Antisense nucleic acid

Abstract

To provide a novel agent for treating Duchenne muscular dystrophy (DMD) caused by mutations of the dystrophin gene.SOLUTION: Provided are antisense oligomers having specific sequences that strongly induce skipping of exons 55, 45, 50, and 44 of a human dystrophin gene, and a muscular dystrophy therapeutic agent comprising the oligomers.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for skipping exons 55, 45, 50 and 44 of the human dystrophin gene. The present invention relates to antisense oligomers that enable binding to a target gene and pharmaceutical compositions containing the oligomers. [Background technology]

[0002] Duchenne muscular dystrophy (DMD) is the most common type of muscular dystrophy, occurring in approximately 1 in 3,500 male births. It is a highly inherited progressive muscular atrophy. During infancy, the patient's condition is almost the same as that of normal humans. However, muscle weakness begins to appear from around the age of 4-5 years. Muscle weakness then progresses until around the age of 12 years. It is a serious disease that can lead to the inability to walk and death from heart or respiratory failure in the 20s. Currently, there is no effective treatment for DMD, and there is a strong demand for the development of new therapeutic agents.

[0003] DMD is known to be caused by mutations in the dystrophin gene. The gene is located on the X chromosome and is a huge gene consisting of 2.2 million base pairs of DNA. Transcribed into a precursor, then spliced ​​to remove introns and combine 79 exons This mRNA is translated into 3,685 amino acids, and the dystrophin protein is synthesized. The dystrophin protein is involved in maintaining membrane stability in muscle cells. The dystrophin gene in DMD patients is mutated. Therefore, functional dystrophin protein is hardly expressed in muscle cells. Therefore, in DMD patients, the structure of muscle cells cannot be maintained, and a large amount of calcium Ions flow into muscle cells, causing an inflammation-like reaction and fibrosis, which leads to muscle damage. Cells become less able to regenerate.

[0004] Becker muscular dystrophy (BMD) is also caused by mutations in the dystrophin gene, Although the symptoms are muscle weakness due to muscle atrophy, they are generally milder than DMD. The progression is slow, and in most cases, the disease develops in adulthood. The clinical symptoms of DMD and BMD differ depending on the mutation. Amino acid sequence during translation of dystrophin mRNA into dystrophin protein It is thought that the cause of this is whether the framework is destroyed or maintained (Non-Patent Document 1 In other words, DMD has a mutation that shifts the amino acid reading frame, which prevents it from retaining its function. In BMD, a mutation in one of the exons causes dystrophin protein expression. Although a portion of the gene is deleted, the amino acid reading frame is maintained, so the gene is functional, albeit incomplete. A dystrophin protein having the gene is produced.

[0005] Exon skipping is a promising treatment for DMD. By modifying the isotyping, the amino acid reading frame of dystrophin mRNA was restored, resulting in partial This method induces the expression of dystrophin protein with restored function (Non-patent Document 2 ) The amino acid sequence portion targeted by exon skipping will be lost. The dystrophin protein expressed in this treatment is shorter than normal, but the amino acid sequence is The function of stabilizing muscle cells is partially preserved because the acid reading frame is maintained. Exon skipping can cause DMD to present symptoms similar to milder forms of BMD. The exon skipping method has been demonstrated in animal experiments using mice and dogs. Clinical trials are currently underway in human DMD patients.

[0006] Exon skipping can involve either the 5' or 3' splice site or both, or This can be induced by binding of antisense nucleic acids that target the inside of the exon. Chron is expressed as mRNA only when both splice sites are recognized by the spliceosome complex. Therefore, targeting splice sites with antisense nucleic acids can By using this method, exon skipping can be induced. SR tagging to exon splicing enhancers (ESEs) is required for recognition by the splicing machinery. Protein binding is thought to be necessary, and targeting ESEs can also enhance ESE activity. It can induce Kusun to skip.

[0007] The location and type of dystrophin gene mutations vary among DMD patients. Antisense nucleic acids that correspond to the target gene are needed. lton et al. have developed an antisense oligonucleotide that induces exon skipping for all 79 exons. A nucleic acid has been prepared by Annemieke Aartsma-Rus et al. in the Netherlands (Non-Patent Document 3). Antisense nucleic acids that induce exon skipping have been created for 39 types of exons. (Non-Patent Document 4).

[0008] Approximately 20% of all DMD patients have mutations in exons 55, 45, 50, and 44 (hereinafter referred to as "exons") Skipping exons (referred to as "exon 55," "exon 45," "exon 50," and "exon 44"). In recent years, the treatment has been improved by modifying exon 55 of the dystrophin gene. Research targeting exon skipping of 45, 50, and 44 has been conducted by several research institutes. However, exons 55, 45, 50, and 44 have been reported (Patent Documents 1 to 8). A technology for highly efficient skipping in patient cells has yet to be established. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication WO 2006 / 000057 [Patent Document 2] International Publication WO 2004 / 048570 [Patent Document 3] U.S. Patent Publication US 2010 / 0168212 [Patent Document 4] International Publication WO 2010 / 048586 [Patent Document 5] International Publication WO 2004 / 083446 [Patent Document 6] International Publication WO 2010 / 050801 [Patent Document 7] International Publication WO 2009 / 139630 [Non-patent literature]

[0010] [Non-Patent Document 1] Monaco AP et al., Genomics 1988; 2: p. 90-95 [Non-patent document 2] Matsuo M., Brain Dev 1996; 18: p. 167-172 [Non-patent document 3] Wilton SD, et al., Molecular Therapy 2007: 15: p. 1288-96 [Non-patent document 4] Annemieke Aartsma-Rus et al., (2002) Neuromuscular Disorders 12: S71-S77 [Non-patent document 5] Linda J. Popplewell et al., (2010) Neuromuscular Disorders , vol. 20, no. 2, p. 102-10 DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0011] In the above situation, the skipping of exons 55, 45, 50 and 44 of the dystrophin gene Antisense oligomers that strongly induce mitochondrial cloning and muscular dystrophy containing the oligomers There is a need for a drug to treat this condition. [Means for solving the problem]

[0012] As a result of detailed research into the structure of the dystrophin gene, the present inventors have discovered that the dystrophin gene The mRNA precursor (hereinafter referred to as "pre-mRNA") of the gene is the sequence 1 to 21 from the 5' end of exon 55. The sequences consisting of nucleotides around positions 11-31 and 14-34 were used as antisense oligonucleotides. Ligomer targeting induces highly efficient exon 55 skipping I discovered that it was possible. Furthermore, the present inventors have identified a region from the 5' end of exon 45 of the dystrophin gene pre-mRNA. The sequences consisting of nucleotides around positions 1 to 25 and 6 to 30 are used as antisense oligonucleotides. By targeting exon 45, we were able to induce exon 45 skipping with high efficiency. I found that... Furthermore, the present inventors have identified a region from the 5' end of exon 50 of the dystrophin gene pre-mRNA. The sequence consisting of nucleotides around positions 107 to 127 was targeted with an antisense oligomer. We found that exon 50 skipping can be induced with high efficiency by activating Ta. Furthermore, the present inventors have found that the 5' end of exon 44 of the dystrophin gene pre-mRNA The sequences consisting of nucleotides around 11 to 32 and 26 to 47 from the end were used as antisense oligonucleotides. Ligomer targeting induces highly efficient exon 44 skipping I discovered that it was possible. Based on this finding, the present inventors have completed the present invention.

[0013] That is, the present invention is as follows. [1] An algorithm that enables skipping of the 55th exon of the human dystrophin gene. An antisense oligomer 5' of the 55th exon of the human dystrophin gene From the end, -2 to 19th, -2 to 20th, -2 to 21st, -2 to 22nd, -2 to 23rd, -1st to 19th, -1st to 20th, -1st to 21st, -1st to 22nd, -1st to 23rd, 1st to 19th , 1st to 20th, 1st to 21st, 1st to 22nd, 1st to 23rd, 2nd to 19th, 2nd to 20th, 2nd-21st, 2nd-22nd, 2nd-23rd, 3rd-19th, 3rd-20th, 3rd-21st, 3rd to 22nd, 3rd to 23rd, 9th to 29th, 9th to 30th, 9th to 31st, 9th to 32nd, 9th ~33rd, 10th~29th, 10th~30th, 10th~31st, 10th~32nd, 10th~33rd, Nos. 11-29, 11-30, 11-31, 11-32, 11-33, 12-29 12th, 12th to 30th, 12th to 31st, 12th to 32nd, 12th to 33rd, 13th to 29th, 13th to 30th, 13th-31st, 13th-32nd, 13th-33rd, 12th-34th, 12th-35th, 12th to 36th, 13th to 34th, 13th to 35th, 13th to 36th, 14th to 32nd, 14th to 33rd , 14th-34th, 14th-35th, 14th-36th, 15th-32nd, 15th-33rd, 15th-34th th, 15th-35th, 15th-36th, 16th-32nd, 16th-33rd, 16th-34th, 16th A base sequence complementary to either one of the sequences consisting of nucleotides 16 to 36 or nucleotides 17 to 35 A string of antisense oligomers. [2] An algorithm that enables skipping of the 45th exon of the human dystrophin gene An antisense oligomer 5' of the 45th exon of the human dystrophin gene From the end, -3 to 19th, -3 to 20th, -3 to 21st, -3 to 22nd, -3 to 23rd, -2nd to 19th, -2nd to 20th, -2nd to 21st, -2nd to 22nd, -2nd to 23rd, -1st to 19th , -1st to 20th, -1st to 21st, -1st to 22nd, -1st to 23rd, 1st to 19th, 1st to 20th , 1st to 21st, 1st to 22nd, 1st to 23rd, 2nd to 19th, 2nd to 20th, 2nd to 21st , 2nd to 22nd, 2nd to 23rd, -2nd to 24th, -2nd to 25th, -2nd to 26th, -2nd to 27th th, -1st to 24th, -1st to 25th, -1st to 26th, -1st to 27th, 1st to 24th, 1st to 25th th, 1st-26th, 1st-27th, 2nd-24th, 2nd-25th, 2nd-26th, 2nd-27th , 3rd-23rd, 3rd-24th, 3rd-25th, 3rd-26th, 3rd-27th, 4th-28th , 4th to 29th, 4th to 30th, 4th to 31st, 4th to 32nd, 5th to 28th, 5th to 29th, 5th-30th, 5th-31st, 5th-32nd, 6th-28th, 6th-29th, 6th-30th, 6th-31st, 6th-32nd, 7th-28th, 7th-29th, 7th-30th, 7th-31st, 7th ~32nd, 8th to 28th, 8th to 29th, 8th to 30th, 8th to 31st or 8th to 32nd An antisense oligonucleotide consisting of a base sequence complementary to any one of the sequences consisting of nucleotides. Gomer. [3] An algorithm that enables skipping of the 50th exon of the human dystrophin gene. An antisense oligomer 5' of the 50th exon of the human dystrophin gene From the end, 105th to 125th, 105th to 126th, 105th to 127th, 105th to 128th, 105th to 1 29th, 106th-125th, 106th-126th, 106th-127th, 106th-128th, 106th-1 29th, 107th-125th, 107th-126th, 107th-127th, 107th-128th, 107th-1 29th, 108th-125th, 108th-126th, 108th-127th, 108th-128th, 108th-1 29th, 109th to 125th, 109th to 126th, 109th to 127th, 109th to 128th or 109th A sequence complementary to any one of the sequences consisting of nucleotides up to 129th position. Chisense oligomer. [4] An algorithm that enables skipping of the 44th exon of the human dystrophin gene. An antisense oligomer of the 5' end of exon 44 of the human dystrophin gene From the end, 9th to 30th, 9th to 31st, 9th to 32nd, 9th to 33rd, 9th to 34th, 10th to 3 0th, 10th to 31st, 10th to 32nd, 10th to 33rd, 10th to 34th, 11th to 30th, 1st 1st to 31st, 11th to 32nd, 11th to 33rd, 11th to 34th, 12th to 30th, 12th to 31st , 12th to 32nd, 12th to 33rd, 12th to 34th, 13th to 30th, 13th to 31st, 13th to 32nd th, 13th-33rd, 13th-34th, 24th-45th, 24th-46th, 24th-47th, 24th ~48th, 24th~49th, 25th~45th, 25th~46th, 25th~47th, 25th~48th, Nos. 25-49, 26-45, 26-46, 26-47, 26-48, 26-49 , 27th-45th, 27th-46th, 27th-47th, 27th-48th, 27th-49th, 28th- 45th, 28th-46th, 28th-47th, 28th-48th, 28th-49th, 29th-45th, A sequence consisting of nucleotides 29 to 46, 29 to 47, 29 to 48, or 29 to 49 An antisense oligomer consisting of a base sequence complementary to one of the following: [5] The 1st to 21st, 11th to 31st, or 5'-end of the 55th exon of the trophin gene The nucleic acid sequence according to [1] above, which is complementary to a sequence consisting of nucleotides 14 to 34. Antisense oligomers. [6] Nucleotides 170 to 190, 160 to 180, or 157 to 177 of SEQ ID NO: 5 The antisense oligonucleotide according to [1] above, which consists of any one of the base sequences of the sequences Mah. [7] The 5' end of exon 45 of the human dystrophin gene, from positions -2 to 19, and from position 1 A base sequence complementary to a sequence consisting of nucleotides 1 to 21, 1 to 25, or 6 to 30 The antisense oligomer according to [2] above, [8] Positions 158 to 178, 156 to 176, 152 to 176, or 147 to 171 of SEQ ID NO: 6 The nucleic acid sequence according to [2] above, which comprises any one of the sequences of nucleotides Antisense oligomers. [9] Positions 106-126 from the 5' end of the 50th exon of the human dystrophin gene [3] above, which comprises a base sequence complementary to a sequence consisting of nucleotides 107 to 127. 1. The antisense oligomer described herein.

[10] Either the sequence consisting of nucleotides 4 to 24 or 3 to 23 of SEQ ID NO: 7 The antisense oligomer according to [3] above, which consists of one base sequence.

[11] The 5' end of exon 44 of the human dystrophin gene, from positions 11 to 32, Complementary to a sequence consisting of nucleotides 25 to 45, 26 to 46, 26 to 47, or 27 to 47 The antisense oligomer according to [4] above, which consists of a base sequence similar to that of the first oligonucleotide.

[12] Positions 117 to 138, 104 to 124, 103 to 123, and 102 to 123 of SEQ ID NO: 8 or a sequence consisting of nucleotides 102 to 122, The antisense oligomer according to [4] above.

[13] The antibody according to any one of [1] to

[12] above, which is an oligonucleotide. Sense oligomer.

[14] a sugar moiety of at least one nucleotide constituting the oligonucleotide; and The antisense oligomer according to

[13] above, wherein the sugar moiety of at least one nucleotide constituting the oligonucleotide is modified and / or the phosphate linkage is modified. The -OH group at position 1 is selected from the group consisting of OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br and I.

[0033] The ribose substituted with any group selected from the group consisting of: Chisense oligomer. (The above R represents alkyl or aryl, and the above R' represents alkylene.)

[16] A phosphate bond in at least one nucleotide constituting the oligonucleotide. The moiety is a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, or In this case, the bond is selected from the group consisting of a phosphoramidate bond and a boranophosphate bond. The antisense oligomer according to

[14] or

[15] above, which is any one of the following:

[17] The anion according to any one of [1] to

[12] , which is a morpholino oligomer. Chisense oligomer.

[18] The anthraquinone according to

[17] , which is a phosphorodiamidate morpholino oligomer. Chisense oligomer.

[19] The antisense oligomer according to

[17] or

[0018] , wherein the 5'-end is a group represented by any one of the following chemical formulas (1) to (3): [ka]

[20] The antisense oligomer according to any one of [1] to

[19] above, and a pharmaceutical composition thereof A pharmaceutical composition for treating muscular dystrophy, comprising a therapeutically acceptable salt or hydrate thereof as an active ingredient. [Effects of the Invention]

[0014] The antisense oligomers of the present invention target exons 55 and 45 of the human dystrophin gene. It is possible to induce skipping of 50 and 44 with high efficiency. By administering this composition, the symptoms of Duchenne muscular dystrophy can be effectively alleviated. Furthermore, the antisense oligomer of the present invention can detect only the exon sequence of the patient. In order to target the target, antisense oligomers targeting introns are used. In contrast, the target sequence is conserved between individuals (individuals), and therefore individual differences (individual Furthermore, the present invention is characterized in that good skipping efficiency can be obtained regardless of the difference. Antisense oligomers have a short chain length of around 20 bp, which is not suitable for conventional DMD treatment. Compared with the antisense oligomer (approximately 25 bp), the target sequence contains a single nucleotide polymorphism (SNP). It is unlikely that variations between individuals (individuals) such as octopide polymorphism are included. It is characterized by the fact that good skipping efficiency can be obtained regardless of differences (individual differences). Generally, short antisense oligomers are relatively unlikely to induce immunity. Sense oligomers are characterized by their low risk of side effects due to the induction of cytokines, etc. . Furthermore, the antisense oligomer of the present invention has a short chain length, and therefore the production cost is relatively low. It is a price. [Brief explanation of the drawings]

