Antisense nucleic acid inducing skipping of exon 51

Abstract

Provided is a drug that allows efficient skipping of the 51st exon in human dystrophin gene. Also provided is an antisense oligomer that has an activity of inducing skipping of the 51st exon in human dystrophin gene.

Description

Antisense nucleic acid that induces exon 51 skipping

[0001] The present invention relates to an antisense oligomer that induces skipping of exon 51 of the human dystrophin gene, and a pharmaceutical composition containing the antisense oligomer.

[0002] Duchenne muscular dystrophy (DMD) is the most common severe hereditary progressive muscular atrophy, occurring in approximately 1 in 3,500 male births. During infancy, patients exhibit motor function almost identical to that of healthy individuals, but muscle weakness begins to decline from around age 4 to 5. DMD patients subsequently experience progressive muscle weakness, becoming unable to walk by around age 12 and eventually dying from heart or respiratory failure in their 20s. Currently, there is no satisfactory treatment for DMD, and there is a strong need for the development of effective therapeutic agents.

[0003] DMD is known to be caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome and is a large gene consisting of 2.2 million base pairs of DNA. It is transcribed from DNA into pre-mRNA, which is then spliced ​​to remove introns and join 79 exons, resulting in an mRNA of 11,058 bases corresponding to the translated region. This mRNA is translated into 3,685 amino acids to produce dystrophin protein. Dystrophin protein is involved in maintaining muscle cell membrane stability and is necessary for preventing muscle cell breakdown. Because DMD patients have mutations in the dystrophin gene, functional dystrophin protein is barely expressed in muscle cells. As a result, muscle cell structure cannot be maintained in DMD patients, resulting in the influx of large amounts of calcium ions into muscle cells. This results in an inflammation-like response, leading to fibrosis and making muscle cell regeneration more difficult.

[0004] Becker muscular dystrophy (BMD) is also caused by mutations in the dystrophin gene. Although symptoms include muscle atrophy and muscle weakness, they are generally milder than DMD, the progression of muscle weakness is slower, and BMD often develops in adulthood. The difference in clinical symptoms between DMD and BMD is thought to be due to whether the mutation disrupts or maintains the amino acid reading frame during translation of dystrophin mRNA into dystrophin protein (Non-Patent Document 1). In other words, DMD has a mutation that shifts the amino acid reading frame, resulting in the expression of almost no functional dystrophin protein. However, BMD has a mutation that deletes part of an exon, but maintains the amino acid reading frame, resulting in the production of incomplete but functional dystrophin protein.

[0005] Exon skipping is a promising treatment for DMD. This method involves modifying splicing to restore the amino acid reading frame of dystrophin mRNA, thereby inducing the expression of a partially functional dystrophin protein (Non-Patent Document 2). The amino acid sequence targeted by exon skipping is lost. Therefore, the dystrophin protein expressed by this treatment is shorter than normal, but the amino acid reading frame is maintained, thereby partially retaining its function of stabilizing muscle cells. Therefore, exon skipping is expected to result in DMD exhibiting symptoms similar to milder forms of BMD. Following animal experiments in mice and dogs, exon skipping is currently undergoing clinical trials in human DMD patients.

[0006] Exon skipping can be induced by binding of an antisense nucleic acid that targets either or both of the 5' or 3' splice sites, or the interior of an exon. An exon is included in mRNA only when both splice sites are recognized by the spliceosome complex. Therefore, exon skipping can be induced by targeting a splice site with an antisense nucleic acid. Furthermore, it is believed that binding of an SR protein to an exon splicing enhancer (ESE) is required for an exon to be recognized by the splicing machinery, and exon skipping can also be induced by targeting the ESE.

[0007] Since mutations in the dystrophin gene differ among DMD patients, antisense nucleic acids appropriate for the location and type of gene mutation are required. To date, Steve Wilton et al. of the University of Western Australia have created antisense nucleic acids that induce exon skipping for all 79 exons (Non-Patent Document 3), and Annemieke Aartsma-Rus et al. of the Netherlands have created antisense nucleic acids that induce exon skipping for 39 types of exons (Non-Patent Document 4).

[0008] It is believed that approximately 13% of all DMD patients can be treated by skipping the 51st exon (hereinafter referred to as "exon 51"). In recent years, several reports have been published on research targeting exon 51 of the dystrophin gene for exon skipping (Patent Documents 1 to 10 and Non-Patent Documents 3 to 7).

[0009] International Publication No. WO 2015 / 137409, International Publication No. WO 2019 / 241385, International Publication No. WO 2002 / 024906, International Publication No. WO 2004 / 048570, International Publication No. WO 2004 / 083432, International Publication No. WO 2006 / 000057, International Publication No. WO 2010 / 048586, International Publication No. WO 2009 / 054725, International Publication No. WO 2010 / 050801, International Publication No. WO 2010 / 050802

[0010] Monaco AP et al., Genomics 2:90-95 (1988)Matsuo M., Brain and Development 18:167-172 (1996)Wilton SD et al., Molecular Therapy 15:1288-1296 (2007)Annemieke Aartsma-Rus et al., Neuromuscular Disorders 12:S71-S77 (2002) Aoki Y. et al., Molecular Therapy 18:1995-2005 (2010) Nakano S. et al., Pediatrics International 53:524-529 (2011) Echigoya Y et al., Molecular Therapy 25:2561-2572 (2017)

[0011] In light of the above-mentioned circumstances, there is a need for a novel antisense oligomer that can highly efficiently induce skipping of exon 51 of the dystrophin gene. There is also a need for an antisense oligomer that maintains the activity of highly efficiently inducing skipping of exon 51 of the dystrophin gene and has excellent properties (e.g., solubility and safety) as a pharmaceutical.

[0012] The present inventors have conducted detailed studies of the technical details described in the above-mentioned documents and the structure of the dystrophin gene, and have found that administration of an antisense oligomer having a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 89 and 91 to 93 highly efficiently induces exon 51 skipping of the human dystrophin gene. Furthermore, as a result of their research, they have discovered an antisense oligomer that highly efficiently induces exon 51 skipping of the human dystrophin gene while also having excellent solubility and safety. Based on this finding, the present inventors have completed the present invention.

[0013] That is, the present invention is as follows. [1] The following (a1) to (d1): (a1) an antisense oligomer comprising a nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93; (b1) an antisense oligomer comprising a nucleotide sequence in which 1 to 5 nucleotides have been deleted, substituted, inserted, and / or added relative to the nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93, and having the activity of inducing skipping of exon 51 of the human dystrophin gene; (c1) an antisense oligomer comprising a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93, and having the activity of inducing skipping of exon 51 of the human dystrophin gene; and (d1) an antisense oligomer that hybridizes under stringent conditions to an oligonucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93, and having the activity of inducing skipping of exon 51 of the human dystrophin gene. An antisense oligomer selected from the group consisting of (excluding antisense oligomers consisting of any of the base sequences of SEQ ID NOs: 90 and 97 to 126), or a pharmaceutically acceptable salt thereof, or a hydrate thereof.[2] The following (e) to (h): (e) an antisense oligomer consisting of a nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93; (f) an antisense oligomer consisting of a nucleotide sequence in which 1 to 5 bases are deleted and / or substituted with respect to the nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93, and having the activity of inducing skipping of exon 51 of the human dystrophin gene; (g) an antisense oligomer consisting of a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93, and having the activity of inducing skipping of exon 51 of the human dystrophin gene; and (h) an antisense oligomer that hybridizes under highly stringent conditions to an oligonucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93, and having the activity of inducing skipping of exon 51 of the human dystrophin gene. an antisense oligomer selected from the group consisting of (excluding antisense oligomers consisting of any of the nucleotide sequences of SEQ ID NOs: 90 and 97 to 126), or a pharmaceutically acceptable salt thereof, or a hydrate thereof. [3] The antisense oligomer according to [1] or [2] above, which is an antisense oligomer having a nucleotide sequence that has 90% or more sequence identity to any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and which has the activity of inducing skipping of exon 51 of the human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.[4] The antisense oligomer is: (a2) an antisense oligomer comprising any one of the nucleotide sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76; (b2) an antisense oligomer comprising a nucleotide sequence in which 1 to 5 nucleotides have been deleted, substituted, inserted, and / or added relative to any one of the nucleotide sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and having the activity of inducing exon 51 skipping of the human dystrophin gene; (c2) an antisense oligomer comprising a nucleotide sequence having 80% or more sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and having the activity of inducing exon 51 skipping of the human dystrophin gene; and (d2) The antisense oligomer according to [1] above, which is an antisense oligomer selected from the group consisting of: (a) an antisense oligomer that hybridizes under stringent conditions to an oligonucleotide consisting of a nucleotide sequence complementary to any of the nucleotide sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and which has the activity of inducing skipping of exon 51 of the human dystrophin gene, or a pharmaceutically acceptable salt thereof, or a hydrate thereof. [5] The antisense oligomer according to any of [1] to [4] above, which is an oligonucleotide, or a pharmaceutically acceptable salt thereof, or a hydrate thereof. [6] The antisense oligomer according to [5] above, which has a modified sugar moiety and / or phosphate linkage moiety of at least one nucleotide constituting the oligonucleotide, or a pharmaceutically acceptable salt thereof, or a hydrate thereof. [7] The sugar moiety of at least one nucleotide constituting the oligonucleotide has an —OH group at the 2′-position that is OR, R, R′OR, SH, SR, or NH. ２ , N.H.R., N.R. ２ , N ３, CN, F, Cl, Br and I (wherein R represents alkyl or aryl, and R' represents alkylene). [8] The antisense oligomer or a pharmaceutically acceptable salt thereof or a hydrate of either of them according to any of [5] to [7] above, wherein the phosphate bond of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of phosphorothioate bond, phosphorodithioate bond, alkylphosphonate bond, phosphoramidate bond, and boranophosphate bond. [9] The antisense oligomer or a pharmaceutically acceptable salt thereof or a hydrate of either of them according to any of [1] to [4] above, which is a morpholino oligomer.

