consisting of SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554; (b) a base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554; (c) a base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1611 to 1654, 1664 to 1707, 1718 to 1761, 1773 to 1816, 1829 to 1872, 1886 to 1929, 1944 to 1987, 2003 to 2046, 2063 to 2106, 2124 to 2167, 2186 to 2229, 2249 to 2292, 2313 to 2356, 2378 to 2421, 2444 to 2487, and 2511 to 2554 and having a length within ±15% compared to the length of any one base sequence so selected, or A base sequence that is part of any one base sequence selected from the group consisting of (d) the above (a), (b), and (c). Comprising a base sequence complementary to the base sequence The combination according to claim 14.

16. The third antisense oligomer is (a) Any one base sequence selected from the group consisting of SEQ ID NOs: 1617 to 1654, 1670 to 1707, 1724 to 1761, 1779 to 1816, 1835 to 1872, 1892 to 1929, 1950 to 1987, 2009 to 2046, 2069 to 2106, 2130 to 2167, 2192 to 2229, 2255 to 2292, 2319 to 2356, 2384 to 2421, 2450 to 2487, and 2517 to 2554, (b) A base sequence that hybridizes under stringent conditions with a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 1617 to 1654, 1670 to 1707, 1724 to 1761, 1779 to 1816, 1835 to 1872, 1892 to 1929, 1950 to 1987, 2009 to 2046, 2069 to 2106, 2130 to 2167, 2192 to 2229, 2255 to 2292, 2319 to 2356, 2384 to 2421, 2450 to 2487, and 2517 to 2554, (c) A base sequence having 85% or more sequence identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1617 to 1654, 1670 to 1707, 1724 to 1761, 1779 to 1816, 1835 to 1872, 1892 to 1929, 1950 to 1987, 2009 to 2046, 2069 to 2106, 2130 to 2167, 2192 to 2229, 2255 to 2292, 2319 to 2356, 2384 to 2421, 2450 to 2487, and 2517 to 2554, and having a length within ±15% compared to the length of any one of the selected base sequences, or A base sequence that is part of any one base sequence selected from the group consisting of (d) the above (a), (b), and (c). Comprising a base sequence complementary to the base sequence The combination according to claim 15.

17. The combination according to claim 12 or 13, wherein the third antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 3060, 3065, 3077, 3082, 3087, 3090, 3096, 3108, 3119, and 3320.

18. The first antisense oligomer includes the first unit oligomer and the second unit oligomer in this order from the 5'-end, the first unit oligomer includes any of the base sequences of SEQ ID NOs: 907 to 1602, the second unit oligomer includes any of the base sequences of SEQ ID NOs: 106 to 210, the second antisense oligomer includes any of the base sequences of SEQ ID NOs: 4299 to 5090, and the third antisense oligomer includes any of the base sequences of SEQ ID NOs: 2555 to 3506. The combination according to claim 12 or 13.

19. The first antisense oligomer includes the first unit oligomer and the second unit oligomer in this order from the 5'-end, and the first unit oligomer includes the base sequence of SEQ ID NO: 1201, the second unit oligomer includes the base sequence of SEQ ID NO: 151, the second antisense oligomer includes the base sequence of SEQ ID NO: 4950, and the third antisense oligomer includes the base sequence of SEQ ID NO: 3082, or the first unit oligomer includes the base sequence of SEQ ID NO: 1201, the second unit oligomer includes the base sequence of SEQ ID NO: 201, the second antisense oligomer includes the base sequence of SEQ ID NO: 4950, and the third antisense oligomer includes the base sequence of SEQ ID NO: 3082, or the first unit oligomer includes the base sequence of SEQ ID NO: 1201, the second unit oligomer includes the base sequence of SEQ ID NO: 203, the second antisense oligomer includes the base sequence of SEQ ID NO: 4950, and the third antisense oligomer includes the base sequence of SEQ ID NO: 3082, or the first unit oligomer includes the base sequence of SEQ ID NO: 1201, the second unit oligomer includes the base sequence of SEQ ID NO: 205, the second antisense oligomer includes the base sequence of SEQ ID NO: 4950, and the third antisense oligomer includes the base sequence of SEQ ID NO: 3082, or the first unit oligomer includes the base sequence of SEQ ID NO: 1239, the second unit oligomer includes the base sequence of SEQ ID NO: 114, the second antisense oligomer includes the base sequence of SEQ ID NO: 4950, and the third antisense oligomer includes the base sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1224, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 124, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1180, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1190, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1212, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1222, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3060, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3065, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3077, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3087, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3090, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3096, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3108, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3119, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3320, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4698, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4702, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4752, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4923, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4926, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4936, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4977, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, or The first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4977, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3096, or the first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1180, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4977, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3096, The combination according to claim 12 or 13.