[0015] [Figure 1]FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene by 2′-OMe-S-RNA oligomers in a human rhabdomyosarcoma cell line (RD cells). [Figure 2] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene by 2′-OMe-S-RNA oligomers in a human rhabdomyosarcoma cell line (RD cells). [Figure 3] FIG. 1 shows the efficiency of PMO-mediated exon 45 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 4] FIG. 1 shows the efficiency of PMO-mediated exon 45 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 5] FIG. 1 shows the efficiency of PMO-mediated skipping of exon 45 of the human dystrophin gene in cells induced to differentiate into muscle cells by introducing the human MyoD gene into fibroblasts (GM05017 cells) derived from a human DMD patient. [Figure 6] FIG. 1 shows the efficiency of exon 55 skipping of the human dystrophin gene by 2′-OMe-S-RNA oligomers in a human rhabdomyosarcoma cell line (RD cells). [Figure 7] FIG. 1 shows the efficiency of exon 55 skipping of the human dystrophin gene by 2′-OMe-S-RNA oligomers in a human rhabdomyosarcoma cell line (RD cells). [Figure 8] FIG. 1 shows the efficiency of PMO-mediated exon 55 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 9] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene by 2′-OMe-S-RNA oligomers in a human rhabdomyosarcoma cell line (RD cells). [Figure 10] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene by 2′-OMe-S-RNA oligomers in a human rhabdomyosarcoma cell line (RD cells). [Figure 11]FIG. 1 shows the efficiency of PMO-mediated exon 44 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 12] FIG. 1 shows the efficiency of PMO-mediated exon 44 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 13] FIG. 1 shows the efficiency of PMO-mediated exon 50 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 14] FIG. 1 shows the efficiency of PMO-mediated exon 45 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 15] FIG. 1 shows the efficiency of PMO-mediated exon 45 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 16] FIG. 1 shows the efficiency of PMO-mediated exon 55 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 17] FIG. 1 shows the efficiency of PMO-mediated exon 55 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 18] FIG. 1 shows the efficiency of PMO-mediated exon 44 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 19] FIG. 1 shows the efficiency of PMO-mediated exon 50 skipping of the human dystrophin gene in a human rhabdomyosarcoma cell line (RD cells). [Figure 20] FIG. 1 shows the efficiency of PMO-mediated exon 44 skipping of the human dystrophin gene in exon 45-deficient DMD patient-derived fibroblasts (GM05112 cells). [Figure 21] FIG. 1 shows the results of PMO-mediated skipping of exon 44 of the human dystrophin gene in exon 45-deficient DMD patient-derived fibroblasts (GM05112 cells) (Western blotting). [Figure 22]FIG. 1 shows the results (RT-PCR) of PMO-mediated exon 50 skipping of the human dystrophin gene in exon 45-deficient DMD patient-derived fibroblasts (GM05112 cells). [Figure 23] FIG. 1 shows the efficiency of PMO-mediated exon 50 skipping of the human dystrophin gene in exon 45-deficient DMD patient-derived fibroblasts (GM05112 cells). [Figure 24] FIG. 1 shows the results (RT-PCR) of PMO-mediated skipping of exon 55 of the human dystrophin gene in exon 45-deficient DMD patient-derived fibroblasts (GM05112 cells). [Figure 25] FIG. 1 shows the efficiency of PMO-mediated exon 55 skipping of the human dystrophin gene in exon 45-deficient DMD patient-derived fibroblasts (GM05112 cells). [Figure 26] FIG. 1 shows the results (RT-PCR) of PMO-mediated exon 50 skipping of the human dystrophin gene in fibroblasts (11-0627 cells) derived from a DMD patient with exon 8-9 duplication. [Figure 27] FIG. 1 shows the efficiency of PMO-mediated exon 50 skipping of the human dystrophin gene in fibroblasts (11-0627 cells) derived from a DMD patient with exon 8-9 duplication. [Figure 28] FIG. 1 shows the results (RT-PCR) of PMO-mediated exon 50 skipping of the human dystrophin gene in fibroblasts (GM04364 cells) derived from a DMD patient lacking exons 51-55. [Figure 29] FIG. 1 shows the efficiency of PMO-mediated exon 50 skipping of the human dystrophin gene in exon 51-55 deleted DMD patient-derived fibroblasts (GM04364 cells). [Figure 30] FIG. 1 shows the results (RT-PCR) of PMO-mediated exon 55 skipping of the human dystrophin gene in cells (04-035 cells) derived from a DMD patient with a deletion in exon 54 alone. [Figure 31]FIG. 1 shows the efficiency of PMO-mediated exon 55 skipping of the human dystrophin gene in cells derived from a DMD patient with a deletion in exon 54 alone (04-035 cells). MODE FOR CARRYING OUT THE INVENTION

[0016] The present invention will be described in detail below. The following embodiments are examples for explaining the present invention. The present invention is not intended to be limited to the embodiments. Unless otherwise specified, it can be implemented in various forms. All documents cited in this specification, as well as publications, patent publications and other patents, The documents are incorporated herein by reference and are not intended to be limiting unless expressly stated otherwise. The Japanese patent application (patent application No. 2011-288040) filed on December 28, 2011, which is the basis of the Described in the specification and drawings of the Japanese patent application (patent application no. 2012-043092) filed on February 29, 2012 Includes the contents of. The present invention will be described in detail below. The following embodiments are examples for explaining the present invention. The present invention is not intended to be limited to the embodiments. Unless otherwise specified, it can be implemented in various forms. Unless otherwise specified, the amino acid sequence has the amino terminus at the left end and the carboxyl terminus at the right end. The left end is the 5' end and the right end is the 3' end of the base sequence.

[0017] 1. Antisense oligomers The present invention enables skipping of the 55th exon of the human dystrophin gene. an antisense oligomer of the 55th exon of the human dystrophin gene; -2 to 19, -2 to 20, -2 to 21, -2 to 22, and -2 to 23 from the 5' end , -1st to 19th, -1st to 20th, -1st to 21st, -1st to 22nd, -1st to 23rd, 1st to 19th th, 1st-20th, 1st-21st, 1st-22nd, 1st-23rd, 2nd-19th, 2nd-20th , 2nd-21st, 2nd-22nd, 2nd-23rd, 3rd-19th, 3rd-20th, 3rd-21st , 3rd to 22nd, 3rd to 23rd, 9th to 29th, 9th to 30th, 9th to 31st, 9th to 32nd, Nos. 9-33, 10-29, 10-30, 10-31, 10-32, 10-33 11th, 11th to 29th, 11th to 30th, 11th to 31st, 11th to 32nd, 11th to 33rd, 12th 29th, 12th-30th, 12th-31st, 12th-32nd, 12th-33rd, 13th-29th, 13th to 30th, 13th to 31st, 13th to 32nd, 13th to 33rd, 12th to 34th, 12th to 35th , 12th to 36th, 13th to 34th, 13th to 35th, 13th to 36th, 14th to 32nd, 14th to 33rd th, 14th-34th, 14th-35th, 14th-36th, 15th-32nd, 15th-33rd, 15th ~34th, 15th~35th, 15th~36th, 16th~32nd, 16th~33rd, 16th~34th, A sequence consisting of nucleotides 16 to 35 or 16 to 36 (hereinafter referred to as "exon 55 target sequence") Antisense oligomers are those consisting of a base sequence complementary to one of the (hereinafter referred to as "the exon 55 skipping oligomer of the present invention").

[0018] The present invention also provides a method for skipping the 45th exon of the human dystrophin gene. an antisense oligomer that targets the 45th exon of the human dystrophin gene; From the 5' end of the nucleotides, positions -3 to 19, -3 to 20, -3 to 21, -3 to 22, and -3 to 2 3rd, -2nd to 19th, -2nd to 20th, -2nd to 21st, -2nd to 22nd, -2nd to 23rd, - 1st to 19th, -1st to 20th, -1st to 21st, -1st to 22nd, -1st to 23rd, 1st to 19th, 1st to 20th, 1st to 21st, 1st to 22nd, 1st to 23rd, 2nd to 19th, 2nd to 20th, 2nd to 21st, 2nd to 22nd, 2nd to 23rd, -2nd to 24th, -2nd to 25th, -2nd to 26th, -2~27th, -1~24th, -1~25th, -1~26th, -1~27th, 1~24th , 1st to 25th, 1st to 26th, 1st to 27th, 2nd to 24th, 2nd to 25th, 2nd to 26th , 2nd to 27th, 3rd to 23rd, 3rd to 24th, 3rd to 25th, 3rd to 26th, 3rd to 27th, 4th-28th, 4th-29th, 4th-30th, 4th-31st, 4th-32nd, 5th-28th, 5th-29th, 5th-30th, 5th-31st, 5th-32nd, 6th-28th, 6th-29th, 6th ~30th, 6th~31st, 6th~32nd, 7th~28th, 7th~29th, 7th~30th, 7th~ 31st, 7th to 32nd, 8th to 28th, 8th to 29th, 8th to 30th, 8th to 31st or 8th Any of the sequences consisting of the 32nd nucleotide (hereinafter also referred to as "exon 45 target sequence") An antisense oligomer (hereinafter referred to as the "extract of the present invention") having a base sequence complementary to one of the bases The company provides a "Son 45 Skipping Oligomer."

[0019] Furthermore, the present invention provides a method for skipping the 50th exon of the human dystrophin gene. an antisense oligomer that targets the 50th end of the human dystrophin gene; Positions 105-125, 105-126, 105-127, and 105-128 from the 5' end of chthon 105th-129th, 106th-125th, 106th-126th, 106th-127th, 106th-128th , 106th-129th, 107th-125th, 107th-126th, 107th-127th, 107th-128th 107th~129th, 108th~125th, 108th~126th, 108th~127th, 108th~128th , 108th-129th, 109th-125th, 109th-126th, 109th-127th, 109th-128th A sequence consisting of nucleotides 109 to 129 of exon 50 (hereinafter referred to as the “exon 50 target sequence”) An antisense oligomer (hereinafter referred to as The present invention provides a method for the preparation of exon 50 skipping oligomers of the present invention.

[0020] Furthermore, the present invention provides a method for the skipping of exon 44 of the human dystrophin gene. an antisense oligomer capable of targeting the 44th amino acid sequence of the human dystrophin gene; From the 5' end of the exon, positions 9-30, 9-31, 9-32, 9-33, and 9- 34th, 10th-30th, 10th-31st, 10th-32nd, 10th-33rd, 10th-34th, 11th to 30th, 11th to 31st, 11th to 32nd, 11th to 33rd, 11th to 34th, 12th to 30th , 12th to 31st, 12th to 32nd, 12th to 33rd, 12th to 34th, 13th to 30th, 13th to 31st th, 13th-32nd, 13th-33rd, 13th-34th, 24th-45th, 24th-46th, 24th ~47th, 24th~48th, 24th~49th, 25th~45th, 25th~46th, 25th~47th, Nos. 25-48, 25-49, 26-45, 26-46, 26-47, 26-48 , 26th-49th, 27th-45th, 27th-46th, 27th-47th, 27th-48th, 27th- 49th, 28th-45th, 28th-46th, 28th-47th, 28th-48th, 28th-49th, Nucleotides 29-45, 29-46, 29-47, 29-48, or 29-49 The sequence is complementary to one of the sequences consisting of exon 44 (hereinafter referred to as "exon 44 target sequence"). The antisense oligomer (hereinafter referred to as "the exon 44 skipping method of the present invention") consisting of the base sequence oligomers). Hereinafter, the exon 55, 45, 50, and 44 skipping oligomers of the present invention will be referred to as "oligomers of the present invention." They are sometimes collectively referred to as "gomers."

[0021] [Exons 55, 45, 50, and 44 of the human dystrophin gene] In the present invention, the term "gene" includes not only genomic genes but also cDNA, mRNA precursors, and mRNA. Preferably, the gene is a precursor to mRNA, i.e., pre-mRNA.

[0022] In the human genome, the human dystrophin gene is located at locus Xp21.2. The strophin gene is 3.0 Mbp in size, the largest known human gene. However, the coding region of the human dystrophin gene is only 14 kb, The coding region is distributed within the dystrophin gene as 79 exons (Roberts, RG., et al., Genomics, 16: 536-538 (1993)). A pre-mRNA is spliced ​​to generate a mature mRNA of 14 kb. The nucleotide sequence of the trophin gene is known (GenBank Accession No. NM_004006). Nucleotides -2 to 190 from the 5' end of exon 55 of the wild-type human dystrophin gene The base sequence of the nucleic acid fragment is shown in SEQ ID NO: 1. The base sequence consisting of nucleotides -3 to 176 from the 5' end of exon 45 of the guinea pig is denoted by SEQ ID NO: The sequence of the first to fifth sequences from the 5' end of exon 50 of the wild-type human dystrophin gene is shown in No. 2. The base sequence consisting of nucleotides 109 and 1 to 20 from the 5' end of intron 50 Shown in sequence number 3. In addition, the 1st to 148th residues from the 5' end of exon 44 of the wild-type human dystrophin gene The base sequence consisting of nucleotides is shown in SEQ ID NO:4.

[0023] The oligomers of the present invention are intended to target exons 55, 45, 50, or 44 of the human dystrophin gene. By mapping, the protein encoded by the DMD-type dystrophin gene is converted to BMD-type dystrophin. It was produced for the purpose of modifying it into a lophin protein. Exon 55 of the dystrophin gene is targeted by oligomeric exon skipping. 45, 50 and 44 include not only the wild type but also their respective mutant forms. Specifically, exons 55, 45, 50, and 44 of the mutant human dystrophin gene are The polynucleotide is the polynucleotide described in (a) or (b). (a) SEQ ID NO: 1 (or a sequence consisting of nucleotides 3 to 192 of SEQ ID NO: 1), 2( or a sequence consisting of nucleotides 4 to 179 of SEQ ID NO: 2), 3 (or SEQ ID NO: A sequence consisting of the 1st to 109th nucleotides of 3) or a base sequence complementary to the base sequence of 4 A polynucleotide that hybridizes under stringent conditions with a polynucleotide that is Do; (b) SEQ ID NO: 1 (or a sequence consisting of nucleotides 3 to 192 of SEQ ID NO: 1), 2( or a sequence consisting of nucleotides 4 to 179 of SEQ ID NO: 2), 3 (or SEQ ID NO: 90% or more identity to the base sequence of 3) consisting of the 1st to 109th nucleotides of 4) or the base sequence of 4 Polynucleotides consisting of identical base sequences

[0024] As used herein, the term "polynucleotide" refers to DNA or RNA. As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to a polynucleotide that hybridizes under stringent conditions. For example, SEQ ID NO: 1 (or a sequence consisting of nucleotides 3 to 192 of SEQ ID NO: 1) ), 2 (or a sequence consisting of nucleotides 4 to 179 of SEQ ID NO: 2), 3 (or A sequence consisting of nucleotides 1 to 109 of SEQ ID NO: 3) or a base sequence complementary to the base sequence of 4 Colony hybridization is performed using all or part of the polynucleotide consisting of the sequence as a probe. hybridization, plaque hybridization, or Southern hybridization Hybridization refers to polynucleotides obtained by using For example, see "Molecular Cloning: A Laboratory Manual" by Sambrook & Russell. ual Vol. 3, Cold Spring Harbor, Laboratory Press 2001" and "Ausubel, Current Prot. Those who are listed in "Collections in Molecular Biology, John Wiley & Sons 1987-1997" The law can be used.

[0025] In the present specification, the term "complementary base sequence" refers to a base sequence that is complementary to the target base sequence and has a Watson-Crick pair structure. It is not limited to base sequences that form a wobble base pair. Watson-Crick pairs include adenine-thymine and adenine- The base pairs formed by hydrogen bonds between guanine and uracil and between guanine and cytosine are called fluctuating base pairs. The base pairs are guanine-uracil, inosine-uracil, inosine-adenine and inosine-cysteine. The term "complementary base sequence" refers to a base pair in which a hydrogen bond is formed between the nucleotides. It is not necessary to have 100% complementarity with the target base sequence. For example, The base sequence may contain 1 to 3, 1 to 2, or 1 non-complementary base.

[0026] As used herein, "stringent conditions" refers to low stringent conditions, medium stringent conditions, The conditions may be either low stringency or high stringency. "Optimal conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, 32 The "moderate stringent conditions" are, for example, conditions of 5x SSC, 5x denaturing acid, and 5x ethanol. soln, 0.5% SDS, 50% formamide, 42°C or 5x SSC, 1% SDS, 50 mM Tris-HCl ( The conditions are pH 7.5, 50% formamide, and 42°C. For example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, 50°C or 0.2x SSC, 0 The conditions were 0.1% SDS and 65°C. Under these conditions, the higher the temperature, the higher the identity. However, it is expected that polynucleotides that can be obtained efficiently will be hybridized. Factors that affect the stringency of the reaction include temperature, probe concentration, and probe length. Several factors, such as the temperature, ionic strength, time, and salt concentration, can be considered. Similar stringency can be achieved by appropriately selecting

[0027] When using a commercially available kit for hybridization, for example, Alkphos Direct The Labelling and Detection System (GE Healthcare) can be used. Incubation with the labeled probe was performed according to the protocol provided with the kit. After overnight washing, the membrane was washed with the first washing buffer containing 0.1% (w / v) SDS at 55°C. After washing, the hybridized polynucleotides can be detected. No. 1 (or a sequence consisting of nucleotides 3 to 192 of SEQ ID NO: 1), 2 (or a sequence consisting of nucleotides 4 to 179 of SEQ ID NO: 2), 3 (or a sequence consisting of nucleotides 1 to 3 of SEQ ID NO: 3) A sequence consisting of the 109th nucleotide) or all or part of the base sequence complementary to the base sequence of 4 When preparing probes based on the above, commercially available reagents (e.g., PCR labeling mix ( The probe was labeled with digoxigenin (DIG) using a reagent such as Roche Diagnostics. In this case, hybridization was performed using the DIG Nucleic Acid Detection Kit (Roche Diagnostics). It is possible to detect

[0028] Polynucleotides other than the above hybridizable polynucleotides include homologous polynucleotides. The results were calculated using the BLAST software, which is a serologic search software, with default parameters. SEQ ID NO: 1 (or a sequence consisting of nucleotides 3 to 192 of SEQ ID NO: 1), (or a sequence consisting of nucleotides 4 to 179 of SEQ ID NO: 2), 3 (or SEQ ID NO: a sequence consisting of nucleotides 1 to 109 of No. 3) or a sequence consisting of a polynucleotide of No. 4 and 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, and 97% or more , 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more , 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity to the polynucleotide. You can give them chid.

[0029] The identity of the base sequences was evaluated using the BLAST algorithm by Carlin and Arthur (Bas ic Local Alignment Search Tool)(Proc. Natl. Acad. Sci. USA 872264-2268, 1990; P roc Natl Acad Sci USA 90: 5873, 1993). Programs called BLASTN and BLASTX based on this method have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing nucleotide sequences using BLASTN, use the parameter For example, the score is 100 and the word length is 12. When used, the default parameters of each program are used.