[10] The antisense oligomer according to [9] above, which is a phosphorodiamidate morpholino oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate of the foregoing.

[11] The 5'-end of the antisense oligomer is a group represented by the following chemical formulas (1) to (3):

[12] The antisense oligomer according to [9] or

[10] above, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the antisense oligomer is any one of the groups shown below.

[13] The pharmaceutical composition according to

[12] above, further comprising a pharmaceutically acceptable carrier.

[14] The pharmaceutical composition according to

[12] or

[13] above, for administration to a patient with muscular dystrophy, wherein the patient has a mutation in the dystrophin gene that is subject to exon 51 skipping.

[15] The pharmaceutical composition according to

[14] above, wherein the patient has a dystrophin gene that has a frameshift mutation due to deletion of at least an exon near exon 51 and in which the amino acid reading frame is corrected by exon 51 skipping.

[16] The pharmaceutical composition according to

[14] or

[15] above, wherein the patient has a frameshift mutation in the dystrophin gene due to a deletion of exons 13-50, 29-50, 40-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52, or 52-63.

[17] The pharmaceutical composition according to any of

[14] to

[16] above, wherein the patient is human.

[18] Use of the antisense oligomer according to any of [1] to

[11] above, or a pharmaceutically acceptable salt thereof, or a hydrate of these, in the manufacture of a medicament for treating muscular dystrophy.

[19] A method for treating muscular dystrophy, comprising the step of administering to a patient with muscular dystrophy an effective amount of the antisense oligomer according to any of [1] to

[11] above, or a pharmaceutically acceptable salt thereof, or a hydrate of these.

[20] The method of treating a muscular dystrophy according to

[19] above, wherein the patient is a human.

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

[11] above, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or the pharmaceutical composition according to any one of

[12] to

[16] above, for use in treating a muscular dystrophy.

[22] The antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate of the foregoing, or the pharmaceutical composition according to

[21] above, wherein the patient with muscular dystrophy in the treatment is a human.

[0014] The present invention provides an antisense oligomer that highly efficiently induces skipping of exon 51 of the human dystrophin gene. The present invention also provides an antisense oligomer that maintains the activity of highly efficiently inducing skipping of exon 51 of the human dystrophin gene and has excellent solubility. Furthermore, the present invention also provides an antisense oligomer that maintains the activity of highly efficiently inducing skipping of exon 51 of the human dystrophin gene and has excellent solubility and safety (e.g., no effect on kidney and liver function or an extremely low likelihood of an effect).

[0015] Figure 1 shows the exon 51 skipping efficiency of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells) for antisense oligomers of PMO Nos. 43, 44, 45, and 46. Figure 2 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 42, 45, 47, 48, 49, and 50. Figure 3 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 42, 62, 63, and 89. Figure 4 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 83 and 85. Figure 1 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers PMO Nos. 33, 34, 35, 36, 37, and 38. Figure 2 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers PMO Nos. 83, 85, and 90. Figure 3 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers PMO Nos. 82, 84, 86, and 87. Figure 4 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers PMO Nos. 45, 51, 52, 56, 57, 58, 59, 60, and 61. Figure 1 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 42, 64, 65, 66, and 85. Figure 2 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 1, 2, 42, 66, 67, 85, 88, 92, and 93. Figure 3 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 3, 4, 5, 6, 7, 8, 42, 68, 69, 70, and 85. Figure 4 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 8, 9, 10, 11, 12, 13, 14, 42, 71, 72, 73, and 91.Figure 1 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 8, 15, 16, 17, 42, 74, 75, and 76. Figure 2 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 8, 18, 19, 20, 42, 63, 75, 76, and 77. Figure 3 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers of PMO Nos. 8, 21, 22, 23, 24, 25, 26, 42, 77, and 78. 1 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers PMO Nos. 8, 21, 27, 28, 29, 30, 42, 79, 80, and 81. 1 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers PMO Nos. 8, 16, 21, 31, 32, and 67. 1 shows the exon 51 skipping efficiency of the human dystrophin gene in RD cells for antisense oligomers PMO Nos. 16, 21, and 94. 1 shows the results of a safety test in mice for antisense oligomer PMO No. 42. From the left, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine values ​​are shown as mean ± standard deviation (Student's t-test significance level: p<0.05). The results of safety tests in mice for antisense oligomers PMO No. 16 and 90 are shown. From the left, AST, ALT, BUN, and creatinine values ​​are shown as mean ± standard deviation, with p values ​​indicating significant increases (Dunnett's test significance level: p<0.05). The results of safety tests in mice for antisense oligomers PMO No. 21 are shown. From the left, AST, ALT, BUN, and creatinine values ​​are shown as mean ± standard deviation (Student's t-test significance level: p<0.05).

[0016] The present invention will be described in detail below. The following embodiments are merely examples for explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be embodied in various forms without departing from the gist of the present invention.

[0017] 1. Antisense Oligomer The present invention provides an antisense oligomer that efficiently skips the 51st exon of the human dystrophin gene (hereinafter referred to as the "antisense oligomer of the present invention").

[0018] [51st Exon of the Human Dystrophin Gene] In the present invention, the term "gene" includes not only genomic genes but also cDNA, pre-mRNA, and mRNA. Preferably, the gene is a pre-mRNA, i.e., pre-mRNA. In the human genome, the human dystrophin gene is located at locus Xp21.2. The human dystrophin gene is 2.2 million base pairs in size, making it the largest known human gene. However, the coding region of the human dystrophin gene is only 14 kb, and this coding region is distributed within the dystrophin gene as 79 exons (Roberts, R.G., et al., Genomics, 16: 536-538 (1993); Koenig, M., et al., Cell 53: 219-228 (1988)). The pre-mRNA, which is a transcript of the human dystrophin gene, undergoes splicing to generate a 14 kb mature mRNA. The nucleotide sequence of the wild-type human dystrophin gene is known (GenBank Accession No. NM_004006). The nucleotide sequence of exon 51 of the wild-type human dystrophin gene is shown in SEQ ID NO: 127.

[0019] [Antisense Oligomer] The antisense oligomer of the present invention was produced for the purpose of modifying a protein encoded by a DMD-type dystrophin gene into a BMD-type dystrophin protein by skipping exon 51 of the human dystrophin gene. Therefore, exon 51 of the dystrophin gene that is the target of exon skipping by the antisense oligomer includes not only wild-type but also mutant-type dystrophin genes.

[0020] Specifically, the antisense oligomer of the present invention is any one of antisense oligomers selected from the group consisting of (a1) to (d1) below: (a1) an antisense oligomer comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93; (b1) an antisense oligomer comprising a nucleotide sequence in which 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide has been deleted, substituted, inserted, and / or added to the nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93, and having the activity of inducing skipping of exon 51 of the human dystrophin gene; (c1) an antisense oligomer comprising a nucleotide sequence having 80% or more, 84% or more, 85% or more, 89% or more, 90% or more, 94% or more, or 95% or more sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1 to 89 and 91 to 93, and having the activity of inducing skipping of exon 51 of the human dystrophin gene; and (d1) An antisense oligomer that hybridizes under stringent conditions to an oligonucleotide consisting of a base sequence complementary to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and has the activity of inducing skipping of exon 51 of the human dystrophin gene.

[0021] In another embodiment, the antisense oligomer of the present invention is specifically any antisense oligomer selected from the group consisting of the following (e) to (h): (e) an antisense oligomer consisting of the nucleotide sequence of any of SEQ ID NOS: 1 to 89 and 91 to 93; (f) an antisense oligomer consisting of a nucleotide sequence in which 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 base is deleted and / or substituted with respect to the nucleotide sequence of any of SEQ ID NOS: 1 to 89 and 91 to 93, and which has the activity of inducing skipping of exon 51 of the human dystrophin gene; (g) an antisense oligomer consisting of a nucleotide sequence which has 80% or more, 84% or more, 85% or more, 89% or more, 90% or more, 94% or more, or 95% or more sequence identity to the nucleotide sequence of any of SEQ ID NOS: 1 to 89 and 91 to 93, and which has the activity of inducing skipping of exon 51 of the human dystrophin gene; and (h) an antisense oligomer that hybridizes under highly stringent conditions to an oligonucleotide consisting of a base sequence complementary to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and has the activity of inducing skipping of exon 51 of the human dystrophin gene.