20. The combination according to claim 18, wherein the first unit oligomer contains the nucleotide sequence of SEQ ID NO: 1201, the second unit oligomer contains the nucleotide sequence of SEQ ID NO: 151, the second antisense oligomer contains the nucleotide sequence of SEQ ID NO: 4950, and the third antisense oligomer contains the nucleotide sequence of SEQ ID NO: 3082, 3090 or 3096.

21. The combination according to claim 1 or 2, which skips all of the 45th exon to the 55th exon in human dystrophin pre-mRNA.

22. The combination according to claim 1, wherein the first to second antisense oligomers are oligonucleotides.

23. The combination according to claim 12, wherein the first to third antisense oligomers are oligonucleotides.

24. The combination according to claim 22 or 23, wherein the sugar moiety and / or phosphate linkage moiety of at least one nucleotide constituting the oligonucleotide is modified.

25. The sugar moiety of at least one nucleotide constituting the oligonucleotide has an -OH group at the 2'-position replaced by any group selected from the group consisting of -OR, -R, -R'OR, -SH, -SR, -NH ２ , -NHR, -NR ２ , -N ３ , -CN, -F, -Cl, -Br, and -I, and is ribose substituted with any group selected from the group consisting of -OR, -R, -R'OR, -SH, -SR, -NH, -NHR, -NR, -N, -CN, -F, -Cl, -Br, and -I, and is ribose substituted with any group selected from the group consisting of -OR, -R, -R'OR, -SH, -SR, -NH, -NHR, -NR, -N, -CN, -F, -Cl, -Br, and -I. The combination according to claim 22 or 23 (wherein R represents alkyl or aryl, and R' represents alkylene).

26. The combination according to claim 22 or 23, wherein the phosphate linkage moiety of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of phosphorothioate linkage, phosphorodithioate linkage, alkylphosphonate linkage, phosphoramidate linkage, and boranophosphate linkage.

27. The combination according to claim 1 or 2, wherein the first to second antisense oligomers are morpholino oligomers.

28. The combination according to claim 12, wherein the first to third antisense oligomers are morpholino oligomers.

29. The combination according to claim 27, wherein the first to second antisense oligomers are phosphorodiamidate morpholino oligomers.

30. The combination according to claim 28, wherein the first to third antisense oligomers are phosphorodiamidate morpholino oligomers.

31. The combination according to claim 27, wherein the 5'-end of the first to second antisense oligomers is a group of any one of the following chemical formulas (1) to (3). 【Chemical 1】

32. The combination according to claim 28, wherein the 5'-end of the first to third antisense oligomers is a group of any one of the following chemical formulas (1) to (3). 【Chemical 2】

33. (a)A pharmaceutical composition comprising the first and second antisense oligomers according to claim 1, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or (b)A combined pharmaceutical of (i) a pharmaceutical composition comprising the first antisense oligomer according to claim 1, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, and (ii) a pharmaceutical composition comprising the second antisense oligomer according to claim 1, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.

34. (a)A pharmaceutical composition comprising the first to third antisense oligomers according to claim 12, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or (b)A combined pharmaceutical of (i) a pharmaceutical composition comprising the first antisense oligomer according to claim 1, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, (ii) a pharmaceutical composition comprising the second antisense oligomer according to claim 1, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, and (iii) a pharmaceutical composition comprising the third antisense oligomer according to claim 12, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.

35. The pharmaceutical composition or combined pharmaceutical according to claim 33 or 34, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

36. The pharmaceutical composition or combined pharmaceutical according to claim 33 or 34, for the treatment of muscular dystrophy.

37. The pharmaceutical composition or combined pharmaceutical according to claim 33 or 34, for administration to a human patient.

38. A method for treating muscular dystrophy, comprising the step of administering to a patient with muscular dystrophy: (i) the first and second antisense oligomers according to claim 1, or a pharmaceutically acceptable salt thereof, or a hydrate thereof; (ii) the first to third antisense oligomers according to claim 12, or a pharmaceutically acceptable salt thereof, or a hydrate thereof; or (iii) the pharmaceutical composition or combination medicament according to claim 33 or 34.

39. The method for treatment according to claim 38, wherein the patient with muscular dystrophy has a mutation targeted for exon 45-55 skipping in the dystrophin gene.

40. The method for treatment according to claim 38, wherein the patient is a human.

Images