[0030] The sequence complementary to the sequence from -2 to 190 nucleotides from the 5' end of exon 55 is designated as SEQ ID NO: The sequence consisting of the -2nd to -1st nucleotides from the 5' end of exon 55 is shown in No. 5. The sequence (the sequence consisting of the first and second nucleotides of SEQ ID NO: 1) is exon 54 and exon 55. The sequence consisting of the two most downstream nucleotides of the 3' end of intron 54 between That is, the base sequence of exon 55 consists of nucleotides 3 to 192 of SEQ ID NO: 1. The sequence complementary to the base sequence of exon 55 is the 1st to 190th nucleotides of SEQ ID NO: 5. It is a sequence consisting of leutides.

[0031] Here, the -2 to 19 positions from the 5' end of the 55th exon of the human dystrophin gene , -2nd to 20th, -2nd to 21st, -2nd to 22nd, -2nd to 23rd, -1st to 19th, -1st to 20th th, -1st to 21st, -1st to 22nd, -1st to 23rd, 1st to 19th, 1st to 20th, 1st to 2nd 1st, 1st-22nd, 1st-23rd, 2nd-19th, 2nd-20th, 2nd-21st, 2nd-22nd th, 2nd-23rd, 3rd-19th, 3rd-20th, 3rd-21st, 3rd-22nd, 3rd-23rd , 9th-29th, 9th-30th, 9th-31st, 9th-32nd, 9th-33rd, 10th-29th 10th, 10th-30th, 10th-31st, 10th-32nd, 10th-33rd, 11th-29th, 11th- 30th, 11th-31st, 11th-32nd, 11th-33rd, 12th-29th, 12th-30th, 12th to 31st, 12th to 32nd, 12th to 33rd, 13th to 29th, 13th to 30th, 13th to 31st , 13th to 32nd, 13th to 33rd, 12th to 34th, 12th to 35th, 12th to 36th, 13th to 34th th, 13th-35th, 13th-36th, 14th-32nd, 14th-33rd, 14th-34th, 14th ~35th, 14th~36th, 15th~32nd, 15th~33rd, 15th~34th, 15th~35th, 15th to 36th, 16th to 32nd, 16th to 33rd, 16th to 34th, 16th to 35th or 16th to 36th The sequence complementary to the base sequence consisting of the 172nd nucleotide of SEQ ID NO: 5 is ~192nd, 171st~192nd, 170th~192nd, 169th~192nd, 168th~192nd, 172nd ~191st, 171st~191st, 170th~191st, 169th~191st, 168th~191st, 172nd ~190th, 171st~190th, 170th~190th, 169th~190th, 168th~190th, 172nd ~189th, 171st~189th, 170th~189th, 169th~189th, 168th~189th, 172nd ~188th, 171st~188th, 170th~188th, 169th~188th, 168th~188th, 162nd ~182nd, 161st~182nd, 160th~182nd, 159th~182nd, 158th~182nd, 162nd ~181st, 161st~181st, 160th~181st, 159th~181st, 158th~181st, 162nd ~180th, 161st~180th, 160th~180th, 159th~180th, 158th~180th, 162nd ~179th, 161st~179th, 160th~179th, 159th~179th, 158th~179th, 162nd ~178th, 161st~178th, 160th~178th, 159th~178th, 158th~178th, 157th ~179th, 156th~179th, 155th~179th, 157th~178th, 156th~178th, 155th ~178th, 159th~177th, 158th~177th, 157th~177th, 156th~177th, 155th ~177th, 159th~176th, 158th~176th, 157th~176th, 156th~176th, 155th ~176th, 159th~175th, 158th~175th, 157th~175th, 156th~175th or 1st It is identical to the sequence consisting of nucleotides 55 to 175.

[0032] A sequence complementary to the sequence from -3 to 176 nucleotides from the 5' end of exon 45 is sequenced. The sequence is shown in number 6. Here, the sequence consisting of the -3rd to -1st nucleotides from the 5' end of exon 45 is The sequence (the sequence consisting of the first to third nucleotides of SEQ ID NO: 2) is exon 44 and exon 4 The sequence consisting of the three most downstream nucleotides at the 3' end of intron 44, which exists between That is, the base sequence of exon 45 consists of nucleotides 4 to 179 of SEQ ID NO: 2. The sequence complementary to the base sequence of exon 45 is the sequence from nucleotides 1 to 176 of SEQ ID NO: 6. It is a sequence consisting of nucleotides.

[0033] Here, the -3 to 19 positions from the 5' end of the 45th exon of the human dystrophin gene , -3~20th, -3~21st, -3~22nd, -3~23rd, -2~19th, -2~20th th, -2nd to 21st, -2nd to 22nd, -2nd to 23rd, -1st to 19th, -1st to 20th, -1st ~21st, -1st to 22nd, -1st to 23rd, 1st to 19th, 1st to 20th, 1st to 21st, 1st ~22nd, 1st~23rd, 2nd~19th, 2nd~20th, 2nd~21st, 2nd~22nd, 2nd~ 23rd, -2nd to 24th, -2nd to 25th, -2nd to 26th, -2nd to 27th, -1st to 24th, -1st to 25th, -1st to 26th, -1st to 27th, 1st to 24th, 1st to 25th, 1st to 26th, 1st to 27th, 2nd to 24th, 2nd to 25th, 2nd to 26th, 2nd to 27th, 3rd to 23rd, 3rd to 24th, 3rd to 25th, 3rd to 26th, 3rd to 27th, 4th to 28th, 4th to 29th, 4th ~30th, 4th~31st, 4th~32nd, 5th~28th, 5th~29th, 5th~30th, 5th~ 31st, 5th-32nd, 6th-28th, 6th-29th, 6th-30th, 6th-31st, 6th-32nd th, 7th-28th, 7th-29th, 7th-30th, 7th-31st, 7th-32nd, 8th-28th a sequence consisting of nucleotides at positions 8 to 29, 8 to 30, 8 to 31, or 8 to 32 The base sequences complementary to the base sequences are the base sequences 158 to 179, 157 to 179, and 159 of SEQ ID NO: 6, respectively. 156th to 179th, 155th to 179th, 154th to 179th, 158th to 178th, 157th to 178th, 156th to 178th, 155th to 178th, 154th to 178th, 158th to 177th, 157th to 177th, 156th to 177th, 155th to 177th, 154th to 177th, 158th to 176th, 157th to 176th, 156th to 176th, 155th to 176th, 154th to 176th, 158th to 175th, 157th to 175th, 156th to 175th, 155th to 175th, 154th to 175th, 153rd to 178th, 152nd to 178th, 151st to 178th, 150th to 178th, 153rd to 177th, 152nd to 177th, 151st to 177th, 150th to 177th, 153rd to 176th, 152nd to 176th, 151st to 176th, 150th to 176th, 153rd to 175th, 152nd to 175th, 151st to 175th, 150th to 175th, 154th to 174th, 153rd to 174th, 152nd to 174th, 151st to 174th, 150th to 174th, 149th to 173rd, 148th to 173rd, 147th to 173rd, 146th to 173rd, 147th to 173rd, 149th to 172nd, 148th to 172nd, 147th to 172nd, 146th to 172nd, 145th to 172nd, 149th to 171st, 148th to 171st, 147th to 171st, 146th to 171st, 145th to 171st, 149th to 170th, 148th to 170th, 147th to 170th, 146th to 170th, 145th to 170th, 149th to 169th, Nucleotides 148-169, 147-169, 146-169, or 145-169 is identical to a sequence consisting of:

[0034] Nucleotides 1 to 109 from the 5' end of exon 50 and 5' end of intron 50 The sequence complementary to the base sequence consisting of nucleotides 1 to 20 is shown in SEQ ID NO: 7. , a sequence consisting of the 1st to 20th nucleotides from the 5' end of intron 50 (the first nucleotide of SEQ ID NO: 3) The sequence consisting of nucleotides 10 to 129) is located between exon 50 and exon 51. The sequence of the 20 most upstream nucleotides of the 5' end of intron 50 is shown. The base sequence of ctxon 50 is a sequence consisting of nucleotides 1 to 109 of SEQ ID NO: 3, The sequence complementary to the base sequence of exon 50 is from nucleotides 21 to 129 of SEQ ID NO: 7. It is an array as follows.

[0035] Here, the 50th exon of the human dystrophin gene is located between positions 105 and 125 from the 5' end. 105th~126th, 105th~127th, 105th~128th, 105th~129th, 106th~125th , 106th-126th, 106th-127th, 106th-128th, 106th-129th, 107th-125th , 107th-126th, 107th-127th, 107th-128th, 107th-129th, 108th-125th , 108th-126th, 108th-127th, 108th-128th, 108th-129th, 109th-125th Nucleotides 109-126, 109-127, 109-128, or 109-129 The sequences complementary to the base sequence consisting of the 5th to 25th bases and the 4th to 25th bases of SEQ ID NO: 7 are respectively th, 3rd-25th, 2nd-25th, 1st-25th, 5th-24th, 4th-24th, 3rd-24th , 2nd to 24th, 1st to 24th, 5th to 23rd, 4th to 23rd, 3rd to 23rd, 2nd to 23rd , 1st to 23rd, 5th to 22nd, 4th to 22nd, 3rd to 22nd, 2nd to 22nd, 1st to 22nd, Nucleotides 5 to 21, 4 to 21, 3 to 21, 2 to 21, or 1 to 21 is identical to the sequence consisting of

[0036] The sequence complementary to the sequence of nucleotides 1 to 148 from the 5' end of exon 44 is denoted by SEQ ID NO: Shown in No. 8.

[0037] Here, the 9th to 30th positions from the 5' end of the 44th exon of the human dystrophin gene, 9th to 31st, 9th to 32nd, 9th to 33rd, 9th to 34th, 10th to 30th, 10th to 31st, Nos. 10-32, 10-33, 10-34, 11-30, 11-31, 11-32 11th, 11th to 33rd, 11th to 34th, 12th to 30th, 12th to 31st, 12th to 32nd, 12th to 33rd, 12th-34th, 13th-30th, 13th-31st, 13th-32nd, 13th-33rd, 13th-34th, 24th-45th, 24th-46th, 24th-47th, 24th-48th, 24th-49th , 25th-45th, 25th-46th, 25th-47th, 25th-48th, 25th-49th, 26th-45th th, 26th-46th, 26th-47th, 26th-48th, 26th-49th, 27th-45th, 27th ~46th, 27th~47th, 27th~48th, 27th~49th, 28th~45th, 28th~46th, Nos. 28-47, Nos. 28-48, Nos. 28-49, Nos. 29-45, Nos. 29-46, Nos. 29-47 A sequence complementary to the base sequence consisting of nucleotides 29 to 48 or 29 to 49 is , respectively, the 119th to 140th positions, the 118th to 140th positions, the 117th to 140th positions, and the 116th to 14th positions of SEQ ID NO: 8. 0th, 115th to 140th, 119th to 139th, 118th to 139th, 117th to 139th, 116th to 13 9th, 115th-139th, 119th-138th, 118th-138th, 117th-138th, 116th-13 8th, 115th-138th, 119th-137th, 118th-137th, 117th-137th, 116th-13 7th, 115th-137th, 119th-136th, 118th-136th, 117th-136th, 116th-13 6th, 115th-136th, 104th-125th, 103rd-125th, 102nd-125th, 101st-12th 5th, 100th-125th, 104th-124th, 103rd-124th, 102nd-124th, 101st-12th 4th, 100th-124th, 104th-123rd, 103rd-123rd, 102nd-123rd, 101st-12th 3rd, 100th-123rd, 104th-122nd, 103rd-122nd, 102nd-122nd, 101st-12th 2nd, 100th-122nd, 104th-121st, 103rd-121st, 102nd-121st, 101st-12 1st, 100th-121st, 104th-120th, 103rd-120th, 102nd-120th, 101st-12th It is identical to the sequence consisting of nucleotides 0 or 100 to 120.

[0038] The correspondence between the base sequences of exons 55, 45, 50, and 44 and SEQ ID NOs: 5 to 8 is as shown in the table below. It can be stopped.

[0039] [Table 1-1] [Table 1-2] [Table 1-3] [Table 2-1] [Table 2-2] [Table 2-3] [Table 3] [Table 4-1] [Table 4-2]

[0040] The exon 55 skipping oligomer of the present invention is preferably derived from the human dystrophin gene. Nucleotides 1-21, 11-31, or 14-34 from the 5' end of the 55th exon of the child a base sequence complementary to any one of the sequences consisting of nucleotides (for example, the 170th base of SEQ ID NO: 5) any one of the sequences consisting of nucleotides 1 to 190, 160 to 180, or 157 to 177 It consists of two base sequences.

[0041] The exon 45 skipping oligomer of the present invention is preferably derived from the human dystrophin gene. -2 to 19, 1 to 21, 1 to 25, or 6 to 19 from the 5' end of the 45th exon of the offspring A base sequence complementary to any one of the sequences consisting of the 30th nucleotide (e.g., SEQ ID NO: Nucleotides 158 to 178, 156 to 176, 152 to 176, or 147 to 171 of No. 6 It consists of a base sequence consisting of one of the base sequences.

[0042] The exon 50 skipping oligomer of the present invention is preferably derived from the human dystrophin gene. Nucleotides 106-126 or 107-127 from the 5' end of the 50th exon of the child a base sequence complementary to any one of the sequences consisting of (for example, the 4th to 24th positions of SEQ ID NO: 7 or It consists of any one of the base sequences consisting of the 3rd to 23rd nucleotides.

[0043] The exon 44 skipping oligomer of the present invention is preferably derived from the human dystrophin gene. From the 5' end of the 44th exon of the child, positions 11 to 32, 25 to 45, 26 to 46, and 26 to 47 A base sequence complementary to either the sequence consisting of nucleotides 27 to 47 (e.g. For example, positions 117 to 138, positions 104 to 124, positions 103 to 123, and positions 102 to 123 of SEQ ID NO: 8 or a sequence consisting of nucleotides 102 to 122).

[0044] "Enabling skipping of the 55th exon of the human dystrophin gene" , a region corresponding to exon 55 of the transcript (e.g., pre-mRNA) of the human dystrophin gene When the oligomer of the present invention binds to the transcript, For example, in a DMD patient with a deletion of exon 54, the nucleotide sequence corresponding to the 3' end of exon 53 The nucleotide sequence corresponding to the 5' end of exon 56 is linked to the 3' side, causing a codon frameshift. This means that untouched mature mRNA is formed.

[0045] Similarly, "enables skipping of the 45th exon of the human dystrophin gene" " corresponds to exon 45 of the transcript (e.g., pre-mRNA) of the human dystrophin gene. The oligomer of the present invention binds to the spliced ​​region of the transcript, thereby preventing the transcript from undergoing splicing. For example, in the case of a DMD patient with a deletion of exon 44, the base corresponding to the 3' end of exon 43 The nucleotide sequence corresponding to the 5' end of exon 46 is linked to the 3' side of the sequence, resulting in a codon frameshift. This means that mature mRNA is formed without any cross-reaction.

[0046] It also "enables skipping of the 50th exon of the human dystrophin gene." " corresponds to exon 50 of the transcript (e.g., pre-mRNA) of the human dystrophin gene. The oligomer of the present invention binds to the spliced ​​region of the transcript, thereby preventing the transcript from undergoing splicing. For example, in the case of a DMD patient with a deletion of exon 51, the base corresponding to the 3' end of exon 49 The nucleotide sequence corresponding to the 5' end of exon 52 is linked to the 3' side of the sequence, resulting in a codon frameshift. This means that mature mRNA is formed without any cross-reaction.

[0047] Similarly, "skipping of the 44th exon of the human dystrophin gene is possible." "To make" refers to a mutation in exon 44 of the transcript (e.g., pre-mRNA) of the human dystrophin gene. The oligomer of the present invention binds to the corresponding site, thereby preventing the transcript from undergoing splicing. For example, in a DMD patient with a deletion of exon 45, the 3' end of exon 43 is The base sequence corresponding to the 5' end of exon 46 is linked to the 3' side of the base sequence corresponding to the codon This means that mature mRNA is formed without any domain shift. Thus, the oligomers of the present invention are intended to be used in conjunction with exons 55, 45, 50 and 60 of the human dystrophin gene. Each target sequence (exons 55, 45, 50, and 44) ​​was included as long as skipping of exon 44 was possible. For example, the oligomer of the present invention may not have a nucleotide sequence that is 100% complementary to the base sequence of the oligomer. may contain 1-3, 1-2, or 1 non-complementary base to the target sequence. good.

[0048] Here, the "binding" refers to the binding of the oligomer of the present invention to a transcript of the human dystrophin gene. When these two are mixed, they hybridize to form a double strand under physiological conditions. The above "physiological conditions" refers to conditions adjusted to pH, salt composition, and temperature similar to those in the body. For example, the temperature is 25 to 40°C, preferably 37°C, and the pH is 5 to 8, preferably 7.4. For example, a sodium chloride concentration of 150 mM is used.

[0049] Whether skipping of exons 55, 45, 50, and 44 of the human dystrophin gene occurs The oligomers of the present invention are introduced into dystrophin-expressing cells (e.g., human rhabdomyosarcoma cells). and extracting mRNA of the human dystrophin gene from the total RNA of the dystrophin-expressing cells. The regions surrounding exons 55, 45, 50, and 44 of the nucleotide sequence were amplified by RT-PCR, and the PCR amplification products were subjected to nested PCR. This can be confirmed by PCR or sequence analysis. The method involves recovering mRNA of the human dystrophin gene from test cells, and determining exon 55 of the mRNA. , 45, 50 and 44 skipped band "A" and exon 55, 45, 5 The amount of polynucleotide "B" in the bands that were not skipped by 0 and 44 was measured, and these "A" Based on the measured values ​​of "A" and "B", it can be calculated according to the following formula: Skipping efficiency (%) = A / ( A + B ) x 100

[0050] The oligomer of the present invention may be, for example, an oligonucleotide having a length of 18 to 28 bases. tides, morpholino oligomers, or peptide nucleic acid (PNA) oligomers The length of the morpholino acid is preferably 15 to 30 bases or 20 to 25 bases. Gomer is preferred.

[0051] The oligonucleotide (hereinafter referred to as "the oligonucleotide of the present invention") is The oligomer of the present invention is composed of nucleotides as building blocks, and such nucleotides are ribonucleic acids. The nucleotides may be nucleotides, deoxyribonucleotides or modified nucleotides. Modified nucleotides are nucleotides that are ribonucleotides or deoxyribonucleotides. It refers to a molecule in which all or part of the acid-base, sugar moiety, and phosphate binding moiety are modified.