[0022] The antisense oligomer of the present invention is more preferably any one of the antisense oligomers selected from the group consisting of (a2) to (d2) below: (a2) an antisense oligomer comprising any one of the nucleotide sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76; (b2) an antisense oligomer comprising a nucleotide sequence in which 1 to 5 nucleotides have been deleted, substituted, inserted, and / or added relative to any one of the nucleotide sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and which has the activity of inducing exon 51 skipping of the human dystrophin gene; (c2) an antisense oligomer comprising a nucleotide sequence having 80% or more sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and which has the activity of inducing exon 51 skipping of the human dystrophin gene; and (d2) An antisense oligomer selected from the group consisting of: an antisense oligomer that hybridizes under stringent conditions to an oligonucleotide consisting of a base sequence complementary to any of the base sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and that has the activity of inducing skipping of exon 51 of the human dystrophin gene.

[0023] The antisense oligomers (b1) to (d1), (f) to (h), and (b2) to (d2) above are specifically variants of the antisense oligomers (a1), (e), and (a2), respectively, and are intended to correspond to mutations (e.g., polymorphisms) in the patient's dystrophin gene.

[0024] However, the antisense oligomer of the present invention excludes (does not include) the antisense oligomer having the following base sequence described in WO 2015 / 137409:

[0025] As used herein, the term "antisense oligomer that hybridizes under stringent conditions" refers to an antisense oligomer obtained by colony hybridization, plaque hybridization, Southern hybridization, or the like, using as a probe, for example, all or part of an oligonucleotide consisting of a base sequence complementary to any of the base sequences of SEQ ID NOS: 1 to 89 and 91 to 93. Hybridization methods that can be used include those described in, for example, "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001" and "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997."

[0026] As used herein, "stringent conditions" may refer to low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Moderate stringency conditions" refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C, or 5x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formamide, and 42°C. "Highly stringent conditions" include, but are not limited to, the following: (1) 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, 50°C; (2) 0.2xSSC, 0.1% SDS, 60°C; (3) 0.2xSSC, 0.1% SDS, 62°C; (4) 0.2xSSC, 0.1% SDS, 65°C; or (5) 0.1xSSC, 0.1% SDS, 65°C. Under these conditions, the higher the temperature, the more efficiently antisense oligomers with higher sequence identity can be obtained. However, several factors, such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, can affect the stringency of hybridization. Those skilled in the art can achieve similar stringency by appropriately selecting these factors. Here, "sequence identity" refers to the identity of two nucleic acid pairs over the entire range of the base sequences being compared, and is expressed as the percentage (%) of matching bases in an optimal alignment of the base sequences created using a mathematical algorithm known in the technical field of the present invention. For example, an antisense oligomer consisting of a base sequence having "80% sequence identity" with respect to a 20-base antisense oligomer means an antisense oligomer having 16 or more identical bases to the 20-base antisense oligomer.

[0027] Sequence identity can be determined using FASTA (Science 227 (4693): 1435-1441, (1985)) or the BLAST (Basic Local Alignment Search Tool) algorithm by Carlin and Altschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993). Programs based on the BLAST algorithm, such as blastn, blastx, tblastn, and tblastx, have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using blastn, the parameters are, for example, score = 100 and wordlength = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

[0028] When a commercially available kit is used for hybridization, for example, the Alkphos Direct Labeling and Detection System (GE Healthcare) can be used. In this case, after incubation with the labeled probe overnight according to the protocol provided with the kit, the membrane is washed with a primary wash buffer containing 0.1% (w / v) SDS at 55°C, and the hybridized antisense oligomer can be detected. Alternatively, when a probe is prepared based on all or part of the base sequence complementary to any of the base sequences of SEQ ID NOS: 1 to 89 and 91 to 93, if the probe is labeled with digoxigenin (DIG) using a commercially available reagent (e.g., PCR Labeling Mix (Roche Diagnostics)), hybridization can be detected using a DIG Nucleic Acid Detection Kit (Roche Diagnostics).

[0029] Other hybridizable antisense oligomers include those that have 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 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 sequence identity to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93, when calculated using default parameters with homology search software such as FASTA or BLAST.

[0030] "Inducing skipping of the 51st exon of the human dystrophin gene" means that the antisense oligomer of the present invention binds to a site corresponding to exon 51 and / or its adjacent intron in a transcript (e.g., pre-mRNA) of the human dystrophin gene, thereby excluding exon 51 when the transcript is spliced; for example, in the case of a DMD patient in which exon 52 is deleted, the nucleotide sequence corresponding to the 5' end of exon 53 is linked to the nucleotide sequence corresponding to the 3' end of exon 50, resulting in the formation of a mature mRNA in which no codon frameshift has occurred.

[0031] Therefore, DMD patients who have a mutation in the dystrophin gene that is subject to skipping of exon 51 can be treated by skipping of exon 51. Examples of such DMD patients include DMD patients who have a frameshift mutation due to deletion of at least an exon near exon 51 and who have a dystrophin gene in which the amino acid reading frame is corrected by skipping of exon 51, and more specifically, examples of such DMD patients include DMD patients who have a frameshift mutation due to deletion of exons 13-50, 29-50, 40-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52, 52-63, etc. of the dystrophin gene.

[0032] Here, the term "binding" refers to the hybridization of the antisense oligomer of the present invention with a transcript of the human dystrophin gene under physiological conditions to form a double-stranded chain. The term "under physiological conditions" refers to conditions adjusted to pH, salt composition, and temperature similar to those in vivo. Examples of such conditions include a temperature of 25 to 40°C, preferably 37°C, a pH of 5 to 8, preferably pH 7.4, and a sodium chloride concentration of 150 mM.

[0033] Whether or not exon 51 skipping of the human dystrophin gene has occurred can be confirmed by introducing the antisense oligomer of the present invention into dystrophin-expressing cells (e.g., human rhabdomyosarcoma cells), RT-PCR amplifying the region surrounding exon 51 of the mRNA of the human dystrophin gene from the total RNA of the dystrophin-expressing cells, and performing nested PCR or sequence analysis on the PCR amplification product. The skipping efficiency (ES) (unit: %) can be calculated by recovering human dystrophin gene mRNA from test cells, measuring the polynucleotide amount "A" of the band in which exon 51 was skipped and the polynucleotide amount "B" of the band in which exon 51 was not skipped, and then calculating the skipping efficiency (ES) according to the following formula (1) based on the measured values ​​of "A" and "B." For calculation of skipping efficiency, reference can be made to International Publication No. 2012 / 029986.

[0034] ES=100×A / (A+B)...(1)

[0035] Preferably, the antisense oligomers of the present invention skip exon 51 with an efficiency of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0036] The antisense oligomer of the present invention preferably also has high solubility in physiological saline. Unlike antisense oligomers with low solubility, antisense oligomers with high solubility in physiological saline are extremely unlikely to precipitate and become unusable during storage of the formulation, and are also extremely unlikely to precipitate and become unusable when used in an infusion solution containing salt. Furthermore, because antisense oligomers with high solubility in physiological saline are less likely to precipitate, they are also extremely unlikely to exhibit toxicity upon administration (Bulletin of Osaka University of Pharmaceutical Sciences 1, 91-99 (2007)). Therefore, antisense oligomers with high solubility in physiological saline are highly useful as active ingredients in pharmaceuticals.

[0037] The solubility in physiological saline is preferably 20 mg / mL or more, more preferably 30 mg / mL or more, even more preferably 40 mg / mL or more, and particularly preferably 50 mg / mL or more. The solubility of an antisense oligomer in physiological saline can be evaluated by dissolving the antisense oligomer in physiological saline at a desired concentration and visually checking for the presence or absence of precipitation after a certain period of time.

[0038] In addition, antisense oligomer is preferably highly safe as the active ingredient of medicine.Safety can be evaluated, for example, by using the aspartate aminotransferase (AST) value, alanine aminotransferase (ALT) value, urea nitrogen (BUN) value and creatinine value in blood after antisense oligomer administration as index.AST value increases when liver damage occurs, ALT value increases when liver problems occur, BUN value increases when kidney function decreases, and creatinine value tends to increase when kidney glomerular filtration function decreases.Therefore, these values ​​can be used as index to evaluate the effect of antisense oligomer on kidney and liver function.

[0039] Specifically, for example, after administering antisense oligomer to healthy mice, measure the AST value, ALT value, BUN value and creatinine value in blood, and carry out statistical significance test.Compared with the measured value of control group (solvent administration or untreated), if there is a significant increase, it can be judged as abnormal value, and it can be judged that the administered antisense oligomer has an effect on kidney and liver function, or is highly likely to have an effect.On the other hand, if there is no significant increase, it can be judged that there is no effect on kidney and liver function, or is highly likely to have an effect.In addition, compared with the measured value of control group, if there is a certain increase rate, specifically, for example, an increase rate of 30% or more, it can be judged as abnormal value.