[0052] Examples of nucleic acid bases include adenine, guanine, hypoxanthine, cytosine, and thymine. Examples of such modified bases include uracil, uracil, and modified bases thereof. For example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g. 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halo Uracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), uracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydrogen (hydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil hydroxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-di Methylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyl Adenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylamino 5-methylmethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl 2-methylthiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetone Acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, Examples of suitable amines include, but are not limited to, benzophenone, imidazole, xanthine, etc. stomach.

[0053] Modifications of the sugar moiety include, for example, modification of the 2'-position of ribose and modifications of other parts of the sugar. Examples of modifications at the 2'-position of ribose include -O at the 2'-position of ribose. Modifications that replace H groups with OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, and I are listed. Here, R represents alkyl or aryl, and R' represents alkylene. Modifications of other sugar moieties include, for example, O at the 4' position of ribose or deoxyribose. The sugars are substituted with S, and the 2' and 4' positions of the sugars are linked together. For example, LNA (Locked Nucleic Acid Examples include ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acid) and ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acid). However, the present invention is not limited to the above.

[0054] Modification of the phosphate bond moiety includes, for example, changing the phosphodiester bond to a phosphorothioate bond. bond, phosphorodithioate bond, alkylphosphonate bond, phosphoramidate bond , boranophosphate bond (Enya et al: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (for example, Patent Republished Publication No. 2006 (See Nos. 2006 / 129594 and 2006 / 038608).

[0055] The alkyl is preferably a linear or branched alkyl having 1 to 6 carbon atoms. Examples of the alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, se c-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n- The alkyl may be substituted, and such substitution may be Examples of the substituent include halogen, alkoxy, cyano, and nitro. These may be substituted by 1 to 3 groups. The cycloalkyl is preferably a cycloalkyl having 5 to 12 carbon atoms. For example, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl Examples include cyclododecyl and cyclododecyl. Examples of halogen include fluorine, chlorine, bromine, and iodine. The alkoxy includes linear or branched alkoxy having 1 to 6 carbon atoms, for example, meth Oxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy oxy, tert-butoxy, n-pentyloxy, isopentyl oxy, n-hexyloxy, isohexyloxy, etc. Alkoxy having 1 to 3 prime numbers is preferred. The aryl is preferably an aryl having 6 to 10 carbon atoms. Examples of the alkyl group include phenyl, α-naphthyl, and β-naphthyl. Phenyl is particularly preferred. The aryl may be substituted, and such substituents include, for example, alkyl, halogen, and the like. Examples of the substituted alkyl include alkoxy, cyano, and nitro. good. The alkylene is preferably a straight-chain or branched-chain alkylene having 1 to 6 carbon atoms. Specifically, for example, methylene, ethylene, trimethylene, tetramethylene, pentamethylene Examples include ethylene, hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene. The acyl may be a straight-chain or branched alkanoyl or an alloy. Examples of the alkanoyl include formyl, acetyl, 2-methyl, and the like. ethylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentyl Examples of the aryl groups include pentanoyl, 2,2-dimethylpropionyl, and hexanoyl. Examples of such alkyl esters include benzoyl, toluoyl, and naphthoyl. The aryl may be substituted at any substitutable position, and may be substituted with alkyl. stomach.

[0056] The oligonucleotide of the present invention preferably has a structure in which the -OH group at the 2'-position of ribose is substituted with methoxy. The phosphate bond moiety is a phosphorothioate bond. The oligomer of the present invention has the following structural units. [ka] (In the formula, Base represents a nucleic acid base.)

[0057] The oligonucleotides of the present invention can be synthesized using various automated synthesizers (e.g., AKTA oligopilot plus 1 0 / 100 (GE Healthcare)) or can be easily synthesized by a third party. Alternatively, the preparation can be outsourced to a research institution (for example, Promega or Takara).

[0058] The morpholino oligomer of the present invention is a morpholino oligomer of the present invention having a group represented by the following general formula as a constituent unit: It is an oligomer. [ka] (wherein Base has the same meaning as defined above; W represents a group represented by any of the following formulas:

[0059] [ka]

[0060] (Wherein, X is —CHR 1 , -O-CH2R 1 , -S-CH2R 1 , -NR 2 R 3 or represents F; R 1 represents H, alkyl; R 2 and R 3 are the same or different and represent H, alkyl, cycloalkyl, or aryl. death; Y1 is 0, S, CH2 or NR 1 represents; Y2 is 0, S or NR 1 represents; Z represents 0 or S.

[0061] 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") . [ka] (In the formula, Base, R 2 , R 3 has the same meaning as above.)

[0062] Morpholino oligomers can be prepared, for example, from the compounds described in International Publication No. WO 1991 / 009033 or WO 2004 / 022664. In particular, PMOs can be prepared according to WO 2009 / 064471. It can be prepared according to the method described in No. 471 or the method described below. Cut.

[0063] [PMO manufacturing method] One embodiment of PMO is, for example, a compound represented by the following general formula (I) (hereinafter referred to as PMO(I)): ) can be mentioned. [ka]

[0064] [In the formula, each Base, R 2 , R 3 has the same meaning as above; n is any integer in the range of 1 to 99, and preferably any integer in the range of 18 to 28. is an integer.]

[0065] PMO(I) can be produced according to known methods, for example, by carrying out the following steps: The film can be produced by carrying out the process. The compounds and reagents used in the following steps are those commonly used in the production of PMOs. If so, there is no particular limitation. All of the following steps can be carried out using either liquid phase or solid phase synthesis (manual or commercially available solid phase automated synthesis). When PMO is produced by the solid phase method, the operation procedure can be simplified. In terms of accuracy of synthesis, a method using an automatic synthesizer is preferred.

[0066] (1) Process A: A compound represented by the following general formula (II) (hereinafter referred to as compound (II)) is reacted with an acid. By this, a compound represented by the following general formula (III) (hereinafter referred to as compound (III)) can be obtained. Manufacturing process. [ka]

[0067] [where n, R 2 , R 3 has the same meaning as above; Each B P independently represent an optionally protected nucleobase; T represents a trityl group, a monomethoxytrityl group, or a dimethoxytrityl group; L is hydrogen, acyl, or a group represented by the following general formula (IV) (hereinafter referred to as group (IV)). represents.] [ka]

[0068] B P The "nucleobase" in this case can be the same as the "base". , B P The amino group or hydroxyl group of the nucleic acid base may be protected. There are no particular restrictions on such amino group-protecting groups as long as they are used as protecting groups for nucleic acids. Specific examples include, but are not limited to, benzoyl, 4-methoxybenzoyl, acetyl, and propyl. Onyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butyl 4-Isopropylphenoxyacetyl, (dimethylamino)methyl Examples of the protecting group for the hydroxyl group include 2-cyanoethyl, 4-nitroethyl, and the like. phenylsulfonylethyl, methylsulfonylethyl, trimethylsilyl ethyl, phenyl optionally substituted with 1 to 5 electron-withdrawing groups at any substitutable position; , diphenylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, methylphenyl 1-pyrrolidinylcarbamoyl, morpholinocarbamoyl, 4-(tert- butylcarboxy)benzyl, 4-[(dimethylamino)carboxy]benzyl, 4-(phenyl carboxy)benzyl (see, for example, International Publication No. 2009 / 064471 reference). The "solid phase carrier" is not particularly limited as long as it can be used in a solid phase reaction of nucleic acid. For example, (i) a reagent (e.g., dichloromethane) that can be used in the synthesis of a morpholino nucleic acid derivative acetonitrile, tetrazole, N-methylimidazole, pyridine, acetic anhydride, (ii) It is used for the synthesis of morpholino nucleic acid derivatives. (iii) be chemically stable to available reagents; (iv) be chemically modifiable; and (v) have sufficient strength to withstand the high pressures applied during processing; (vi) It is desirable that the particle size range and distribution are constant. Specifically, swellable polystyrene (For example, aminomethyl polystyrene resin 1% dibenzylbenzene cross-linked (200-400 mesh) (2.4-3.0 mmol / g) (Tokyo Chemical Industry Co., Ltd.), Aminomethylated Polystyrene Resin·HCl [ Dibenzylbenzene 1%, 100-200 mesh (Peptide Institute Co., Ltd.), non-swelling polymer polyethylene (e.g., Primer Support (GE Healthcare)), PEG-linked polystyrene ( For example, NH2-PEG resin (manufactured by Watanabe Chemical Co., Ltd.), TentaGel resin, controlled pore glass (controlled pore glass (CPG) (e.g., manufactured by CPG), oxalyl-pore glass (e.g., manufactured by Alul et al., N (See Nucleic Acids Research, Vol. 19, 1527 (1991)), TentaGel support - aminopolyethylene ethylene glycol-derivatized supports (e.g., Wright et al., Tetrahedron Letters, Vol. 34, 3373 ( 1993), and examples thereof include Poros-polystyrene / divinylbenzene copolymers. do. The "linker" is a compound that is usually used to link nucleic acids or morpholino nucleic acid derivatives. Known alkyl groups can be used, such as 3-aminopropyl, succinyl, 2,2'-diaminopropyl, and the like. Examples include ethanol sulfonyl and long chain alkylamino (LCAA).

[0069] This step can be carried out by reacting compound (II) with an acid.

[0070] The "acid" that can be used in this step is, for example, trifluoroacetic acid, dichloroacetic acid, or trifluoroacetic acid. The amount of the acid used is, for example, 1 mol of Compound (II) The amount is suitably in the range of 0.1 to 1000 molar equivalents, preferably 1 to 100 It is within the range of molar equivalents. In addition, an organic amine can be used together with the acid. Although not limited to, for example, triethylamine can be mentioned. The amount of amine used is, for example, within the range of 0.01 to 10 molar equivalents per mole of acid. and preferably in the range of 0.1 molar equivalents to 2 molar equivalents. When a salt or a mixture of an acid and an organic amine is used in this step, for example, triflate Examples of the salt or mixture of fluoroacetic acid and triethylamine include triethylamine and more specifically, triethylamine and A mixture of 2 equivalents of trifluoroacetic acid and 1 equivalent of triethylamine can be given. do. The acid that can be used in this process is diluted with an appropriate solvent to a concentration within the range of 0.1% to 30%. The solvent is not particularly limited as long as it does not participate in the reaction. For example, dichloromethane, acetonitrile, alcohols (ethanol, isopropanol) , trifluoroethanol, etc.), water or a mixture thereof.

[0071] The reaction temperature in the above reaction is preferably within the range of, for example, 10°C to 50°C, more preferably The temperature is preferably in the range of 20°C to 40°C, and more preferably in the range of 25°C to 35°C. The reaction time varies depending on the type of acid used and the reaction temperature, but is usually in the range of 0.1 minutes to 24 hours. Preferably, it is within the range of 1 minute to 5 hours.

[0072] After this step is completed, if necessary, a base may be added to neutralize the acid present in the system. The "base" is not particularly limited, but for example, diisopropyl The base should be added at a concentration in the range of 0.1% (v / v) to 30% (v / v). It can also be used after diluting with an appropriate solvent. The solvent used in this step is not particularly limited as long as it does not participate in the reaction. ethane, acetonitrile, alcohols (ethanol, isopropanol, trifluoroethanol The reaction temperature can be, for example, 10 The temperature is preferably in the range of 20°C to 50°C, more preferably in the range of 20°C to 40°C, and even more preferably Preferably, it is in the range of 25°C to 35°C. The reaction time varies depending on the type of base used and the reaction temperature, but is usually between 0.1 minutes and 24 hours. The range is appropriate, and preferably, it is in the range of 1 minute to 5 hours.

[0073] In addition, in the compound (II), n=1 and L is a group (IV), the following general formula (IIa) The compound represented by the formula (hereinafter referred to as compound (IIa)) can be produced by the following method. This can be done. [ka] [In the formula, B P , T, linker, and solid phase support are as defined above.]

[0074] Step 1: The compound represented by the following general formula (V) is reacted with an acylating agent to give the compound represented by the following general formula (V): A process for producing a compound represented by formula (VI) (hereinafter referred to as compound (VI)). [ka] [In the formula, B P , T, linker are as defined above; R 4 represents a hydroxyl group, a halogen, or an amino group.

[0075] This step is carried out by a known linker introduction reaction using compound (V) as a starting material. It is possible. In particular, the compound represented by the following general formula (VIa) can be prepared by reacting the compound (V) with succinic anhydride: It can be produced by carrying out a process known as an esterification reaction. [ka] [In the formula, B P , T has the same meaning as above.]

[0076] Step 2: Compound (VI) is reacted with a solid support by reacting with a condensing agent or the like to give compound (II a) A process for producing the product. [ka] [In the formula, B P , R 4 , T, linker, and solid phase support are as defined above.] This step is carried out by a method known as a condensation reaction using compound (VI) and a solid support. It is possible. In the compound (II), n is 2 to 99, and L is a group (IV), and the compound is represented by the following general formula (IIa2): The compound (IIa) can be produced by the steps A and B of the method for producing PMO described herein, using compound (IIa) as a starting material. The process can be produced by repeating steps A and B a desired number of times. [ka] [In the formula, B P , R 2 , R 3 , T, linker, and solid support are as defined above; n' represents 1 to 98.

[0077] In addition, in the compound (II), n=1 and L is hydrogen, and the compound (II) is represented by the following general formula (IIb): The compound can be produced by the method described in, for example, WO 1991 / 009033. can. [ka] [In the formula, B P , T has the same meaning as above.]

[0078] In the compound (II), n is 2 to 99, and L is hydrogen, and the compound is represented by the following general formula (IIb2): The compound to be produced is a compound (IIb) starting from compound (IIb) and produced by the process for producing PMO described herein. The production can be carried out by repeating steps A and B a desired number of times. [ka] [In the formula, B P , n', R 2 , R 3 , T has the same meaning as above.]

[0079] In addition, in the compound (II), n=1 and L is acyl, the compound represented by the following general formula (IIc) The compound represented by the formula (IIb) is subjected to a process known as an acylation reaction. It can be produced by [ka] [In the formula, B P , T is as defined above; R 5 represents acyl.]

[0080] In the compound (II), n is 2 to 99, and L is acyl, and the compound is represented by the following general formula (IIc2): The compound to be produced is a compound (IIc) starting from compound (IIc) and produced by the process for producing PMO described herein. The production can be carried out by repeating steps A and B a desired number of times. [ka] [In the formula, B P , n', R 2 , R 3 , R 5, T has the same meaning as above.]

[0081] (2) Process B: Compound (III) is reacted with a morpholino monomer compound in the presence of a base, A process for producing a compound represented by the following general formula (VII) (hereinafter referred to as compound (VII)): [ka] [In the formula, each B P , L, n, R 2 , R 3 , T has the same meaning as above.]

[0082] This step involves reacting compound (III) with a morpholino monomer compound in the presence of a base. This can be carried out by:

[0083] As the morpholino monomer compound, for example, a compound represented by the following general formula (VIII) Some examples include: [ka] [In the formula, B P , R 2 , R 3 , and T has the same meaning as above.]

[0084] Examples of the "base" that can be used in this step include diisopropylamine, triethylamine, and the like. Examples of the base include N-ethylmorpholine and N-ethylmorpholine. The amount is preferably in the range of 1 to 1000 molar equivalents relative to 1 mole of compound (III). Alternatively, it is in the range of 10 molar equivalents to 100 molar equivalents. The morpholino monomer compound and base that can be used in this step have a concentration of 0.1% to 30%. The solvent may be diluted with a suitable solvent as long as it is not involved in the reaction. Examples of the methylimidazolidone include, but are not limited to, N,N-dimethylimidazolidone, N-methylpiperidone, DM F, dichloromethane, acetonitrile, tetrahydrofuran, or a mixture thereof. It is possible.

[0085] The reaction temperature is preferably within the range of, for example, 0°C to 100°C, more preferably 10°C to 50°C. It is within range. The reaction time varies depending on the type of base used and the reaction temperature, but is usually in the range of 1 minute to 48 hours. The suitable time is within this range, and preferably within the range of 30 minutes to 24 hours.

[0086] Furthermore, after the completion of this step, an acylating agent can be added as needed. Examples of the "acidifier" include acetic anhydride, acetic acid chloride, and phenoxyacetic anhydride. The acylating agent can be dissolved in a suitable solvent to give a concentration in the range of, for example, 0.1% to 30%. The solvent is not particularly limited as long as it is not involved in the reaction. However, for example, dichloromethane, acetonitrile, alcohols (ethanol, isopropyl alcohol, trifluoroethanol, etc.), water, or a mixture thereof. . If necessary, an acylating agent such as pyridine, lutidine, collidine, Use a base such as triethylamine, diisopropylethylamine, or N-ethylmorpholine. The amount of the acylating agent used is in the range of 0.1 to 10,000 molar equivalents. The amount of the base used is preferably in the range of 1 molar equivalent to 1000 molar equivalents, and more preferably in the range of 1 molar equivalent to 1000 molar equivalents. For example, the amount is suitably within the range of 0.1 molar equivalents to 100 molar equivalents relative to 1 mole of the acylating agent. The amount is preferably within the range of 1 molar equivalent to 10 molar equivalents. The reaction temperature of this reaction is preferably within the range of 10°C to 50°C, more preferably 10°C to 50°C. The temperature is preferably within the range of 20°C to 40°C, and more preferably within the range of 25°C to 40°C. The reaction time varies depending on, for example, the type of acylating agent used and the reaction temperature. Although it varies depending on the circumstances, a range of 0.1 minutes to 24 hours is usually appropriate, and preferably 1 minute to 5 hours. is within the range.

[0087] (3) Process C: In the compound (VII) produced in step B, the protecting group is removed using a deprotecting agent, A process for producing a compound represented by general formula (IX). [ka] [In the formula, Base, B P , L, n, R 2 , R 3 , T has the same meaning as above.]

[0088] This step can be carried out by reacting compound (VII) with a deprotecting agent.

[0089] Examples of the "deprotecting agent" include concentrated aqueous ammonia and methylamine. Examples of the deprotecting agent that can be used in this step include water, methanol, ethanol, isopropyl alcohol, and the like. Phenyl alcohol, acetonitrile, tetrahydrofuran, DMF, N,N-dimethylimidazoline It can also be used by diluting it with N-methylpiperidone, N-methylpiperidone, or a mixture of these solvents. However, ethanol is preferred. The amount of the deprotecting agent used is, for example, 1 mol of compound (VII). For example, the range of 1 to 100,000 molar equivalents is suitable, and preferably 10 molar equivalents. The range is 1 molar equivalent to 1000 molar equivalents.

[0090] The reaction temperature is, for example, suitably in the range of 15°C to 75°C, preferably in the range of 40°C to 70°C. The deprotection reaction time is within the range of 50°C to 60°C, more preferably within the range of 50°C to 60°C. Although it varies depending on the type of the compound, reaction temperature, etc., it is suitable to use a time period ranging from 10 minutes to 30 hours, preferably The time is within a range of 30 minutes to 24 hours, and more preferably within a range of 5 hours to 20 hours.

[0091] (4) Process D: PMO (I) is prepared by reacting compound (IX) prepared in step C with an acid. The manufacturing process. [ka] [where Base, n, R 2 , R 3 , T has the same meaning as above.] This step can be carried out by adding an acid to compound (IX).

[0092] The "acid" that can be used in this step includes, for example, trichloroacetic acid, dichloroacetic acid, Examples of the acid include acetic acid, phosphoric acid, and hydrochloric acid. The amount of acid used depends on, for example, the pH of the solution. It is suitable to use it so that the value falls within the range of 0.1 to 4.0, and more preferably within the range of 1.0 to 3.0. The solvent is used so as to be within the range. There are no particular restrictions on the solvent as long as it does not participate in the reaction. For example, acetonitrile, water, or a mixed solvent thereof can be used.

[0093] The reaction temperature is preferably in the range of 10°C to 50°C, more preferably in the range of 20°C to 40°C. The deprotection reaction time is preferably within the range of 25° C. to 35° C. Although it varies depending on the type of compound, reaction temperature, etc., a range of 0.1 minutes to 5 hours is suitable, and preferably The time is within a range of 1 minute to 1 hour, and more preferably within a range of 1 minute to 30 minutes.

[0094] PMO(I) can be separated and purified from the reaction mixture obtained in this step by conventional separation and purification methods, such as extraction and concentration. , neutralization, filtration, centrifugation, recrystallization, C8 to C 18 reversed-phase column chromatography, positive ion Ion exchange column chromatography, anion exchange column chromatography, gel filtration column Simple methods such as column chromatography, high performance liquid chromatography, dialysis, and ultrafiltration are used. The desired PMO(I) can be isolated and purified by using these compounds alone or in combination. (See, for example, International Publication WO1991 / 09033). When PMO(I) is purified using reversed-phase chromatography, the elution solvent may be, for example, A mixture of 20 mM triethylamine / acetate buffer and acetonitrile can be used. do. When purifying PMO(I) using ion exchange chromatography, for example, 1M A mixed solution of 10 mM saline and 10 mM sodium hydroxide solution can be used.

[0095] Peptide nucleic acid is an oligomer of the present invention having a constituent unit represented by the following general formula: . [ka] (In the formula, Base has the same meaning as defined above.)

[0096] Peptide nucleic acids 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. Vulpi us, KH Petersen, RH Berg, PE Nielsen, O. Buchardt,J. Org. Chem., 59, 5 767 (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. Or um, J. Pept. Res., 49, 80 (1997)

[0097] The oligomer of the present invention has a 5'-end which is a group represented by any one of the following chemical formulas (1) to (3): It may be present, and is preferably (3)-OH. [ka] Hereinafter, the groups represented by (1), (2), and (3) above will be referred to as "group (1)," "group (2)," and "group (3)," respectively. 3)"

[0098] 2. Pharmaceutical Compositions The oligomers of the present invention have higher efficiency than conventional antisense oligomers. It allows skipping of exons 55, 45, 50 and 44. Thus, the oligomers of the present invention By administering a pharmaceutical composition containing the compound to a DMD patient, the symptoms of muscular dystrophy can be alleviated with high efficiency. For example, it is expected that the use of pharmaceutical compositions containing the oligomers of the present invention will When the oligomer is administered, the same therapeutic effect can be obtained at a smaller dose than that of the oligomer according to the prior art. This reduces side effects and is economical. Thus, in another embodiment, the oligomer of the present invention, its pharmaceutically acceptable salt or water a pharmaceutical composition for treating muscular dystrophy containing a compound thereof as an active ingredient (hereinafter referred to as "the composition of the present invention"); (hereinafter referred to as "the Company")

[0099] Examples of pharmaceutically acceptable salts of the oligomers of the invention that can be included in the compositions of the invention include: Alkali metal salts such as sodium salts, potassium salts, and lithium salts, calcium salts, magnesium salts, Alkaline earth metal salts such as sodium salts; aluminum salts, iron salts, zinc salts, copper salts, nickel salts salts, metal salts such as cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine salts Salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine Amine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroproca amine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, pipet ammonium salt, tetramethylammonium salt, tris(hydroxymethyl)aminomethane salt, etc. organic amine salts such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides; Hydrochlorides; inorganic acid salts such as nitrates, perchlorates, sulfates, and phosphates; methanesulfonates lower alkanesulfonates such as ammonium salts, trifluoromethanesulfonates, and ethanesulfonates; sulfonates; aryl sulfonates such as benzenesulfonates and p-toluenesulfonates Salts: acetate, malate, fumarate, succinate, citrate, tartrate, oxalate Salts, organic acid salts such as maleates; glycine salts, lysine salts, arginine salts, ornithine salts , and amino acid salts such as glutamate and aspartate. can be produced by known methods. Alternatively, the present invention can be The oligomer may be in the form of its hydrate.

[0100] The dosage form of the composition of the present invention is not particularly limited as long as it is a pharmaceutically acceptable dosage form. It can be selected depending on the treatment method, but from the viewpoint of ease of delivery to muscle tissue, intravenous administration is recommended. Intra-arterial administration, intramuscular administration, subcutaneous administration, oral administration, intratissue administration, transdermal administration, etc. are preferred. The dosage form that the composition of the present invention can take is not particularly limited, but may be, for example, various Examples include injections, oral preparations, drip infusions, inhalants, ointments, lotions, and the like.

[0101] When the oligomer of the present invention is administered to a patient with muscular dystrophy, the composition of the present invention Preferably, the ligomer comprises a carrier that facilitates delivery to muscle tissue. There are no particular limitations as long as it is pharmaceutically acceptable, and examples thereof include cationic liposomes, Cationic carriers such as cationic polymers or carriers using viral envelopes Examples of cationic liposomes include 2-O-(2-diethylamino) Ethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids are essential components Liposomes formed by the above procedure (hereinafter referred to as "liposome A"), oligofectamine ( Registered trademark) (manufactured by Invitrogen), Lipofectin (registered trademark) (manufactured by Invitrogen), Lipo Infectamine® (Invitrogen), Lipofectamine 2000® (In (manufactured by Invitrogen), DMRIE-C (registered trademark) (manufactured by Invitrogen), GeneSilencer (registered trademark) (Gene Therapy Systems), TransMessenger (registered trademark) (QIAGEN), TransIT Examples of such products include TKO (registered trademark) (Mirus) and Nucleofector II (Lonza). Among these, liposome A is preferred. Examples of cationic polymers include JetSI (registered trademark). Trademark) (manufactured by Qbiogene), Jet-PEI (registered trademark) (polyethyleneimine, manufactured by Qbiogene) Examples of carriers using a viral envelope include Genome One (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Kaisha, Ltd.) can also be mentioned. The medical device described in Patent No. 2924179, Patent Republished Publication No. 2006 / 129594 and Patent Republished Publication No. It is also possible to use the cationic carriers described in Publication No. 2008 / 096690.

[0102] The concentration of the oligomer of the present invention contained in the composition of the present invention varies depending on the type of carrier, etc. The range of 0.1 nM to 100 μM is appropriate, the range of 1 nM to 10 μM is preferable, and the range of 10 nM to 1 The oligomer of the present invention contained in the composition of the present invention and the carrier are more preferably in the range of 1 μM. The weight ratio (carrier / oligomer of the present invention) of the carrier to the oligomer of the present invention depends on the properties of the oligomer, the type of the carrier, etc. Although it varies depending on the type, a range of 0.1 to 100 is appropriate, a range of 1 to 50 is preferable, and a range of 10 to 20 is preferable. It is more preferable that the range is within the range of .

[0103] The composition of the present invention may contain, in addition to the oligomer of the present invention and the carrier described above, any pharmaceutically acceptable carrier. Such additives include, for example, an emulsifying aid (e.g., For example, fatty acids having 6 to 22 carbon atoms and pharmaceutically acceptable salts thereof, albumin, dextran, stabilizers (e.g., cholesterol, phosphatidic acid), tonicity agents (e.g., sodium chloride), sodium, glucose, maltose, lactose, sucrose, trehalose), pH adjuster (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine) These may be used alone or in combination of two or more. The content of the additive in the composition of the present invention is suitably 90% by weight or less, and preferably 70% by weight or less. It is preferable that the content is 50% by weight or less, and more preferable that the content is 50% by weight or less.

[0104] The composition of the present invention can be prepared by adding the oligomer of the present invention to a dispersion of a carrier and stirring appropriately. The additive may be added either before or after the addition of the oligomer of the present invention. Water that can be used when adding the oligomer of the present invention can also be added at an appropriate stage. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water for injection, Examples include electrolyte solutions such as distilled water for injection, physiological saline solution, and sugar solutions such as glucose solution and maltose solution. In such cases, conditions such as pH and temperature can be appropriately selected by those skilled in the art. Cut.

[0105] The composition of the present invention can be, for example, a liquid formulation or a freeze-dried formulation thereof. The dry preparation can be prepared by freeze-drying the composition of the present invention in the form of a liquid preparation in a conventional manner. For example, the composition of the present invention in the form of a liquid can be prepared by After sterilization, dispense the specified amount into vials and pre-freeze at approximately -40 to -20°C for 2 hours. The drying is carried out for about 1 hour, followed by primary drying under reduced pressure at about 0 to 10°C, and then secondary drying under reduced pressure at about 15 to 25°C. It can be dried and lyophilized, and the inside of the vial is typically flushed with nitrogen gas. The container is then sealed to obtain a freeze-dried preparation of the composition of the present invention.

[0106] The lyophilized formulation of the composition of the present invention can generally be reconstituted by adding any suitable solution (reconstitution solution). Such a reconstitution liquid can be used by reconstitution with water for injection, physiological saline, etc. The amount of the reconstituted solution varies depending on the intended use. Although there are no particular limitations, an amount of 0.5 to 2 times the liquid volume before freeze-drying, or 500 mL or less, is appropriate.

[0107] The dosage when administering the composition of the present invention depends on the type of the oligomer of the present invention contained therein, Prepare the medicine taking into consideration the dosage form, the patient's condition such as age and weight, the route of administration, and the nature and severity of the disease. However, the amount of the oligomer of the present invention for adults is preferably 0.1 mg to 10 g / day. Generally, the dose is within the human range, preferably within the range of 1 mg to 1 g / human. It may also vary depending on the type of disease, the administration method, and the target molecule. In some cases, a dose less than this may be sufficient, but conversely, a dose greater than this may be necessary. The administration can be once or several times a day, or at intervals of one day to several days.

[0108] Another embodiment of the composition of the present invention is a vector capable of expressing the oligonucleotide of the present invention. and the above-mentioned carrier. The composition may be capable of expressing the oligonucleotide of the present invention. As with the compositions of the present invention containing the oligomers of interest, pharmaceutically acceptable additives may be added. The concentration of the expression vector contained in the composition can be adjusted depending on the type of carrier, etc. The concentration varies depending on the individual, but is suitably in the range of 0.1 nM to 100 μM, preferably in the range of 1 nM to 10 μM, and The range of 0 nM to 1 μM is more preferable. The weight ratio (carrier / expression vector) varies depending on the properties of the expression vector, the type of carrier, etc. The range of 0.1 to 100 is suitable, the range of 1 to 50 is preferable, and the range of 10 to 20 is more preferable. The content of the carrier contained in the composition is preferably 100% or more, and more preferably 100% or more, including the oligomer of the present invention. The same applies to the composition of the present invention, and the preparation method thereof is also the same as that of the composition of the present invention. is the same as: [Example]

[0109] The present invention will be described in more detail below with reference to examples and test examples. The ranges shown in the examples are not intended to be limiting.

[0110] [Reference example 1] 4-{[(2S,6R)-6-(4-benzamido-2-one]- Aminopolystyrene Resin-Supported 4-{[(2S,6R)-6-(4-benzamido-2-one]- xopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxo Butanoic acid Step 1: 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl] Preparation of {(4-trityl)morpholin-2-yl]methoxy}-4-oxobutanoic acid Under an argon atmosphere, N-{1-[(2R,6S)-6-(hydroxymethyl)-4-trimethylmo [[ ...(())])((((())])))))) 0.44g and 1.1g of 4-dimethylaminopyridine (4-DMAP) were suspended in 50mL of dichloromethane and 0.90 g of succinic acid was added, and the mixture was stirred at room temperature for 3 hours. 10 mL of methanol was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was extracted with ethyl acetate and 0.5M aqueous potassium dihydrogen phosphate solution. The resulting organic layer was washed with 0.5 M aqueous potassium dihydrogen phosphate solution, water, and saturated saline in this order. The resulting organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain 4.0 g of the desired product.

[0111] Step 2: Aminopolystyrene Resin-Supported 4-{[(2S,6R)-6-(4-benzamide] -2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4 -Production of oxobutanoic acid 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl) 4.0 g of [4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid was dissolved in pyridine ( Dissolve 0.73 g of 4-DMAP and 1-ethyl-3-(3-dimethylaminopropyl) carboxymethylcellulose in 200 mL of dehydrated water. 11.5 g of rubodiimide hydrochloride was added. Then, aminopolystyrene resin Primer support 25.0 g of 200 amino acid (GE Healthcare Japan, 17-5214-97) and 8.5 mL of triethylamine The mixture was added and shaken at room temperature for 4 days. After the reaction, the resin was filtered off. The resulting resin was dissolved in pyridine, methyl The resin was washed with ethanol and dichloromethane in that order and dried under reduced pressure. 200 mL of ran (dehydrated), 15 mL of acetic anhydride, and 15 mL of 2,6-lutidine were added and shaken at room temperature for 2 hours. The resin was collected by filtration, washed with pyridine, methanol, and dichloromethane in that order, and dried under reduced pressure. 0.7g of the desired product was obtained. The loading amount of the target compound can be determined by a known method using the moles of trityl per gram of resin. The amount of resin was determined by measuring the UV absorbance at 409 nm. The concentration was 29.2 μmol / g. UV measurement conditions Equipment: U-2910 (Hitachi) Solvent: methanesulfonic acid Wavelength: 265 nm ε value: 45000

[0112] [Reference example 2] 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)- ... (amino)purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-o xobutanoic acid Process 1:N 2 Preparation of -(phenoxyacetyl)guanosine 100 g of guanosine was dried under reduced pressure at 80°C for 24 hours. Add 500 mL of methane (dehydrated) and 401 mL of chlorotrimethylsilane at 0°C under an argon atmosphere. The mixture was cooled again with ice and 66.3 g of phenoxyacetyl chloride was added dropwise. The reaction mixture was stirred for another 3 hours under ice cooling, and 500 ml of methanol was added to the reaction mixture, which was then stirred overnight at room temperature. The solvent was distilled off under reduced pressure. 500 mL of methanol was added to the residue, and the mixture was concentrated under reduced pressure three times. 4 L of water was added to the residue, and the mixture was stirred under ice-cooling for 1 hour, and the precipitate was collected by filtration. After washing with cold methanol and drying, 150.2 g of the target compound was obtained (yield: 102%) (Reference: Org. Lett. (2004), Vol. 6, No. 15, 2555-2557).

[0113] Process 2:N 9 -{[(2R,6S)-6-(hydroxymethyl)-4-morpholin-2-yl]-6 -Oxo-6,9-dihydro-1H-purin-2-yl}-2-phenoxyacetamide p-to Benzene sulfonate 30 g of the compound obtained in step 1 was suspended in 480 mL of methanol, and 130 mL of 2N hydrochloric acid was added under ice cooling. Next, add 56.8 g of ammonium tetraborate tetrahydrate and 16.2 g of sodium periodate in that order. The reaction mixture was cooled on ice, and insoluble matter was removed by filtration. The filtrate and washings were combined and cooled on ice, and 11.52 g of 2-picoline borane was added. After stirring for 20 minutes, 54.6 g of p-toluenesulfonic acid monohydrate was slowly added and the mixture was stirred overnight at 4°C. The precipitate was collected by filtration, washed with 500 mL of cold methanol, and then dried to obtain 17.7 g of the target compound ( Yield: 43.3%). 1 H NMR(δ,DMSO-d6):9.9-9.2(2H,br), 8.35(1H,s), 7.55(2H,m), 7.35( 2H,m), 7.10(2H,d, J=7.82Hz), 7.00(3H,m), 5.95(1H,dd, J=10.64,2.42H z), 4.85(2H,s), 4.00(1H,m), 3.90-3.60(2H,m), 3.50-3.20(5H,m), 2.9 0 (1H,m), 2.25 (3H,s)

[0114] Process 3:N9 -{(2R,6S)-6-hydroxymethyl-4-tritylmorpholine-2 -Il}-N 2 Preparation of -(phenoxyacetyl)guanine 2.0 g of the compound obtained in step 2 was suspended in dichloromethane (30 ml) and triturated under ice cooling. Add tritylamine (13.9 g) and trityl chloride (18.3 g) and stir at room temperature for 1 hour. The reaction mixture was washed with saturated sodium bicarbonate water and then with water, and the organic layer was recovered and washed with magnesium sulfate. The organic layer was dried and concentrated under reduced pressure. The residue was diluted with 0.2 M sodium citrate buffer (pH 3) / Add methanol (1:4 (v / v), 40 ml) and stir, then add water (40 ml). The mixture was stirred for 1 hour under ice cooling (suspension state), filtered, washed with cold methanol, and dried. 1.84 g of the target compound was obtained (yield: 82.0%).