[0040] Examples of the antisense oligomer of the present invention include oligonucleotides, morpholino oligomers, and peptide nucleic acid (PNA) oligomers having a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases. The length of the antisense oligomer is preferably 20 to 30 bases, 20 to 29 bases, 22 to 30 bases, 22 to 29 bases, or 25 to 29 bases, more preferably 22 to 30 bases, 22 to 29 bases, or 25 to 29 bases, and morpholino oligomers are preferred.

[0041] The above-mentioned oligonucleotide (hereinafter referred to as "the oligonucleotide of the present invention") is an antisense oligomer of the present invention having nucleotides as its constituent units, and such nucleotides may be any of ribonucleotides, deoxyribonucleotides, or modified nucleotides.

[0042] A modified nucleotide refers to a ribonucleotide or deoxyribonucleotide in which all or part of the nucleic acid base, sugar moiety, and phosphate linkage moiety that constitute the ribonucleotide or deoxyribonucleotide have been modified.

[0043] In the present invention, examples of nucleic acid bases include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, and the like. Examples of amino acids that can be used include, but are not limited to, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, and xanthine.

[0044] Modifications of the sugar moiety include, for example, modification of the 2'-position of ribose and modifications of other parts of the sugar. Modifications of the 2'-position of ribose include, for example, replacing the —OH group at the 2'-position of ribose with OR, R, R'OR, SH, SR, NH ２ , N.H.R., N.R. ２ , N ３, CN, F, Cl, Br, or I. Here, R represents alkyl or aryl. R' represents alkylene. Modifications of other sugar moieties include, but are not limited to, substitution of O at the 4'-position of ribose or deoxyribose with S, and bridging of the 2' and 4' positions of the sugar, such as LNA (Locked Nucleic Acid) or ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids).

[0045] Modifications of the phosphate linkage moiety include, for example, substitution of the phosphodiester bond with a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, or a boranophosphate bond (Enya et al.: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (see, for example, Patent Republished Publications Nos. 2006 / 129594 and 2006 / 038608).

[0046] In the present invention, the alkyl is preferably a linear or branched alkyl having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, and isohexyl. The alkyl may be substituted, and examples of such substituents include halogen, alkoxy, cyano, and nitro, and the alkyl may be substituted with 1 to 3 of these. In the present invention, the cycloalkyl is preferably a cycloalkyl having 5 to 12 carbon atoms. Specific examples include cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, and cyclododecyl. In the present invention, the halogen may be fluorine, chlorine, bromine, or iodine. Examples of the alkoxy include straight-chain or branched-chain alkoxy having 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy, etc. In particular, alkoxy having 1 to 3 carbon atoms is preferred.

[0047] In the present invention, the aryl is preferably an aryl having 6 to 10 carbon atoms. Specific examples include phenyl, α-naphthyl, and β-naphthyl. Phenyl is particularly preferred. The aryl may be substituted, and examples of such substituents include alkyl, halogen, alkoxy, cyano, and nitro, and these may be substituted with 1 to 3 of them. In the present invention, the alkylene is preferably a linear or branched alkylene having 1 to 6 carbon atoms. Specific examples include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene. In the present invention, the acyl may be a linear or branched alkanoyl or aroyl. Examples of alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl, and hexanoyl. Examples of aroyl include benzoyl, toluoyl, and naphthoyl. Such aroyl may be substituted at any substitutable position, and may be substituted with alkyl.

[0048] The oligonucleotide of the present invention is preferably an antisense oligomer of the present invention having as a constituent unit a group represented by the following general formula, in which the —OH group at the 2'-position of ribose is substituted with methoxy and the phosphate linkage is a phosphorothioate linkage: (In the formula, Base represents a nucleic acid base.)

[0049] The oligonucleotides of the present invention can be easily synthesized using various automated synthesizers (e.g., AKTA Oligopilot Plus 10 / 100 (GE Healthcare)), or can be produced by outsourcing to a third party (e.g., Promega, Takara, or Japan Bioservices).

[0050] The morpholino oligomer of the present invention is an antisense oligomer of the present invention having a group represented by the following general formula as a constituent unit. (In the formula, Base has the same meaning as defined above; and W represents a group represented by any of the following formulae:

[0051] (Wherein, X is —CH ２ R １ , —O—CH ２ R １ , -S-CH ２ R １ , -NR ２ R ３ or F; R １ represents H or alkyl; R ２ and R ３ are the same or different and represent H, alkyl, cycloalkyl, or aryl; Y １ is O, S, CH ２ or NR １ represents Y ２ is O, S or NR １ Z represents O or S.

[0052] Examples of morpholino monomer compounds used in the synthesis of the morpholino oligomer of the present invention include, but are not limited to, morpholino monomer compound (A), morpholino monomer compound (C), morpholino monomer compound (T), and morpholino monomer compound (G) listed in the table below.

[0053]

[0054] The morpholino oligomer is preferably an oligomer having a group represented by the following formula as a constituent unit (phosphorodiamidate morpholino oligomer (hereinafter referred to as "PMO")). (In the formula, Base, R ２ , R ３ has the same meaning as above.) The morpholino oligomer of the present invention includes those in which the nucleic acid base, morpholino ring moiety, phosphate linkage moiety, 3' end and / or 5' end constituting the oligomer are modified in whole or in part.

[0055] Modifications of the phosphate linkage moiety include, for example, substitution with a phosphorodiamidate linkage, phosphorothioate linkage, phosphorodithioate linkage, alkylphosphonate linkage, phosphoramidate linkage, or boranophosphate linkage (Enya et al., Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (see, for example, Patent Republished Publications Nos. 2006 / 129594 and 2006 / 038608). Morpholino oligomers can be prepared, for example, according to WO 1991 / 009033 or WO 2009 / 064471. In particular, PMOs can be prepared according to the method described in WO 2009 / 064471 or the method described below.

[0056] [Method for Producing PMO] One embodiment of PMO is, for example, a compound represented by the following general formula (I) (hereinafter referred to as PMO(I)). [In the formula, each Base, R ２ , R ３ has the same meaning as above; n is an integer in the range of 1 to 99, preferably an integer in the range of 19 to 29, 19 to 28, 21 to 29, 21 to 28, or 24 to 28, and more preferably an integer in the range of 21 to 29, 21 to 28, or 24 to 28.

[0057] PMO (I) can be produced according to known methods, for example, by carrying out the operations of the following steps. The compounds and reagents used in the following steps are not particularly limited as long as they are commonly used in the production of PMOs. Furthermore, all of the following steps can be carried out by a liquid phase method or a solid phase method (manually or using a commercially available solid phase automated synthesizer). When producing PMO by the solid phase method, a method using an automated synthesizer is desirable from the standpoints of simplifying the operation procedure and ensuring accurate synthesis.

[0058] (1) Step A: A step of producing a compound represented by the following general formula (III) (hereinafter referred to as compound (III)) by allowing an acid to act on a compound represented by the following general formula (II) (hereinafter referred to as compound (II)). [In the formula, n, R ２ , R ３ has the same meaning as above; each B Ｐ each independently represents an optionally protected nucleobase; T represents a trityl group, a monomethoxytrityl group, or a dimethoxytrityl group; and L represents hydrogen, acyl, or a group represented by the following general formula (IV) (hereinafter referred to as group (IV)).

[0059] B Ｐ The "nucleobase" according to the present invention can be the same as the "nucleobase" of Base. Ｐ The amino group or hydroxyl group of the nucleic acid base in the above formula (1) may be protected. The protecting group for such an amino group is not particularly limited as long as it is used as a protecting group for nucleic acids, and specific examples thereof include benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl, and (dimethylamino)methylene. Examples of hydroxyl-protecting groups include 2-cyanoethyl, 4-nitrophenethyl, phenylsulfonylethyl, methylsulfonylethyl, trimethylsilylethyl, phenyl which may be substituted at any substitutable position with 1 to 5 electron-withdrawing groups, diphenylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, methylphenylcarbamoyl, 1-pyrrolidinylcarbamoyl, morpholinocarbamoyl, 4-(tert-butylcarboxy)benzyl, 4-[(dimethylamino)carboxy]benzyl, and 4-(phenylcarboxy)benzyl (see, for example, WO 2009 / 064471 ).