[0115] Process 4:N 9 -[(2R,6S)-6-{(tert-butyldimethylsilyloxy)methyl ethyl}-4-tritylmorpholin-2-yl]-N 2 -(Phenoxyacetyl)guanine Manufacturing The compound obtained in step 3 (38.3 g) was dissolved in dichloromethane (300 mL) and cooled with ice. In the cold, imidazole (4.64 g), t-butyldimethylsilyl chloride (9.47 g) The reaction mixture was added in this order and stirred at room temperature for 1 hour. After washing with saturated saline, the organic layer was collected and dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to give 44.1 g of the desired compound as a crude product.

[0116] Process 5:N 9 -[(2R,6S)-6-{(tert-butyldimethylsilyloxy)methyl ethyl}-4-tritylmorpholin-2-yl]-N 2 -(phenoxyacetyl)-O 6 - Preparation of triisopropylbenzenesulfonylguanine The compound obtained in step 4 (44.1 g) was dissolved in dichloromethane (300 mL) and cooled with ice. Under cooling, 4-dimethylaminopyridine (0.64 g) and triethylamine (29.2 mL) Triisopropylbenzenesulfonyl chloride (19.0 g) was added and stirred at room temperature for 1 hour. The reaction mixture was washed with 1M aqueous sodium dihydrogen phosphate solution, and the organic layer was recovered and mixed with sulfuric acid. The organic layer was dried over magnesium and concentrated under reduced pressure to give 60.5 g of the target compound as a crude product. Ta.

[0117] Step 6: N 9 -[(2R,6S)-6-{(tert-butyldimethylsilyloxy) Methyl}-4-tritylmorpholin-2-yl]-N 2 -(phenoxyacetyl)-O 6 Preparation of -(2-cyanoethyl)guanine The compound obtained in step 5 (60.5 g) was dissolved in dichloromethane (300 mL) and cooled with ice. N-methylpyrrolidine (54.5 mL) was added under cooling and stirred for 1 hour. cyanohydrin (37.2 g), followed by 1,8-diazabicyclo[5.4.0]undecane Carboxy-7-ene (11.96 g) was added, and the mixture was stirred for another 2 hours under ice-cooling. After washing with an aqueous solution of sodium dihydrogen carbonate and then with water, the organic layer was collected and dried over magnesium sulfate. The organic layer was dried and concentrated under reduced pressure to give 72.4 g of the desired compound as a crude product.

[0118] Process 7:N 9 -[(2R,6S)-6-hydroxymethyl-4-tritylmorpholine-2 -Il]-N2 -(phenoxyacetyl)-O 6 Preparation of -(2-cyanoethyl)guanine The compound obtained in step 6 (72.4 g) was dissolved in dichloromethane (300 mL) and Triethylamine trihydrofluoride (21.1 g) was added and the mixture was stirred at room temperature for 17 hours. The reaction mixture was poured into cold saturated sodium bicarbonate water, neutralized, and the dichloromethane layer was collected and dried over magnesium sulfate. The residue was purified by silica gel column chromatography (PSQ100B (rich The resulting mixture was purified by a cyclohexane-1,2-diol (manufactured by Silysia Chemical Ltd., the same applies hereinafter) to obtain 14.3 g of the target compound. (Yield from step 4: 39.2%).

[0119] Step 8: Aminopolystyrene resin supported 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[ (2-phenoxyacetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy ]-4-oxo-butanoic acid The title compound was produced in the same manner as in Reference Example 1. However, the N-{1 -[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-o Instead of N-1,2-dihydropyrimidin-4-yl}benzamide, in this process 9 -[(2R,6S)-6-Hydroxymethyl-4-tritylmorpholin-2-yl]- N 2 -(phenoxyacetyl)-O 6 -(2-cyanoethyl)guanine was used.

[0120] [Reference example 3] Aminopolystyrene Resin-Supported 4-{[(2S,6R)-6-(5-methyl-2,4-dioxa So-3,4-dihydropyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy }-4-oxobutanoic acid The title compound was produced in the same manner as in Reference Example 1. However, the N-{1 -[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-o Instead of 1,2-dihydropyrimidin-4-yl}benzamide, in this step, [(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-5-methyl Irpyrimidine-2,4(1H,3H)-dione was used.

[0121] [Reference example 4] Aminopolystyrene resin-supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4 -tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid The title compound was produced in the same manner as in Reference Example 1. However, the N-{1 -[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-o Instead of N-1,2-dihydropyrimidin-4-yl}benzamide, in this process {9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]purin-6-yl} Benzamide was used.

[0122] [Reference example 5] Aminopolystyrene Resin-Supported 1,12-Dioxo-1-(4-tritylpiperazine-1 -yl)-2,5,8,11-tetraoxa-15-pentadecanoic acid The title compound was produced in the same manner as in Reference Example 1. However, the N-{1 -[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-o Instead of 2-(1,2-dihydropyrimidin-4-yl)benzamide, in this step [2-(2-hydroxyethoxy)ethoxy]ethyl 4-tritylpiperazine-1-carbo Phosphonic acid (a compound described in WO 2009 / 064471) was used.

[0123] About Exon 45 According to the description of Examples 1 to 8 and Comparative Example 1 below, various PMs shown in PMO Nos. 1 to 6 and 8 to 10 in Table 5 were used. The synthesized PMO was dissolved in water for injection (Otsuka Pharmaceutical Factory). was purchased from Gene Tools.

[0124] [Table 5]

[0125] [Example 1] PMO No.1 4-{[(2S,6R)-6-(4-benzamido-2- Oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4 0.2 g (26 μmol) of oxobutanoic acid (Reference Example 1) was packed into a column with a filter, and the nucleic acid was mixed. The synthesis cycle was performed using a synthesis machine (AKTA Oligopilot 10 plus). The desired sequence was obtained in each coupling cycle to obtain the base sequence of the title compound. of morpholino monomer compound was added. [Table 6]

[0126] The deblocking solution was dichloromethane containing 3% (w / v) trifluoroacetic acid. The neutralization and cleaning solution used was a 10% ( 35% (v / v) of tetrahydrofuran to 5% (v / v) The coupling solution was dissolved in dichloromethane containing acetonitrile. A is a morpholino monomer compound dissolved in tetrahydrofuran to a concentration of 0.10M. Coupling solution B was prepared by dissolving N,N-diisopropylethylamine in 2 mL of HCl. acetone to 0% (v / v) and tetrahydrofuran to 10% (v / v). The capping solution was prepared by dissolving the compound in acetonitrile. A solution of 20% (v / v) acetic anhydride and 30% (v / v) 2,6-lutidine was used.

[0127] The aminopolystyrene resin carrying the synthesized PMO was recovered from the reaction vessel and stirred for 2 hours. The dried aminopolystyrene resin-supported PMO was placed in a reaction vessel. 5 mL of 28% aqueous ammonia-ethanol (1 / 4) was added and the mixture was stirred at 55°C for 15 hours. The polystyrene resin was filtered off and washed with 1 mL of water-ethanol (1 / 4). The resulting residue was concentrated under reduced pressure. The resulting solution was dissolved in 10 mL of a mixed solvent of acetone and nitrile (4 / 1) and filtered through a membrane filter. The filtrate was purified by reverse phase HPLC under the following conditions: [Table 7]

[0128] Each fraction was analyzed, and the target substance was collected and concentrated under reduced pressure. 0.5 mL of the solution was added and stirred for 15 minutes. Then, 2 mL of 2 M aqueous sodium hydroxide solution was added to the solution. The solution was diluted with water and filtered through a membrane filter (0.45 μm). The resulting aqueous solution containing the target product was purified using an anion exchange resin column. is as follows:

[0129] [Table 8]

[0130] Each fraction was analyzed (HPLC) to obtain the target substance as an aqueous solution. The solution was neutralized by adding phosphate buffer (pH 6.0). Then, the solution was desalted by reversed-phase HPLC under the following conditions: . [Table 9]

[0131] The target product was collected and concentrated under reduced pressure. The resulting residue was dissolved in water and freeze-dried to give a white flocculent. 1.5 mg of the desired compound was obtained as a solid. ESI-TOF-MS calculated value: 6877.8 Measurement: 6877.4

[0132] [Example 2] PMO.No.3 The title compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 6862.8 Measurement: 6862.5

[0133] [Example 3] PMO.No.2 The title compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 6862.8 Measurement: 6862.3

[0134] [Example 4] PMO.No.4 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6902.8 Measurement: 6902.3

[0135] [Example 5] PMO.No.5 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6902.8 Measurement: 6902.4

[0136] [Example 6] PMO.No.8 The title compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 6547.5 Measurement: 6547.2

[0137] [Example 7] PMO.No.9 The title compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 6547.5 Measurement: 6547.2

[0138] [Example 8] PMO.No.10 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 1,12-Dioxo-1-(4-tritylpiperazine-1-yl)methylpropanol supported on polystyrene resin (I)-2,5,8,11-tetraoxa-15-pentadecanoic acid (Reference Example 5) was used. ESI-TOF-MS calculated value: 7214.1 Measurement: 7213.7

[0139] [Comparative Example 1] PMO.No.6 The title compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 8193.9 Measurement: 8195.3

[0140] [Test Example 1] In vitro assays The 2'-O-methoxy-phosphorothioate (2'-OMe-S-RNA) derivatives shown in SEQ ID NOs: 19 to 35 The experiment was carried out using antisense oligomers. The antisense oligomers were purchased from Japan Bioservices. The sequences of the various antisense oligomers are listed below. show. [Table 10]

[0141] RD cells (human rhabdomyosarcoma cell line) 1 x 10 5 10% fetal bovine serum was added to a 12-well plate. Eagle's minimal essential medium (EMEM) containing FCS (Invitrogen) The exon 4 was cultured overnight at 37°C under 5% CO2 conditions in 1 mL of Sigma-Aldrich (Sigma-Aldrich). 5. Various antisense oligomers for skipping (manufactured by Japan Bioservices Co., Ltd.) (0.3 or 1 μM) and Lipofectamine 2000 (Invitrogen), and 100 μl of this was added to the exchanged RD cells to give a final concentration of 30 or 100 nM.

[0142] After the addition, the cells were cultured overnight. After washing twice with PBS (manufactured by Nissui Co., Ltd., the same applies below), the cells were Add 250 μl of EN (Nippon Gene) to the cells and leave at room temperature for several minutes to lyse the cells. The lysate was collected in an Eppendorf tube according to the protocol provided with ISOGEN. Total RNA was extracted. The concentration of the extracted total RNA was measured using NanoDrop ND-1000 (LMS). Measurements were performed using a meter (manufactured by the company). RT-PCR was performed on 400 ng of extracted total RNA using the QIAGEN OneStep RT-PCR Kit. The reaction mixture was prepared according to the protocol attached to the kit. C-100 (MJ Research) was used. The RT-PCR program used was as follows: 50℃, 30 minutes: reverse transcription 95℃, 15 minutes: heat denaturation PCR amplification: [94°C, 30 seconds; 60°C, 30 seconds; 72°C, 1 minute] x 35 cycles 72°C, 10 min:

[0143] The nucleotide sequences of the forward and reverse primers used for RT-PCR are as follows: is. Forward primer: 5'-GCTCAGGTCGGATTGACATT-3' (SEQ ID NO: 36) Reverse primer: 5'-GGGCAACTCTTCCACCAGTA-3' (SEQ ID NO: 37)

[0144] 1 μl of the PCR reaction product was analyzed using a Bioanalyzer (Agilent). The polynucleotide amount of the band with skipped exon 45 is "A" and the amount of the band with skipped exon 45 is "B". The amount of polynucleotides in the unpaired bands, "B," was measured. Based on the fixed values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / ( A + B ) x 100

[0145] Experimental results The results are shown in Figures 1 and 2. This experiment identified the 45th exon of the human dystrophin gene. When the antisense oligomer is designed at positions 1 to 25 or 6 to 30 from the 5' end of the When an antisense oligomer was designed for the 7th to 31st positions from the 5' end of the 45th exon, It was found that exon 45 was skipped more efficiently than with the α-glucanase.

[0146] [Test Example 2] In vitro assays RD cells (human rhabdomyosarcoma cell line) 3.5 x 10 5 In contrast, the present invention of PMO Nos. 1 to 5 and 8 to 10 The oligomers of PMO No. 1 and PMO No. 7 and the antisense oligomers of PMO No. 6 and 7 were added at 1, 3, and 10 μM each to Amaxa The cells were transfected using the Cell Line Nucleofector Kit L with Nucleofector II (Lonza). Gram was T-030.

[0147] After transfection, the cells were cultured in Eagle's medium containing 10% fetal calf serum (FCS) (Invitrogen). 2 mL of EMEM (Sigma) medium was incubated at 37°C and 5% CO2. The cells were cultured for 3 days under the conditions described above. After washing twice with PBS (manufactured by Nissui Co., Ltd., the same applies hereinafter), the cells were Add 500 μl of GEN (Nippon Gene) to the cells and leave at room temperature for several minutes to lyse the cells. The lysate was collected in an Eppendorf tube according to the protocol provided with ISOGEN. Total RNA was extracted. The concentration of the extracted total RNA was measured using NanoDrop ND-1000 (LMS). Measurements were performed using a meter (manufactured by the company).

[0148] The QIAGEN OneStep RT-PCR Kit (Qiagen) was used for 400 ng of extracted total RNA. RT-PCR was performed using the same DNA fragments. The reaction mixture was prepared according to the protocol provided with the kit. The RT-PCR program used was the following: It is as follows: 50℃, 30 minutes: reverse transcription 95℃, 15 minutes: heat denaturation PCR amplification: [94°C, 30 seconds; 60°C, 30 seconds; 72°C, 1 minute] x 35 cycles 72°C, 10 min:

[0149] The nucleotide sequences of the forward and reverse primers used for RT-PCR are as follows: is. Forward primer: 5'-GCTCAGGTCGGATTGACATT-3' (SEQ ID NO: 36) Reverse primer: 5'-GGGCAACTCTTCCACCAGTA-3' (SEQ ID NO: 37)

[0150] 1 μl of the PCR reaction product was analyzed using a Bioanalyzer (Agilent). The polynucleotide amount of the band with skipped exon 45 is "A" and the amount of the band with skipped exon 45 is "B". The amount of polynucleotides in the unpaired bands, "B," was measured. Based on the fixed values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / ( A + B ) x 100

[0151] Experimental results The results are shown in Figures 3, 4, 14 and 15. From this experiment, it was confirmed that the present invention The oligomers inhibited exonuclease activity in RD cells with the same efficiency as the antisense oligomer of PMO No. 6. It was found that the skipping of PMONo.1, 2, and 3 was caused by the The bright oligomers showed higher activity in RD cells compared to the antisense oligomer of PMO No. 7. It was found that PMONo.3 efficiently skipped exon 45 (Fig. 14). Higher efficiency of exon 45 skipping compared to PMO No. 10, which has a different terminal structure It was found (Figure 15).

[0152] [Test Example 3] In vitro assay using human fibroblasts ZsGreen1 co-expressing retroviral vector was transfected into GM05017 cells (human DMD patient-derived fibroblasts). The human MyoD gene (SEQ ID NO: 38) was introduced into the cells (Coriell Institute for Medical Research). did. After 4 to 5 days of incubation, ZsGreen-positive MyoD-transformed fibroblasts were identified by FACS. Collect, 5 x 10 4 pieces / cm 2The growth medium was 10% FCS and 1% Dulbecco's Modified Penicillin / Streptomycin (P / S) (Sigma-Aldrich) Eagle Medium: Use 1 mL of Nutrient Mixture F-12 (DMEM·F-12) (Invitrogen). Ta.

[0153] After 24 hours, the cells were cultured in differentiation medium (2% horse serum (Invitrogen), 1% P / S, and ITS Liquid Medium). The medium was replaced with DMEM / F-12 containing a supplement (Sigma). The cells were incubated for 14 days from the day they were harvested and allowed to differentiate into myotubes.

[0154] Thereafter, the differentiation medium was replaced with a differentiation medium containing 6 μM Endo-Porter (Gene Tools). Morpholino oligomers were added to a concentration of 10 μM. After 48 hours of incubation, T Total RNA was extracted from the cells using RIzol (Invitrogen). RT-PCR was performed using 50 ng of RNA using the QIAGEN OneStep RT-PCR Kit. The reaction mixture was prepared according to the instructions. The thermal cycler used was an iCycler (Bio-Rad). The RT-PCR program used was as follows: 50℃, 30 minutes: reverse transcription 95℃, 15 minutes: heat denaturation PCR amplification: [94°C, 1 min; 60°C, 1 min; 72°C, 1 min] x 35 cycles 72°C, 7 minutes: Heat inactivation of polymerase

[0155] The primers used were hDMD44F and hDMD46R. hDMD44F: 5'- CCTGAGAATTGGGAACATGC-3' (SEQ ID NO: 39) hDMD46R: 5'- TTGCTGCTCTTTTCCAGGTT-3' (SEQ ID NO: 40) The reaction products of the above RT-PCR reaction were separated by 2% agarose gel electrophoresis and analyzed by GeneFlash. Gel photographs were taken using Image J (National Institutes of Health, USA). The polynucleotide amount of the band with skipped exon 45 is "A" and the amount of the band with skipped exon 45 is "B". The amount of polynucleotides in the unpaired bands, "B," was measured. Based on the fixed values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / ( A + B ) x 100

[0156] Experimental results The results are shown in Figure 5. This experiment demonstrated that the oligomer of the present invention, PMO No. 3, inhibited the growth of GM05017 cells. It was found that exon 45 was skipped with high efficiency.