[0060] The "solid phase carrier" is not particularly limited as long as it is a carrier that can be used in solid phase reactions of nucleic acids, but for example, it is desirable that the carrier (i) is practically insoluble in reagents that can be used in the synthesis of morpholino nucleic acid derivatives (e.g., dichloromethane, acetonitrile, tetrazole, N-methylimidazole, pyridine, acetic anhydride, lutidine, trifluoroacetic acid), (ii) is chemically stable against reagents that can be used in the synthesis of morpholino nucleic acid derivatives, (iii) can be chemically modified, (iv) can be loaded with a desired morpholino nucleic acid derivative, (v) has sufficient strength to withstand high pressures applied during processing, and (vi) has a certain particle size range and distribution. Specifically, swelling polystyrene (e.g., aminomethyl polystyrene resin 1% divinylbenzene crosslinked (200-400 mesh) (2.4-3.0 mmol / g) (Tokyo Chemical Industry Co., Ltd.), Aminomethylated Polystyrene Resin.HCl [divinylbenzene 1%, 100-200 mesh] (Peptide Institute Co., Ltd.)), non-swelling polystyrene (e.g., Primer Support (GE Healthcare)), PEG chain-bonded polystyrene (e.g., NH ２ Examples of such supports include PEG resin (manufactured by Watanabe Chemical Co., Ltd.), TentaGel resin), controlled pore glass (CPG) (e.g., manufactured by CPG Corporation), oxalated controlled pore glass (see, for example, Allul et al., Nucleic Acids Research, Vol. 19, 1527 (1991)), TentaGel support-aminopolyethylene glycol derivatized support (see, for example, Wright et al., Tetrahedron Letters, Vol. 34, 3373 (1993)), and Poros-polystyrene / divinylbenzene copolymer. As the "linker," any known linker that is usually used to link nucleic acids or morpholino nucleic acid derivatives can be used, including, for example, 3-aminopropyl, succinyl, 2,2'-diethanolsulfonyl, and long chain alkylamino (LCAA).

[0061] This step can be carried out by reacting compound (II) with an acid.

[0062] Examples of the "acid" that can be used in this step include trifluoroacetic acid, dichloroacetic acid, and trichloroacetic acid. The amount of the acid used is, for example, within the range of 0.1 to 1,000 molar equivalents relative to 1 mole of compound (II), and preferably within the range of 1 to 100 molar equivalents. An organic amine can also be used together with the acid. The organic amine is not particularly limited, but examples include triethylamine. The amount of the organic amine used is, for example, within the range of 0.01 to 10 molar equivalents relative to 1 mole of the acid, and preferably within the range of 0.1 to 2 molar equivalents. When a salt or mixture of an acid and an organic amine is used in this step, examples include a salt or mixture of trifluoroacetic acid and triethylamine, and more specifically, a mixture of 2 equivalents of trifluoroacetic acid and 1 equivalent of triethylamine. The acid that can be used in this step can also be 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 is inert to the reaction, and examples thereof include dichloromethane, acetonitrile, alcohols (ethanol, isopropanol, trifluoroethanol, etc.), water, and mixtures thereof.

[0063] The reaction temperature in the above reaction is, for example, preferably in the range of 10°C to 50°C, more preferably in the range of 20°C to 40°C, and even 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 minute to 24 hours, preferably in the range of 1 minute to 5 hours.

[0064] Furthermore, after completion of this step, a base can be added, if necessary, to neutralize the acid present in the system. The "base" is not particularly limited, but examples thereof include diisopropylethylamine. The base can also be used after diluting with an appropriate solvent to a concentration within the range of 0.1% (v / v) to 30% (v / v). The solvent used in this step is not particularly limited as long as it is not involved in the reaction, but examples include dichloromethane, acetonitrile, alcohols (ethanol, isopropanol, trifluoroethanol, etc.), water, and mixtures thereof. The reaction temperature is preferably, for example, within the range of 10°C to 50°C, more preferably within the range of 20°C to 40°C, and even more preferably within 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 within the range of 0.1 minute to 24 hours, preferably within the range of 1 minute to 5 hours.

[0065] A compound represented by the following general formula (IIa), in which n=1 and L is group (IV) in compound (II) (hereinafter referred to as compound (IIa)), can be produced by the following method. [In the formula, B Ｐ , T, linker, and solid phase carrier are as defined above.]

[0066] Step 1: A step of producing a compound represented by the following general formula (VI) (hereinafter referred to as compound (VI)) by reacting an acylating agent with a compound represented by the following general formula (V). [In the formula, B Ｐ , T, and linker are as defined above; R ４ represents a hydroxyl group, a halogen, a carboxyl group, or an amino group.

[0067] This step can be carried out by a known linker introduction reaction using compound (V) as a starting material. In particular, a compound represented by the following general formula (VIa) can be produced by carrying out a method known as an esterification reaction using compound (V) and succinic anhydride. [In the formula, B Ｐ , T has the same meaning as above.]

[0068] Step 2: A step of reacting compound (VI) with a solid support by treating it with a condensing agent or the like to produce compound (IIa). [In the formula, B Ｐ , R ４ , T, linker, and solid support are as defined above.] This step can be carried out by a method known as a condensation reaction using compound (VI) and a solid support. A compound represented by the following general formula (IIa2), in which n=1 to 99 in compound (II) (in a specific embodiment, n is, for example, 2 to 29, 2 to 28, 2 to 27, 2 to 26, 2 to 25, 2 to 24, 2 to 23, 2 to 22, 2 to 21, or 2 to 20, preferably 19 to 29, 19 to 28, 21 to 29, 21 to 28, or 24 to 28, and more preferably 21 to 29, 21 to 28, or 24 to 28), and L is group (IV), can be produced by using compound (IIa) as a starting material and repeatedly carrying out step A and step B of the method for producing PMO described in this specification a desired number of times. [In the formula, B Ｐ , n, R ２ , R ３ , T, linker, and solid phase carrier are as defined above.]

[0069] (2) Step B: A step of producing a compound represented by the following general formula (VII) (hereinafter referred to as compound (VII)) by allowing a morpholino monomer compound to act on compound (III) in the presence of a base. [In the formula, each B Ｐ , L, n, R ２ , R ３ , T has the same meaning as above.]

[0070] This step can be carried out by reacting compound (III) with a morpholino monomer compound in the presence of a base.

[0071] The morpholino monomer compound may be, for example, a compound represented by the following general formula (VIII): [In the formula, B Ｐ , R ２ , R３ , and T has the same meaning as above.] Examples of the "base" that can be used in this step include diisopropylethylamine, triethylamine, and N-ethylmorpholine. The amount of base used is, for example, suitably within the range of 1 to 1,000 molar equivalents, and preferably within the range of 10 to 100 molar equivalents, relative to 1 mole of compound (III). The morpholino monomer compound and base that can be used in this step can also be diluted with an appropriate solvent to a concentration of 0.1% to 30% before use. The solvent is not particularly limited as long as it is not involved in the reaction, and examples include N,N-dimethylimidazolidone, N-methylpiperidone, DMF, dichloromethane, acetonitrile, tetrahydrofuran, and mixtures thereof.

[0072] The reaction temperature is, for example, preferably in the range of 0° C. to 100° C., more preferably in the range of 10° C. to 50° C. 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, preferably in the range of 30 minutes to 24 hours.

[0073] Furthermore, after completion of this step, an acylating agent can be added, if necessary. Examples of "acylating agents" include acetic anhydride, acetic acid chloride, and phenoxyacetic anhydride. The acylating agent can also be used after diluting it with an appropriate solvent to a concentration within 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, and examples include dichloromethane, acetonitrile, tetrahydrofuran, alcohols (ethanol, isopropanol, trifluoroethanol, etc.), water, and mixtures thereof. If necessary, a base such as pyridine, lutidine, collidine, triethylamine, diisopropylethylamine, or N-ethylmorpholine can be used together with the acylating agent. The amount of the acylating agent used is preferably within the range of 0.1 to 10,000 molar equivalents, and more preferably within the range of 1 to 1,000 molar equivalents. The amount of base used is, for example, suitably within the range of 0.1 to 100 molar equivalents, and preferably within the range of 1 to 10 molar equivalents, relative to 1 mole of the acylating agent. The reaction temperature for this reaction is preferably within the range of 10 to 50°C, more preferably within the range of 10 to 50°C, more preferably within the range of 20 to 40°C, and even more preferably within the range of 25 to 35°C. The reaction time varies depending on, for example, the type of acylating agent used and the reaction temperature, but is usually suitably within the range of 0.1 minute to 24 hours, and preferably within the range of 1 minute to 5 hours.

[0074] (3) Step C: A step of removing the protecting group from compound (VII) produced in step B using a deprotecting agent to produce a compound represented by general formula (IX). [In the formula, Base, B Ｐ , L, n, R ２ , R ３ , T has the same meaning as above.]

[0075] This step can be carried out by reacting compound (VII) with a deprotecting agent.

[0076] Examples of the "deprotecting agent" include concentrated aqueous ammonia and methylamine. The "deprotecting agent" that can be used in this step can be diluted with, for example, water, methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran, DMF, N,N-dimethylimidazolidone, N-methylpiperidone, or a mixed solvent thereof. Of these, ethanol is preferred. The amount of the deprotecting agent used is, for example, appropriately within the range of 1 to 100,000 molar equivalents, and preferably within the range of 10 to 1,000 molar equivalents, relative to 1 mole of compound (VII).