[0157] About Exon 55 Various PMOs shown in Table 11, PMO Nos. 11 to 14 and 16 to 22, were synthesized according to the following Examples 9 to 19. The synthesized PMO was dissolved in water for injection (Otsuka Pharmaceutical Factory). Purchased from Ntools. [Table 11]

[0158] [Example 9] PMO.No.11 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4- ritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 6807.8 Measurement: 6807.0

[0159] [Example 10] PMO.No.12 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6822.8 Measurement: 6822.5

[0160] [Example 11] PMO.No.13 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6837.8 Measurement: 6837.3

[0161] [Example 12] PMO.No.14 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4- ritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 6861.8 Measurement: 6861.4

[0162] [Example 13] PMO.No.16 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6812.8 Measurement: 6812.7

[0163] [Example 14] PMO.No.17 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6852.8 Measurement: 6852.7

[0164] [Example 15] PMO.No.18 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6901.8 Measurement: 6901.5

[0165] [Example 16] PMO.No.19 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6901.8 Measurement: 6901.7

[0166] [Example 17] PMO.No.20 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6522.5 Measurement: 6522.0

[0167] [Example 18] PMO.No.21 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4- ritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 6546.5 Measurement: 6546.0

[0168] [Example 19] PMO.No.22 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 1,12-Dioxo-1-(4-tritylpiperazine-1-yl)methylpropanol supported on polystyrene resin (I)-2,5,8,11-tetraoxa-15-pentadecanoic acid (Reference Example 5) was used. ESI-TOF-MS calculated value: 7213.1 Measurement: 7212.5

[0169] [Test Example 4] In vitro assays The 2'-O-methoxy-phosphorothioate (2'-OMe-S-RNA) derivatives shown in SEQ ID NOs: 49 to 68 The experiment was carried out using antisense oligomers. The antisense oligomers were purchased from Japan Bioservices. The sequences of the various antisense oligomers are listed below. show. [Table 12]

[0170] RT-PCR was performed using the following primers under the same conditions and procedures as for exon 45 (Test Example 1). I went in order. Forward primer: 5'- CATGGAAGGAGGGTCCCTAT-3' (SEQ ID NO: 69) Reverse primer: 5'-CTGCCGGCTTAATTCATCAT-3' (SEQ ID NO: 70)

[0171] Experimental results The results are shown in Figures 6 and 7. This experiment identified the 55th exon of the human dystrophin gene. When the antisense oligomer is designed at positions 1 to 21 or 11 to 31 from the 5' end of the Antisense oligomers were designed to target positions 104-123 from the 5' end of the 55th exon. It was found that exon 55 skipping was more efficient than when the nucleotide sequence was used.

[0172] [Test Example 5] In vitro assays RT-PCR was performed using the following primers under the same conditions and procedures as for exon 45 (Test Example 2). I went in order. Forward primer: 5'- CATGGAAGGAGGGTCCCTAT-3' (SEQ ID NO: 69) Reverse primer: 5'-CTGCCGGCTTAATTCATCAT-3' (SEQ ID NO: 70)

[0173] Experimental results The results are shown in Figures 8, 16, and 17. This experiment revealed that PMONo.12, 13, 14 (H55_8-28(OH)), The oligomers of the present invention (H55_11-31(OH) and H55_14-34(OH)) showed high efficacy in RD cells. It was found that exon 55 was skipped in PMO Nos. 14, 16, and 17 (Fig. 8). , 18, 19 (H55_14-34(OH), H55_12-32(OH), H55_13-33(OH), H55_15-35(OH) and H55_16-3 The oligomer of the present invention (H55_139-156(GT)) inhibited the activity of PMO No. 15 (H55_139-156(GT)) in RD cells. It can skip exon 55 with significantly higher efficiency compared to the nonsense oligomer. The oligomer of the present invention, PMO No. 14, and PMO No. 21 ( H55_15-34(OH)) was found to skip exon 55 with comparable efficiency (Figure 17 Furthermore, the oligomer of the present invention, PMO No. 14, is similar to PMO No. 22 (H55_14-34), which has a different terminal structure. (TEG)) and was found to skip exon 55 with comparable efficiency (Figure 1 7).

[0174] About Exon 44 According to the description of Examples 20 to 29 below, various PMOs shown in PMO Nos. 23 to 29 and 31 to 33 in the table below were used. The synthesized PMO was dissolved in water for injection (Otsuka Pharmaceutical Factory). It was purchased from Gene Tools. [Table 13]

[0175] [Example 20] PMO.No.23 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6918.9 Measurement: 6918.3

[0176] [Example 21] PMO.No.24 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6903.9 Measurement: 6904.2

[0177] [Example 22] PMO.No.25 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4- ritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 6912.9 Measurement: 6912.4

[0178] [Example 23] PMO.No.26 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6903.9 Measurement: 6904.2

[0179] [Example 24] PMO.No.27 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4- ritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 6927.9 Measurement: 6927.4 [Example 25] PMO.No.28 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6942.9 Measurement: 6942.3

[0180] [Example 26] PMO.No.29 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6917.9 Measurement: 6918.3

[0181] [Example 27] PMO.No.31 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4- ritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 6573.6 Measurement: 6572.4

[0182] [Example 28] PMO.No.32 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6588.6 Measurement: 6588.3

[0183] [Example 29] PMO.No.33 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 1,12-Dioxo-1-(4-tritylpiperazine-1-yl)methylpropanol supported on polystyrene resin (I)-2,5,8,11-tetraoxa-15-pentadecanoic acid (Reference Example 5) was used. ESI-TOF-MS calculated value: 7255.2 Measurement: 7254.7

[0184] [Test Example 6] In vitro assays 2'-O-methoxy-phosphorothioates shown in SEQ ID NOs: 82 to 95 and 109 to 118 The experiment was carried out using an antisense oligomer of (2'-OMe-S-RNA). Various antisense oligomers were purchased from Japan Bioservices. The sequences of the ligomers are shown below. [Table 14]

[0185] The same conditions and procedures were used as for exon 45 (Test Example 1).

[0186] Experimental results The results are shown in Figures 9 and 10. This experiment identified the 44th exon of the human dystrophin gene. Antisense oligomers were designed at positions 11-32 or 26-47 from the 5' end of the nucleotide sequence. In this case, antisense oligomers were designed to address positions 62-81 from the 5' end of exon 44. It was found that exon 44 skipping was achieved with the same efficiency as when the nucleotide sequence was used.

[0187] [Test Example 7] In vitro assays The same conditions and procedures were used as for exon 45 (Test Example 2).

[0188] Experimental results The results are shown in Figures 11, 12, and 18. This experiment revealed that PMONo.24, 26(H44, 25-45(OH), H The oligomer of the present invention (H44_27-47(OH)) inhibited the activity of PMO No. 30 (H44_10-39(OH)) in RD cells. It was found that this oligonucleotide induces exon 44 skipping with the same efficiency as the antisense oligomer of The oligomer of the present invention, PMO No. 26, and the oligomer of PMO No. 31 (H) with a chain length shortened by one base were used (Figs. 11 and 12). 44_27-46(OH)) was found to skip exon 44 with comparable efficiency (Figure 18 Furthermore, the oligomer of the present invention, PMO No. 26, is similar to PMO No. 33 (H44_27-4), which has a different terminal structure. 7(TEG)), it was found to skip exon 44 with similar efficiency (Figure 18).

[0189] About Exon 50 According to the description of Examples 30 to 39 below, various PMOs shown in Table 15, PMO Nos. 34 to 38 and 41 to 45, were synthesized. The synthesized PMO was dissolved in water for injection (Otsuka Pharmaceutical Factory). was purchased from Gene Tools. [Table 15]

[0190] [Example 30] PMO.No.34 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}- 4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6861.8 Measurement: 6861.8

[0191] [Example 31] PMO.No.35 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4- ritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 6885.8 Measurement: 6885.9

[0192] [Example 32] PMO.No.36 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6925.9 Measurement: 6925.9

[0193] [Example 33] PMO.No.37 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6950.9 Measurement: 6950.9

[0194] [Example 34] PMO.No.38 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6990.9 Measurement value: 6991.0

[0195] [Example 35] PMO.No.41 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6635.6 Measurement: 6635.0

[0196] [Example 36] PMO.No.42 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxy)-4-hydroxybenzoate supported on polystyrene resin acetyl)amino]purin-9-yl]-4-tritylmorpholin-2-yl]methoxy]-4-oxo- Butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 6635.6 Measurement: 6634.9

[0197] [Example 37] PMO.No.43 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene resin-supported 4-(((2S,6R)-6-(5-methyl-2,4-dioxo- 3,4-Dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)meth (xi)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 6965.9 Measurement: 6965.2

[0198] [Example 38] PMO.No.44 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. Polystyrene Resin-Supported 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4- ritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 6949.9 Measurement: 6949.2

[0199] [Example 39] PMO.No.45 The title compound was produced in the same manner as in Example 1, except that amine was used as the starting material. 1,12-Dioxo-1-(4-tritylpiperazine-1-yl)methylpropanol supported on polystyrene resin (I)-2,5,8,11-tetraoxa-15-pentadecanoic acid (Reference Example 5) was used. ESI-TOF-MS calculated value: 7342.2 Measurement: 7341.6

[0200] [Test Example 8] In vitro assays RT-PCR was performed with the following primers at concentrations of 0.1, 0.3, and 1 μM: The test was carried out under the same conditions and procedures as in (Test Example 2). Forward primer: 5'-AACAACCGGATGTGGAAGAG-3' (SEQ ID NO: 103) Reverse primer: 5'-TTGGAGATGGCAGTTTCCTT-3' (SEQ ID NO: 104)

[0201] Experimental results The results are shown in Figures 13 and 19. This experiment confirmed the effectiveness of PMONo.38 (H50_107-127(OH)) in the present invention. The oligomers were PMO No. 39 or 40 (H50_90-114(GT), H50_103-127(GT)) in RD cells. It can skip exon 50 with higher efficiency compared to antisense oligomers. In addition, PMO No. 38 was found to have a different terminal structure compared to PMO No. 45 (H50_107-127(TEG)). It was found that exon 50 was skipped with high efficiency (Figure 19).

[0202] Exon 44 skipping study In vitro assay using human fibroblasts [Test Example 9] GM05112 cells (fibroblasts derived from a DMD patient with exon 45 deletion, Coriell Institute for Medical The exon 44 skipping activity was examined using a medium containing 10% FCS and 1% PEG / PPG-1000. Dulbecco's Modification containing Penicillin / Streptomycin (P / S) (Sigma-Aldrich) d Eagle Medium: Nutrient Mixture F-12 (DMEM / F-12) (Invitrogen) was used for 5 The cells were cultured at 37°C in the presence of 100% CO2. Cells were cultured in T225 flasks and 2.5 mL of human MyoD (SEQ ID NO: 3) was added to 35 mL of growth medium. 8) Expression retrovirus (co-expressing ZsGreen1) and polybrene (Sigma) at a final concentration of 8 μg / mL After culturing at 32°C for 2 days, the medium was replaced with fresh growth medium. The cells were further cultured at 37°C for 3 days. ZsGreen was analyzed using a BD FACSAria Cell Sorter (BD Biosciences). MyoD-transformed fibroblasts were collected by selecting 1-positive cells and plated on a collagen-coated 24-well plate. Rate 9 x 10 4 The next day, the cells were cultured in differentiation medium (2% horse serum (Invitrogen) The medium was then replaced with DMEM / F-12 containing 1% P / S and ITS Liquid Media Supplement (Sigma). The cells were cultured with the medium changed every 2 to 3 days to induce differentiation into myotubes. On the 7th day after the change to differentiation medium, Endo-Porter (Gene Tools) was added to a final concentration of 6 μM. The differentiation medium was replaced with a medium containing PMO No. 26 and PMO No. 31, and the final concentrations were 1, 3, and 10 μM. After 7 days of incubation, the cells were harvested and purified using the RNeasy Mini Kit (Qiagen). Total RNA was extracted using the QIAGEN OneStep RT-PCR Kit. RT-PCR was performed using the following. The reaction mixture was prepared according to the attached protocol. The iCycler (Bio-Rad) was used. The RT-PCR program used was as follows: be. 50℃, 30 minutes: reverse transcription 95℃, 15 minutes: heat denaturation PCR amplification: [94°C, 1 min; 60°C, 1 min; 72°C, 1 min] x 35 cycles 72℃, 7 minutes: final extension reaction

[0203] The nucleotide sequences of the forward and reverse primers used for RT-PCR are as follows: is. Forward primer: 5'-GCTCAGGTCGGATTGACATT-3' (SEQ ID NO: 36) Reverse primer: 5'-GGGCAACTCTTCCACCAGTA-3' (SEQ ID NO: 37)

[0204] The reaction products of the above RT-PCR reaction were separated by 2% agarose gel electrophoresis and imaged. Gel photographs were taken using the ImageQuant LAS 4000 mini analyzer (Fujifilm). The software of the genus determines the polynucleotide amount of the band in which exon 44 is skipped, "A", and The amount of polynucleotides in the bands that were not skipped by ctxon 44, "B", was measured. Based on the measured values ​​of "A" and "B", the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / ( A + B ) x 100

[0205] Experimental results The results are shown in Figure 20. This experiment confirmed that the oligomers of the present invention, PMO Nos. 26 and 31, were significantly more potent than GM051. It was found to skip exon 44 with high efficiency in 12 cells.

[0206] [Test Example 10] As in Test Example 9, MyoD-transformed fibroblasts were prepared using GM05112 cells and differentiated into myotubes. On the 6th day after the medium was changed to differentiation medium, Endo-Porter (GeneTwo) was added to a final concentration of 6 μM. The differentiation medium was replaced with a medium containing PMO No. 26 and PMO No. 31 at a final concentration of 10 μM. After 14 days of incubation, the cells were incubated with protease inhibitor cocktail Complete Mini (Ro The scraper was lysed using RIPA buffer (Pierce) containing The cells were then lysed using an ultrasonic homogenizer, Bioruptor UCD-250 (Tosho Denki Co., Ltd.). The supernatant after centrifugation was collected and used as a cell lysate. The protein concentration of the cell lysate was The absorbance at a wavelength of 544 nm was measured using the Pierce BCA protein assay kit (Pierce). Measurements were performed using a rate reader Thermo Appliskan Type 2001 (Thermo Electron). 3 μg of cell lysate was analyzed on a polyacrylamide gel (NuPAGE) Novex Tris-Acetate Gel 3-8%. After electrophoresis, the sample was subjected to semi-dry blotting. The transferred membrane was then washed with 0.1% Tween 20. After washing with PBST containing 5% Amersham ECL Prime Blocking agent (GE Healthcare), The membrane was blocked overnight in a refrigerator with PBST containing 1000 mg of dystrophin. (NCL-Dys1, Novocastra) was diluted 50 times with Can Get Signal1 (TOYOBO) and After washing with PBST, peroxidase-labeled goat anti-mouse IgG was added to the wells. Antibody (170-6516, Bio-Rad) diluted 2,500-fold with Can Get Signal 2 (TOYOBO) After washing with PBST, the plate was incubated with ECL Plus Western Blotting Degreaser (ECL Plus Western Blotting Degreaser) for 10 minutes at room temperature. The DNA was processed using the DNA Removal System (GE Healthcare). The chemiluminescence of dystrophin was measured using the ImageQuant LAS 4000 luminometer and image analyzer. Detection was performed using a NI (Fujifilm Corporation).

[0207] Experimental results The results of Western blotting are shown in Figure 21. In Figure 21, the arrows indicate the genes where expression was confirmed. This experiment confirmed that PMO Nos. 26 and 31 were capable of expressing the dystrophin protein. Akari oligomers induce dystrophin protein expression in GM05112 cells It was discovered that...

[0208] Considering exon 50 skipping In vitro assay using human fibroblasts [Test Example 11] As in Test Example 9, MyoD-transformed fibroblasts were prepared using GM05112 cells and induced to differentiate into myotubes. Led. On the 12th day after the change to differentiation medium, Endo-Porter (Gene Tools) was added to a final concentration of 6 μM. The differentiation medium was replaced with a medium containing PMO No. 38 at final concentrations of 0.1, 0.3, 1, 3, and 10 μM. After incubation for 2 days, the cells were collected. Total RNA was extracted in the same manner as in Test Example 9. After that, RT-PCR was performed to determine the skipping efficiency. The base sequences of the primer and reverse primer are as follows: Forward primer: 5'-AACAACCGGATGTGGAAGAG-3' (SEQ ID NO: 103) Reverse primer: 5'-TTGGAGATGGCAGTTTCCTT-3' (SEQ ID NO: 104)

[0209] Experimental results The results of RT-PCR are shown in Figure 22, and the skipping efficiency is shown in Figure 23. The oligomer of the present invention, 8, efficiently skipped exon 50 in GM05112 cells. The EC50 value was found to be 1.3 μM.

[0210] [Test Example 12] 11-0627 cells (fibroblasts derived from a DMD patient with exon 8-9 duplication, National Center for Neurology and Psychiatry) The final concentration of PMO No. 38 was adjusted to 0.1, 1. A skipping test was carried out under the same conditions and procedures as in Test Example 11, except that the concentration of 10 μM was 10 μM.

[0211] Experimental results The results of RT-PCR are shown in Figure 26, and the skipping efficiency is shown in Figure 27. The oligomers of the present invention (8) efficiently skipped exon 50 in 11-0627 cells. It was found that this was possible.

[0212] [Test Example 13] Generation of pLVX-MyoD-ZsGreen1 lentivirus XhoI (position 2816) in the multiple cloning site of pLVX-puro (8120 bp, Clontech) 1164 bp from the 3' end of the Puromycin resistance gene coding region (position 3890) The linearized vector was then inserted with a sequence encoding the human MyoD gene, IRES sequence, and ZsGreen1 gene in that order. The sequence (2272 bp) was incorporated into the lentiviral expression vector pLVX-MyoD-ZsGreen1 (9210 bp) was prepared according to the protocol of the Lenti-X HTX Packaging System (Clontech). Lenti-X 293T cells were seeded on a 10 cm collagen-coated dish. Three days before the scheduled date, the lentiviral expression vector and packaging vector were transfected. The medium was changed after 4 hours, and the cells were cultured for 3 days without changing the medium. On the day of incubation, the culture supernatant was collected as a virus solution (approximately 9 mL per 10 cm dish). The mixture was filtered through a cell strainer (40 μm) and further centrifuged at 500 × g for 10 minutes. The supernatant was concentrated according to the protocol of the Lenti-X Concentrator (Clontech) and finally recovered. The virus was dissolved in DMEM / F12 medium to a concentration 10 times that of the virus immediately after harvest. Used.

[0213] Viral infection of fibroblasts Before the scheduled infection date, GM04364 cells (fibroblasts derived from a DMD patient lacking exons 51-55, Coriell (Institute for Medical Research) in a collagen-coated 24-well plate, 3x10 4 / wel On the day of infection, 400 μL of differentiation medium and 100 μL of virus solution were added per well. 100 μL of polybrene was added to a final concentration of 8 μg / mL. The medium was replaced with 500 μL. After that, the differentiation medium was replaced every 2-3 days and cultured for 12 days. The cells were induced to differentiate into myotubes. On the 12th day after the change to differentiation medium, Endo-Porter (Gene Tools) was added to a final concentration of 6 μM. The differentiation medium was replaced with a medium containing PMO No. 38 at final concentrations of 0.1, 0.3, 1, 3, and 10 μM. After incubation for 2 days, the cells were collected. The efficiency was calculated using the forward and reverse primers used in RT-PCR. The base sequence is as follows: Forward primer: 5'-AACAACCGGATGTGGAAGAG-3' (SEQ ID NO: 103) Reverse primer: 5'-CTGCCGGCTTAATTCATCAT-3' (SEQ ID NO: 70)

[0214] Experimental results The results of RT-PCR are shown in Figure 28, and the skipping efficiency is shown in Figure 29. The oligomer of the present invention, 8, efficiently skipped exon 50 in GM04364 cells. It was found that this was possible.

[0215] Exon 55 skipping study In vitro assay using human fibroblasts [Test Example 14] PMO Nos. 14 and 21 were used, and RT-PCR was performed using the following primers, in the same manner as in Test Example 11. The experiment was carried out under the conditions and procedures described above. Forward primer: 5'- CATGGAAGGAGGGTCCCTAT-3' (SEQ ID NO: 69) Reverse primer: 5'-CTGCCGGCTTAATTCATCAT-3' (SEQ ID NO: 70)

[0216] Experimental results The results of RT-PCR are shown in Figure 24, and the skipping efficiency is shown in Figure 25. The oligomers of the present invention, 4 and 21, skipped exon 55 with high efficiency in GM05112 cells. The EC50 values ​​were found to be 3.5 μM and 7.5 μM, respectively.

[0217] [Test Example 15] The cells were 04-035 cells (derived from a DMD patient with exon 54 deletion, National Institute of Neurology and Psychiatry). PMO Nos. 14 and 21 were added at final concentrations of 1, 3, and 10 mg / mL using the National Center for Neuromuscular Disease Research Resources Repository (NCRRR). The conditions and procedures were the same as in Test Example 13, except that RT-PCR was performed using the following primers: So I went. Forward primer: 5'- CATGGAAGGAGGGTCCCTAT-3' (SEQ ID NO: 69) Reverse primer: 5'-CTGCCGGCTTAATTCATCAT-3' (SEQ ID NO: 70)

[0218] Experimental results The results of RT-PCR are shown in Figure 30, and the skipping efficiency is shown in Figure 31. The oligomers 4 and 21 of the present invention showed high efficiency in cells derived from a DMD patient with a single deletion of exon 54. It was found that this causes exon 55 skipping. [Industrial Applicability]

[0219] From the experimental results shown in the test examples, it was found that the oligomer of the present invention inhibits the proliferation of both RD cells and DMD patient-derived cells. In this environment, target exons (e.g., exon 55) are skipped with significantly higher efficiency. It has been shown that this can be done. Therefore, the oligomers of the present invention are highly useful in the treatment of DMD. [Sequence List Free Text]

[0220] SEQ ID NO: 9: Synthetic nucleic acid SEQ ID NO: 10: Synthetic nucleic acid SEQ ID NO: 11: Synthetic nucleic acid SEQ ID NO: 12: Synthetic nucleic acid SEQ ID NO: 13: Synthetic nucleic acid SEQ ID NO: 14: Synthetic nucleic acid SEQ ID NO: 15: Synthetic nucleic acid SEQ ID NO: 16: Synthetic nucleic acid SEQ ID NO: 17: Synthetic nucleic acid SEQ ID NO: 18: Synthetic nucleic acid SEQ ID NO: 19: Synthetic nucleic acid SEQ ID NO: 20: Synthetic nucleic acid SEQ ID NO: 21: Synthetic nucleic acid SEQ ID NO: 22: Synthetic nucleic acid SEQ ID NO: 23: Synthetic nucleic acid SEQ ID NO: 24: Synthetic nucleic acid SEQ ID NO: 25: Synthetic nucleic acid SEQ ID NO: 26: Synthetic nucleic acid SEQ ID NO: 27: Synthetic nucleic acid SEQ ID NO: 28: Synthetic nucleic acid SEQ ID NO: 29: Synthetic nucleic acid SEQ ID NO: 30: Synthetic nucleic acid SEQ ID NO: 31: Synthetic nucleic acid SEQ ID NO: 32: Synthetic nucleic acid SEQ ID NO: 33: Synthetic nucleic acid SEQ ID NO: 34: Synthetic nucleic acid SEQ ID NO: 35: Synthetic nucleic acid SEQ ID NO: 36: Synthetic nucleic acid SEQ ID NO: 37: Synthetic nucleic acid SEQ ID NO: 38: Synthetic nucleic acid SEQ ID NO: 39: Synthetic nucleic acid SEQ ID NO: 40: Synthetic nucleic acid SEQ ID NO: 41: Synthetic nucleic acid SEQ ID NO: 42: Synthetic nucleic acid SEQ ID NO: 43: Synthetic nucleic acid SEQ ID NO: 45: Synthetic nucleic acid SEQ ID NO: 46: Synthetic nucleic acid SEQ ID NO: 47: Synthetic nucleic acid SEQ ID NO: 48: Synthetic nucleic acid SEQ ID NO: 49: Synthetic nucleic acid SEQ ID NO: 50: Synthetic nucleic acid SEQ ID NO: 51: Synthetic nucleic acid SEQ ID NO: 52: Synthetic nucleic acid SEQ ID NO: 53: Synthetic nucleic acid SEQ ID NO: 54: Synthetic nucleic acid SEQ ID NO: 55: Synthetic nucleic acid SEQ ID NO: 56: Synthetic nucleic acid SEQ ID NO: 57: Synthetic nucleic acid SEQ ID NO: 58: Synthetic nucleic acid SEQ ID NO: 59: Synthetic nucleic acid SEQ ID NO: 60: Synthetic nucleic acid SEQ ID NO: 61: Synthetic nucleic acid SEQ ID NO: 62: Synthetic nucleic acid SEQ ID NO: 63: Synthetic nucleic acid SEQ ID NO: 64: Synthetic nucleic acid SEQ ID NO: 65: Synthetic nucleic acid SEQ ID NO: 66: Synthetic nucleic acid SEQ ID NO: 67: Synthetic nucleic acid SEQ ID NO: 68: Synthetic nucleic acid SEQ ID NO: 69: Synthetic nucleic acid SEQ ID NO: 70: Synthetic nucleic acid SEQ ID NO: 71: Synthetic nucleic acid SEQ ID NO: 72: Synthetic nucleic acid SEQ ID NO: 73: Synthetic nucleic acid SEQ ID NO: 74: Synthetic nucleic acid SEQ ID NO: 75: Synthetic nucleic acid SEQ ID NO: 76: Synthetic nucleic acid SEQ ID NO: 77: Synthetic nucleic acid SEQ ID NO: 78: Synthetic nucleic acid SEQ ID NO: 79: Synthetic nucleic acid SEQ ID NO: 80: Synthetic nucleic acid SEQ ID NO: 81: Synthetic nucleic acid SEQ ID NO: 82: Synthetic nucleic acid SEQ ID NO: 83: Synthetic nucleic acid SEQ ID NO: 84: Synthetic nucleic acid SEQ ID NO: 85: Synthetic nucleic acid SEQ ID NO: 86: Synthetic nucleic acid SEQ ID NO: 87: Synthetic nucleic acid SEQ ID NO: 88: Synthetic nucleic acid SEQ ID NO: 89: Synthetic nucleic acid SEQ ID NO: 90: Synthetic nucleic acid SEQ ID NO: 91: Synthetic nucleic acid SEQ ID NO: 92: Synthetic nucleic acid SEQ ID NO: 93: Synthetic nucleic acid SEQ ID NO: 94: Synthetic nucleic acid SEQ ID NO: 95: Synthetic nucleic acid SEQ ID NO: 96: Synthetic nucleic acid SEQ ID NO: 97: Synthetic nucleic acid SEQ ID NO: 98: Synthetic nucleic acid SEQ ID NO: 99: Synthetic nucleic acid SEQ ID NO: 100: Synthetic nucleic acid SEQ ID NO: 101: Synthetic nucleic acid SEQ ID NO: 102: Synthetic nucleic acid SEQ ID NO: 103: Synthetic nucleic acid SEQ ID NO: 104: Synthetic nucleic acid SEQ ID NO: 105: Synthetic nucleic acid SEQ ID NO: 106: Synthetic nucleic acid SEQ ID NO: 107: Synthetic nucleic acid SEQ ID NO: 108: Synthetic nucleic acid SEQ ID NO: 109: Synthetic nucleic acid SEQ ID NO: 110: Synthetic nucleic acid SEQ ID NO: 111: Synthetic nucleic acid SEQ ID NO: 112: Synthetic nucleic acid SEQ ID NO: 113: Synthetic nucleic acid SEQ ID NO: 114: Synthetic nucleic acid SEQ ID NO: 115: Synthetic nucleic acid SEQ ID NO: 116: Synthetic nucleic acid SEQ ID NO: 117: Synthetic nucleic acid SEQ ID NO: 118: Synthetic nucleic acid SEQ ID NO: 119: Synthetic nucleic acid SEQ ID NO: 120: Synthetic nucleic acid SEQ ID NO: 121: Synthetic nucleic acid SEQ ID NO: 122: Synthetic nucleic acid

Claims

1. Antisense oligonucleotides that allow skipping of the 55th exon of the human dystrophin gene It is an oligomer derived from the 5' end of the 55th exon of the human dystrophin gene. 14th to 34th, -2nd to 19th, -2nd to 20th, -2nd to 21st, -2nd to 22nd, -2nd to 23rd th, -1st to 19th, -1st to 20th, -1st to 21st, -1st to 22nd, -1st to 23rd, 1st ~19th, 1st-20th, 1st-21st, 1st-22nd, 1st-23rd, 2nd-19th, 2nd- 20th, 2nd-21st, 2nd-22nd, 2nd-23rd, 3rd-19th, 3rd-20th, 3rd-21st th, 3rd-22nd, 3rd-23rd, 9th-29th, 9th-30th, 9th-31st, 9th-32nd , 9th-33rd, 10th-29th, 10th-30th, 10th-31st, 10th-32nd, 10th-3 3rd, 11th-29th, 11th-30th, 11th-31st, 11th-32nd, 11th-33rd, 1st 2nd to 29th, 12th to 30th, 12th to 31st, 12th to 32nd, 12th to 33rd, 13th to 29th , 13th-30th, 13th-31st, 13th-32nd, 13th-33rd, 12th-34th, 12th-35th th, 12th-36th, 13th-34th, 13th-35th, 13th-36th, 14th-32nd, 14th ~33rd, 14th-35th, 14th-36th, 15th-32nd, 15th-33rd, 15th-34th, No. 15-35, No. 15-36, No. 16-32, No. 16-33, No. 16-34, No. 16-35 A base sequence complementary to either the sequence consisting of nucleotides 16 to 36 of the An antisense oligomer.

2. Antisense oligonucleotides that allow skipping of the 45th exon of the human dystrophin gene It is an oligomer derived from the 5' end of the 45th exon of the human dystrophin gene. -3rd to 19th, -3rd to 20th, -3rd to 21st, -3rd to 22nd, -3rd to 23rd, -2nd to 19th th, -2nd to 20th, -2nd to 21st, -2nd to 22nd, -2nd to 23rd, -1st to 19th, -1st ~20th, -1st to 21st, -1st to 22nd, -1st to 23rd, 1st to 19th, 1st to 20th, 1st to 21st, 1st to 22nd, 1st to 23rd, 2nd to 19th, 2nd to 20th, 2nd to 21st, 2nd ~22nd, 2nd to 23rd, -2nd to 24th, -2nd to 25th, -2nd to 26th, -2nd to 27th, -1st to 24th, -1st to 25th, -1st to 26th, -1st to 27th, 1st to 24th, 1st to 25th , 1st to 26th, 1st to 27th, 2nd to 24th, 2nd to 25th, 2nd to 26th, 2nd to 27th, 3rd to 23rd, 3rd to 24th, 3rd to 25th, 3rd to 26th, 3rd to 27th, 4th to 28th, 4th to 29th, 4th to 30th, 4th to 31st, 4th to 32nd, 5th to 28th, 5th to 29th, 5th ~30th, 5th-31st, 5th-32nd, 6th-28th, 6th-29th, 6th-30th, 6th- 31st, 6th-32nd, 7th-28th, 7th-29th, 7th-30th, 7th-31st, 7th-32nd 8th, 8th to 28th, 8th to 29th, 8th to 30th, 8th to 31st or 8th to 32nd nuclei An antisense oligomer consisting of a base sequence complementary to any one of the sequences consisting of nucleotides. -.

3. Antisense oligonucleotides that allow skipping of the 50th exon of the human dystrophin gene It is an oligomer derived from the 5' end of the 50th exon of the human dystrophin gene. 105th to 125th, 105th to 126th, 105th to 127th, 105th to 128th, 105th to 129th , 106th to 125th, 106th to 126th, 106th to 127th, 106th to 128th, 106th to 129th , 107th-125th, 107th-126th, 107th-127th, 107th-128th, 107th-129th , 108th-125th, 108th-126th, 108th-127th, 108th-128th, 108th-129th , 109th to 125th, 109th to 126th, 109th to 127th, 109th to 128th or 109th to 129th Antisense oligonucleotides consist of a base sequence complementary to one of the sequences consisting of the first nucleotide. So-oligomer.

4. Antisense oligonucleotides that allow skipping of exon 44 of the human dystrophin gene It is an oligomer derived from the 5' end of exon 44 of the human dystrophin gene. 9th to 30th, 9th to 31st, 9th to 32nd, 9th to 33rd, 9th to 34th, 10th to 30th , 10th-31st, 10th-32nd, 10th-33rd, 10th-34th, 11th-30th, 11th-31st th, 11th-32nd, 11th-33rd, 11th-34th, 12th-30th, 12th-31st, 12th ~32nd, 12th to 33rd, 12th to 34th, 13th to 30th, 13th to 31st, 13th to 32nd, Nos. 13-33, 13-34, 24-45, 24-46, 24-47, 24-48 , 24th-49th, 25th-45th, 25th-46th, 25th-47th, 25th-48th, 25th- 49th, 26th-45th, 26th-46th, 26th-47th, 26th-48th, 26th-49th, 27th-45th, 27th-46th, 27th-47th, 27th-48th, 27th-49th, 28th-45th , 28th-46th, 28th-47th, 28th-48th, 28th-49th, 29th-45th, 29th-46th a sequence consisting of nucleotides 29-47, 29-48, or 29-49; An antisense oligomer is one that consists of a base sequence complementary to one of the two.

5. Positions 1-21 and 11-31 from the 5' end of exon 55 of the human dystrophin gene nucleotide sequence complementary to the sequence consisting of the 14th or 15th to 34th nucleotides of the 2. The antisense oligomer according to claim 1.

6. From nucleotides 170 to 190, 160 to 180, or 157 to 177 of SEQ ID NO:5 2. The antisense oligomer according to claim 1, comprising any one of the base sequences:

7. Positions -2 to 19 and 1 to 21 from the 5' end of exon 45 of the human dystrophin gene A base sequence complementary to a sequence consisting of nucleotides 1 to 25 or 6 to 30 of the base sequence. The antisense oligomer of claim 2.

8. The 158th to 178th, 156th to 176th, 152nd to 176th, or 147th to 171st of SEQ ID NO: 6 The antisense oligonucleotide according to claim 2, which comprises any one of the base sequences of the sequence consisting of nucleotides. Ins oligomer.

9. Positions 106-126 or 107 from the 5' end of the 50th exon of the human dystrophin gene The antigen according to claim 3, which comprises a base sequence complementary to a sequence consisting of nucleotides 1 to 127. Antisense oligomers.

10. Any one of the sequences consisting of nucleotides 4 to 24 or 3 to 23 of SEQ ID NO: 7 4. The antisense oligomer according to claim 3, which consists of a base sequence.

11. Positions 11-32 and 25-45 from the 5' end of exon 44 of the human dystrophin gene A salt complementary to the sequence consisting of nucleotides 26-46, 26-47, or 27-47 The antisense oligomer according to claim 4, comprising a base sequence.

12. positions 117 to 138, positions 104 to 124, positions 103 to 123, positions 102 to 123 of SEQ ID NO: 8, or Claim 4: consisting of any one of the base sequences consisting of nucleotides 102 to 122 The antisense oligomer according to claim 1.

13. The antisense oligomer according to any one of claims 1 to 12, which is an oligonucleotide. -.

14. The sugar moiety and / or linker of at least one nucleotide constituting the oligonucleotide 14. The antisense oligomer of claim 13, wherein the phosphate binding moiety is modified.

15. The sugar moiety of at least one nucleotide constituting the oligonucleotide is -OH group is OR, R, R'OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F, Cl, Br and I 15. The antisense oligonucleotide of claim 14, wherein the ribose is substituted with any one of the groups selected from the group consisting of: Oligomer. (The above R represents alkyl or aryl, and the above R' represents alkylene.)

16. The phosphate binding moiety of at least one nucleotide constituting the oligonucleotide , phosphorothioate bond, phosphorodithioate bond, alkylphosphonate bond, any one selected from the group consisting of a phosphoamidate bond and a boranophosphate bond; 16. The antisense oligomer of claim 14 or 15, which is one of:

17. The antisense oligo of any one of claims 1 to 12, which is a morpholino oligomer. Mah.

18. 18. The antisense oligonucleotide of claim 17, which is a phosphorodiamidate morpholino oligomer. Ligomar.

19. The 5'-end of claim 17 or 18 is a group represented by any one of the following chemical formulas (1) to (3): Antisense oligomers. 【Chemistry 24】

20. The antisense oligomer according to any one of claims 1 to 19, its pharmaceutically acceptable salt A pharmaceutical composition for treating muscular dystrophy, comprising a salt or hydrate thereof as an active ingredient.

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