[0077] The reaction temperature is suitably within the range of, for example, 15° C. to 75° C., preferably within the range of 40° C. to 70° C., and more preferably within the range of 50° C. to 60° C. The deprotection reaction time varies depending on the type of compound (VII), the reaction temperature, etc., but is suitably within the range of 10 minutes to 30 hours, preferably within the range of 30 minutes to 24 hours, and more preferably within the range of 5 hours to 20 hours.

[0078] (4) Step D: A step of preparing PMO (I) by reacting compound (IX) prepared in Step C with an acid. [where Base, n, R ２ , R ３ , T has the same meaning as above.]

[0079] This step can be carried out by adding an acid to compound (IX).

[0080] Examples of the "acid" that can be used in this step include trichloroacetic acid, dichloroacetic acid, acetic acid, phosphoric acid, and hydrochloric acid. The amount of acid used is, for example, such that the pH of the solution 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 not particularly limited as long as it is not involved in the reaction, and examples thereof include acetonitrile, water, and a mixed solvent thereof.

[0081] The reaction temperature is preferably within the range of 10° C. to 50° C., more preferably within the range of 20° C. to 40° C., and even more preferably within the range of 25° C. to 35° C. The deprotection reaction time varies depending on the type of compound (IX), the reaction temperature, etc., but is suitably within the range of 0.1 minute to 5 hours, preferably within the range of 1 minute to 1 hour, and more preferably within the range of 1 minute to 30 minutes.

[0082] PMO (I) can be separated and purified from the reaction mixture obtained in this step by conventional separation and purification means, such as extraction, concentration, neutralization, filtration, centrifugation, recrystallization, C ８ From C １８ The desired PMO (I) can be isolated and purified by using techniques such as reverse-phase column chromatography, cation exchange column chromatography, anion exchange column chromatography, gel filtration column chromatography, high-performance liquid chromatography, dialysis, and ultrafiltration, either alone or in combination (see, for example, International Publication No. 1991 / 09033). When purifying PMO (I) using reverse-phase chromatography, a mixed solution of 20 mM triethylamine / acetic acid buffer and acetonitrile can be used as the elution solvent. When purifying PMO (I) using ion exchange chromatography, a mixed solution of 1 M saline and 10 mM aqueous sodium hydroxide can be used.

[0083] Peptide nucleic acids are antisense oligomers of the present invention that have as their constituent units groups represented by the following general formula: (In the formula, Base has the same meaning as defined above.)

[0084] Peptide nucleic acids can be produced, for example, according to the following documents: 1) PE Nielsen, M. Egholm, RH Berg, O. Buchardt, Science, 254, 1497 (1991) 2) M. Egholm, O. Buchardt, PE Nielsen, RH Berg, Jacs., 114, 1895 (1992) 3) KL Dueholm, M. Egholm, C. Behrens, L. Christensen, HF Hansen, T. Vulpius, KH Petersen, RH Berg, PE Nielsen, O. Buchardt, J. Org. Chem., 59, 5767 (1994) 4) L. Christensen, R. Fitzpatrick, B. Gildea, KH Petersen, HF Hansen, T. Koch, M. Egholm, O. Buchardt, PE Nielsen, J. Coull, R.H. Berg, J. Pept. Sci., 1, 175 (1995) 5) T. Koch, HF Hansen, P. Andersen, T. Larsen, HG Batz, K. Otteson, H. Orum, J. Pept. Res., 49, 80 (1997)

[0085] Furthermore, the 5'-end of the antisense oligomer of the present invention may be any of the groups represented by the following chemical formulae (1) to (3), preferably (3) -OH. Hereinafter, the groups represented by (1), (2) and (3) above will be referred to as "group (1)," "group (2)" and "group (3)," respectively.

[0086] The antisense oligomers of the present invention may include compounds in which the phosphorus atom in the phosphate binding moiety is an asymmetric center and the stereochemistry of the phosphorus atom is optically pure. Those skilled in the art can obtain pure optically active compounds from mixtures of isomers (WO 2017 / 024264). Alternatively, the antisense oligomers of the present invention may be synthesized as pure optically active compounds. Those skilled in the art can control the synthesis reaction to obtain pure optically active compounds (Patent Publication No. 2018-537952).

[0087] 2. Peptide-linked antisense oligomers The antisense oligomers of the present invention may be conjugated with functional peptides (e.g., membrane-permeable peptides for improving transport efficiency into target cells) to improve efficacy (WO 2008 / 036127, WO 2009 / 005793, WO 2012 / 150960, WO 2016 / 187425, WO 2018 / 118662, WO 2018 / 118599, WO 2018 / 118627, JD Ramsey, NH Flynn, Pharmacology & Therapeutics 154, 78-86 (2015), MK Tsoumpra et al., EBioMedicine, 45, 630-645 (2019)). The binding site is not particularly limited, but preferably the 5' or 3' end of the antisense oligomer is bound to the amino or carboxyl end of the functional peptide. In another embodiment, the antisense oligomer of the present invention and the functional peptide may form a complex via a linker. The linker is not particularly limited, but preferably the 5' or 3' end of the antisense oligomer is bound to one end of the linker, and the amino or carboxyl end of the functional peptide is bound to the other end of the linker. An additional amino acid may be present between the functional peptide and the linker.

[0088] 3. Pharmaceutical Compositions The antisense oligomers of the present invention can induce exon 51 skipping with high efficiency, even when their length is shorter than that of conventional antisense oligomers. Furthermore, the antisense oligomers of the present invention have excellent solubility while maintaining the activity of inducing exon 51 skipping with high efficiency. Furthermore, the antisense oligomers of the present invention have excellent solubility and safety while maintaining the activity of inducing exon 51 skipping with high efficiency. Therefore, it is expected that administration of the antisense oligomers of the present invention to DMD patients who have a mutation in the dystrophin gene that is subject to exon 51 skipping (e.g., a frameshift mutation, a missense mutation / nonsense mutation in exon 51, etc.) can highly efficiently alleviate the symptoms of muscular dystrophy. For example, it is expected that administration of the antisense oligomers of the present invention to DMD patients who have a specific mutant dystrophin gene lacking at least an exon near exon 51 can highly efficiently alleviate the symptoms of muscular dystrophy. The predetermined mutant dystrophin gene refers to a dystrophin gene that has a frameshift mutation due to a deletion of at least an exon near exon 51, and in which the amino acid reading frame is corrected when exon 51 is omitted (skipped). Examples of DMD patients include DMD patients with a frameshift mutation due to a deletion of exons 13-50, 29-50, 40-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52, 52-63, etc.More specifically, it is predicted that administration of a pharmaceutical composition containing the antisense oligomer of the present invention to DMD patients (patients with an in-frame mutation due to exon 51 skipping, for example, patients with exon 13-50 deletion, patients with exon 29-50 deletion, patients with exon 40-50 deletion, patients with exon 43-50 deletion, patients with exon 45-50 deletion, patients with exon 47-50 deletion, patients with exon 48-50 deletion, patients with exon 49-50 deletion, patients with exon 50 deletion, patients with exon 52 deletion, patients with exon 52-63 deletion, etc.) will enable highly efficient alleviation of the symptoms of muscular dystrophy. For example, when using a pharmaceutical composition containing the antisense oligomer of the present invention, a therapeutic effect comparable to that of oligomers according to conventional techniques can be obtained even at a lower dose, thereby reducing side effects and being more economical. Furthermore, the antisense oligomers of the present invention are useful in the preparation of pharmaceutical compositions because they maintain the activity of inducing exon 51 skipping with high efficiency and have excellent solubility. Furthermore, the antisense oligomers of the present invention are useful as pharmaceutical compositions because they maintain the activity of inducing exon 51 skipping with high efficiency and have excellent solubility and safety. Therefore, in another embodiment, a pharmaceutical composition for treating muscular dystrophy (hereinafter referred to as the "composition of the present invention") containing the antisense oligomer of the present invention or a pharmaceutically acceptable salt or hydrate thereof as an active ingredient is provided. The present invention also provides a method for treating muscular dystrophy, comprising administering the antisense oligomer of the present invention to a DMD patient. In this method of treatment, the antisense oligomer of the present invention may be administered as the pharmaceutical composition for treating muscular dystrophy. Furthermore, the present invention provides use of the antisense oligomer of the present invention in the preparation of a pharmaceutical composition for treating muscular dystrophy, and the antisense oligomer of the present invention for use in treating muscular dystrophy.

[0089] Examples of pharmaceutically acceptable salts of the antisense oligomers of the present invention contained in the compositions of the present invention include alkali metal salts such as sodium salt, potassium salt, and lithium salt, and alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; ammonium salt; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, and the like. Examples of suitable salts include organic amine salts such as amine salts, piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; hydrohalide salts such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonate salts such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonate salts such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. These salts can be prepared by known methods. Alternatively, the antisense oligomer of the present invention contained in the composition of the present invention may be in the form of a hydrate.

[0090] The administration form of the composition of the present invention is not particularly limited as long as it is a pharmaceutically acceptable administration form and can be selected depending on the treatment method, but from the viewpoint of ease of delivery to muscle tissue, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, oral administration, intratissue administration, transdermal administration, etc. Furthermore, the dosage form that the composition of the present invention can take is not particularly limited, and examples thereof include various injections, oral preparations, infusions, inhalants, ointments, lotions, etc.

[0091] When the antisense oligomer of the present invention is administered to a patient with muscular dystrophy, the composition of the present invention may contain a carrier that promotes delivery of the oligomer to muscle tissue. Such a carrier is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include cationic carriers such as cationic liposomes and cationic polymers, or carriers that utilize viral envelopes. Examples of cationic liposomes include liposomes formed from 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids as essential components (hereinafter referred to as "Liposome A"), Oligofectamine (registered trademark) (manufactured by Invitrogen), Lipofectin (registered trademark) (manufactured by Invitrogen), Lipofectamine (registered trademark) (manufactured by Invitrogen), Lipofectamine 2000 (registered trademark) (manufactured by Invitrogen), DMRIE-C (registered trademark) (manufactured by Invitrogen), GeneSilencer (registered trademark) (manufactured by Gene Therapy Systems), TransMessenger (registered trademark) (manufactured by QIAGEN), and TransIT Examples of suitable carriers include TKO (registered trademark) (Mirus) and Nucleofector II (Lonza). Of these, Liposome A is preferred. Examples of cationic polymers include JetSI (registered trademark) (Qbiogene) and Jet-PEI (registered trademark) (polyethyleneimine, Qbiogene). Examples of carriers utilizing viral envelopes include GenomeOne (registered trademark) (HVJ-E liposome, Ishihara Sangyo Kaisha). Alternatively, the pharmaceutical device described in Japanese Patent No. 2924179 and the cationic carriers described in Japanese Patent Republication Nos. 2006 / 129594 and 2008 / 096690 can also be used. For details, reference can be made to U.S. Pat. Nos. 4,235,871 and 4,737,323, International Publication No. 96 / 14057, and "New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990) pages 33-104," etc.

[0092] The concentration of the antisense oligomer of the present invention contained in the composition of the present invention varies depending on the type of carrier, etc., but in one embodiment, it is suitably in the range of 0.1 nM to 100 μM, and preferably in the range of 100 nM to 10 μM. Furthermore, the weight ratio of the antisense oligomer of the present invention to the carrier contained in the composition of the present invention (carrier / antisense oligomer of the present invention) varies depending on the properties of the oligomer, the type of carrier, etc., but it is suitably in the range of 0.1 to 100, and preferably in the range of 0.1 to 10.

[0093] The composition of the present invention may be in the form of an aqueous solution, and in that case, the composition of the present invention may contain the antisense oligomer of the present invention at a concentration of 2.5 to 500 mg / mL, 5 to 450 mg / mL, 10 to 400 mg / mL, 15 to 350 mg / mL, 20 to 300 mg / mL, 20 to 250 mg / mL, 20 to 200 mg / mL, 20 to 150 mg / mL, 20 to 100 mg / mL, 20 to 50 mg / mL, 20 to 40 mg / mL, 20 to 30 mg / mL, 23 to 27 mg / mL, 24 to 26 mg / mL, or 25 mg / mL. Alternatively, the composition of the present invention may contain the antisense oligomer of the present invention at a concentration of 10-100 mg / mL, 15-95 mg / mL, 20-80 mg / mL, 25-75 mg / mL, 30-70 mg / mL, 35-65 mg / mL, 40-60 mg / mL, 45-55 mg / mL, 47-53 mg / mL, 48-52 mg / mL, 49-51 mg / mL, or 50 mg / mL.

[0094] The composition of the present invention may be in a dry form. In this case, to prepare an aqueous solution of the composition of the present invention, for example, a dry form of the composition of the present invention containing 125 mg or 250 mg of the antisense oligomer of the present invention in a dry form may be mixed with 0.5 mL to 100 mL of water (equivalent to a concentration of the antisense oligomer of the present invention of 1.25 mg / mL to 250 mg / mL or 2.5 mg / mL to 500 mg / mL), preferably 1 mL to 50 mL of water (equivalent to a concentration of the antisense oligomer of the present invention of 2.5 mg / mL to 125 mg / mL or 5 mg / mL to 250 mg / mL), more preferably 5 mL to 10 mL of water (equivalent to a concentration of the antisense oligomer of the present invention of 12.5 mg / mL to 25 mg / mL or 25 mg / mL to 50 mg / mL).

[0095] In addition to the antisense oligomer of the present invention and the carrier described above, the composition of the present invention can optionally contain pharmaceutically acceptable additives. Examples of such additives include emulsifiers (e.g., fatty acids having 6 to 22 carbon atoms or pharmaceutically acceptable salts thereof, albumin, and dextran), stabilizers (e.g., cholesterol, phosphatidic acid, sucrose, mannitol, sorbitol, and xylitol), isotonicity agents (e.g., sodium chloride, glucose, maltose, lactose, sucrose, trehalose, mannitol, sorbitol, and xylitol), and pH adjusters (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, and triethanolamine). These additives can be used alone or in combination. The content of the additive in the composition of the present invention is preferably 90% by weight or less, more preferably 70% by weight or less, and even more preferably 50% by weight or less.

[0096] The composition of the present invention can be prepared by adding the antisense oligomer of the present invention to a dispersion of a carrier and stirring appropriately. The additives can be added at any suitable stage, either before or after the addition of the antisense oligomer of the present invention. When the composition of the present invention is in the form of an aqueous solution, the aqueous solvent that can be used when adding the antisense oligomer of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution. In such cases, conditions such as pH and temperature can be appropriately selected by those skilled in the art.

[0097] The composition of the present invention can be, for example, a liquid formulation or a lyophilized formulation thereof. As one embodiment of the dried form of the composition of the present invention, the lyophilized formulation can be prepared by lyophilizing the composition of the present invention in liquid form using conventional methods. For example, after appropriate sterilization of the composition of the present invention in liquid form, a predetermined amount can be dispensed into vials, pre-frozen at approximately −40 to −20°C for about 2 hours, primary dried under reduced pressure at approximately 0 to 10°C, and then secondary dried under reduced pressure at approximately 15 to 25°C, thereby achieving lyophilization. The interior of the vial can then generally be purged with nitrogen gas and the vial can be stoppered to obtain a lyophilized formulation of the composition of the present invention.

[0098] The lyophilized formulation of the composition of the present invention can generally be reconstituted and used by adding any appropriate solution (reconstitution liquid). Examples of such reconstitution liquid include water for injection, physiological saline, and other general infusion solutions. The volume of this reconstitution liquid varies depending on the intended use and is not particularly limited, but is suitably 0.5 to 2 times the volume of the liquid before lyophilization, or 500 mL or less.

[0099] The dosage of the composition of the present invention is preferably determined taking into consideration the type of antisense oligomer of the present invention contained therein, the dosage form, the patient's condition, such as age and weight, the route of administration, and the nature and severity of the disease. The amount of the antisense oligomer of the present invention for an adult is generally in the range of 0.1 mg to 10 g per person per day, preferably 1 mg to 1 g per person. This value may vary depending on the type of target disease, the administration form, and the target molecule. Therefore, in some cases, a lower dose may be sufficient, while in other cases a higher dose may be required. The composition can be administered once or several times a day, or at intervals of one day to several days.

[0100] Another embodiment of the composition of the present invention is a pharmaceutical composition comprising a vector capable of expressing an oligonucleotide of the present invention and the above-described carrier. Such an expression vector may be capable of expressing multiple oligonucleotides of the present invention. Similar to the composition of the present invention containing the oligomer of the present invention, pharmaceutically acceptable additives may be added to the composition. The concentration of the expression vector contained in the composition varies depending on the type of carrier, etc., but in one embodiment, it is suitably in the range of 0.1 nM to 100 μM, preferably in the range of 100 nM to 10 μM. The weight ratio of the expression vector to the carrier contained in the composition (carrier / expression vector) varies depending on the properties of the expression vector, the type of carrier, etc., but is suitably in the range of 0.1 to 100, preferably in the range of 0.1 to 10. The content of the carrier contained in the composition is the same as in the composition of the present invention containing the antisense oligomer of the present invention, and the preparation method, etc. are also the same as in the composition of the present invention.

[0101] The present invention will be explained in more detail below with reference to examples and test examples, but the present invention is not limited to the scope shown in the examples.

[0102] Example 1: Synthesis of antisense oligomers Antisense oligomers (PMO Nos. 1 to 93 (SEQ ID NOs: 1 to 93)) shown in Table 1 were synthesized according to the method described in International Publication No. 2015 / 137409, targeting a portion of the base sequence of exon 51 of the human dystrophin gene and / or its 5'-adjacent intron, intron 50. The table also shows the theoretical molecular weight of each antisense oligomer and the actual measured value by ESI-TOF-MS. In Table 1, for example, "H51_67-81_131-142" indicates that the antisense oligomer targets the sequence of bases 67 to 81 and the sequence of bases 131 to 142, when the base at the 5' end of exon 51 of the human dystrophin gene is designated as the first base and the bases subsequent to the 3' end are numbered sequentially. The sequence of bases before -1 in the target base sequence is the base sequence in intron 50. A base sequence including the sequence of exon 51 of the human wild-type dystrophin gene and the sequence near the 3' end of intron 50 is shown in SEQ ID NO: 128.

[0103] [Example 2: Exon skipping activity test of antisense oligomers] In vitro test of exon 51 skipping of the human dystrophin gene (1) Test method RD cells (human rhabdomyosarcoma cell line, CCL-136, purchased from ATCC) 3.5 x 10 ５ Each cell was transfected with 0.3 to 120 μM of each of the antisense oligomers in Tables 1 and 2 using Nucleofector II (Lonza) with Amaxa Cell Line Nucleofector Kit L. The pulse program used for transfection was T-030.

[0104] The transfected RD cells were cultured in 2 mL of Eagle's minimal essential medium (EMEM) (Sigma, the same applies below) containing 10% fetal bovine serum (FBS) (Invitrogen) at 37°C and 5% CO ２The cells were cultured under these conditions for three nights. After culturing, the RD cells were washed once with PBS (Nissui, hereinafter the same), and then 350 μL of Buffer RA1 (Takara Bio) containing 1% 2-mercaptoethanol (Nacalai Tesque) was added to the cells. The cells were left at room temperature for several minutes to lyse the cells, and then collected on a NucleoSpin® Filter (Takara Bio). A homogenate was prepared by centrifugation at 11,000 × g for 1 minute. Total RNA was extracted according to the protocol attached to the NucleoSpin® RNA (Takara Bio). The concentration of the extracted total RNA was measured using a NanoDrop ONE (Thermo Fisher).

[0105] One-Step RT-PCR was performed on 400 ng of extracted total RNA using the QIAGEN OneStep RT-PCR Kit (Qiagen) and a thermal cycler. The reaction mixture was prepared according to the protocol attached to the kit. The thermal cycler used was the TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio). The RT-PCR program used was as follows: 50°C, 30 minutes: reverse transcription; 95°C, 15 minutes: polymerase activation, reverse transcriptase inactivation, cDNA thermal denaturation [94°C, 30 seconds; 60°C, 30 seconds; 72°C, 1 minute] x 35 cycles: PCR amplification; 72°C, 10 minutes: final extension reaction

[0106] The nucleotide sequences of the forward and reverse primers used in RT-PCR are as follows: Forward primer: 5'-CTGAGTGGAAGGCGGTAAAC-3' (SEQ ID NO: 95) Reverse primer: 5'-GAAGTTTCAGGGCCAAGTCA-3' (SEQ ID NO: 96)

[0107] 1 μL of the PCR reaction product was analyzed using a Bioanalyzer (Agilent) or a MultiNA (Shimadzu). The polynucleotide amount "A" of the band in which exon 51 was skipped and the polynucleotide amount "B" of the band in which exon 51 was not skipped were measured as band signal intensities. Based on the measured values ​​of "A" and "B," the skipping efficiency was calculated according to the above-mentioned formula (1).

[0108] (2) Test Results The results of exon 51 skipping efficiency obtained for each antisense oligomer are shown in Figures 1 to 18. As a direct or indirect control, an antisense oligomer (PMO No. 94 (SEQ ID NO: 94)) shown in Table 2 was synthesized according to the method described in JP 2015-91229 A. The antisense oligomers of the present invention shown in Table 1 had significantly higher skipping efficiency than the antisense oligomers shown in Table 2 that have the same overall structure as eteplirsen (WHO Drug Information 24, 2, 137-139 (2010), Proposed INN List 103), an exon 51 skipping drug. The oligomers have the same 5'-terminal modification, i.e., the 5'-terminal has the above-described group (1), and therefore have the same overall structure. The oligomers were used.

[0109] [Example 3: Solubility test of antisense oligomers] Solubility test of antisense oligomers in physiological saline To further verify the usefulness for pharmaceutical applications of each of the antisense oligomers PMO Nos. 7, 8, 10, 16, 21, 24, 31, 42, 67, 76, and 90, which showed very high skipping efficiency in Example 2, a solubility test in physiological saline was performed.

[0110] (1) Test Method: 57 μL of water for injection was added to a sample vial containing 5.7 mg of each antisense oligomer, and the solution was dissolved using ultrasound and a vortex mixer. 57 μL of double-strength saline was then added and stirred using a vortex mixer to obtain a 50 mg / mL saline solution. The solution was left to stand at room temperature for 24 hours, after which the presence or absence of precipitation was visually confirmed. Antisense oligomers that showed no precipitation were evaluated as having high solubility.

[0111] (2) Test Results Of the antisense oligomers tested, PMO Nos. 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76 exhibited a solubility of 50 mg / mL or more in physiological saline. These results demonstrate that these antisense oligomers have a significantly high exon 51 skipping efficiency and high solubility in physiological saline, making them highly useful as pharmaceuticals.

[0112] [Example 4: Safety evaluation of antisense oligomers] Of the antisense oligomers for which the solubility test in physiological saline was carried out in Example 3, PMO Nos. 16, 21, 42, and 90 were subjected to a safety evaluation to verify their safety for pharmaceutical use.

[0113] (1) Evaluation Method: Each antisense oligomer was dissolved in saline and administered into the tail vein of 6-week-old male C57BL / 6N mice. The following day, serum was collected from the mice, and blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine levels were measured. Measurements from control groups of mice administered only saline as vehicle or untreated mice were considered normal values. A statistical significance test (Student's t-test or Dunnett's test) was performed, and values ​​significantly elevated at a significance level of p<0.05 in the groups administered with each antisense oligomer were deemed abnormal. Each test was performed using the statistical analysis system SAS (registered trademark, SAS Institute) version 9.3. When no abnormal values ​​were observed in any of the AST, ALT, BUN and creatinine levels at a dose of 1000 mg / kg, the antisense oligomer was determined to be highly safe.

[0114] (2) Evaluation Results For the antisense oligomers PMO No. 16, 21, and 42, no abnormal values ​​were observed in any of the AST, ALT, BUN, and creatinine values ​​at a dose of 1000 mg / kg, confirming that they are highly safe antisense oligomers (specifically, have no effect on kidney and liver function or are highly unlikely to have an effect). The results are shown in Figures 19 to 21. Values ​​that were significantly elevated at a significance level of p<0.05 (abnormal values) are indicated by p-values.

[0115] The above results demonstrate that the antisense oligomer of the present invention exhibits the activity of inducing exon 51 skipping of the dystrophin gene with high efficiency, and also has excellent physical properties and safety as a pharmaceutical.

[0116] SEQ ID NOs: 1 to 126: synthetic nucleic acids

Claims

1. The following (a1) to (d1): (a1) an antisense oligomer containing any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93; (b1) an antisense oligomer containing a base sequence in which 1 to 5 bases are deleted, substituted, inserted, and / or added with respect to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and having an activity of inducing skipping of exon 51 of the human dystrophin gene; (c1) an antisense oligomer containing a base sequence having 80% or more sequence identity with respect to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and having an activity of inducing skipping of exon 51 of the human dystrophin gene; and (d1) an antisense oligomer that hybridizes under stringent conditions with an oligonucleotide consisting of a base sequence complementary to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and having an activity of inducing skipping of exon 51 of the human dystrophin gene, provided that it is selected from the group consisting of antisense oligomers (excluding antisense oligomers consisting of any of the base sequences of SEQ ID NOs: 90 and 97 to 126) or pharmaceutically acceptable salts thereof or hydrates thereof.

2. The following (e) to (h): (e) an antisense oligomer consisting of any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93; (f) consisting of a nucleotide sequence in which 1 to 5 bases are deleted and / or substituted with respect to any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and having an activity of inducing skipping of exon 51 of the human dystrophin gene; (g) consisting of a nucleotide sequence having 80% or more sequence identity with respect to any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and having an activity of inducing skipping of exon 51 of the human dystrophin gene; and (h) an antisense oligomer that hybridizes under highly stringent conditions with an oligonucleotide consisting of a nucleotide sequence complementary to any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and having an activity of inducing skipping of exon 51 of the human dystrophin gene, an antisense oligomer selected from the group consisting of (excluding antisense oligomers consisting of any of the nucleotide sequences of SEQ ID NOs: 90 and 97 to 126) or a pharmaceutically acceptable salt thereof or a hydrate thereof.

3. The antisense oligomer according to claim 1 or 2, which has a nucleotide sequence having 90% or more sequence identity with respect to any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93, and has an activity of inducing skipping of exon 51 of the human dystrophin gene, or a pharmaceutically acceptable salt thereof or a hydrate thereof.

4. The antisense oligomer is selected from the group consisting of: (a2) an antisense oligomer containing the base sequence of any one of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76; (b2) an antisense oligomer containing a base sequence in which 1 to 5 bases are deleted, substituted, inserted, and / or added with respect to the base sequence of any one of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and having an activity of inducing skipping of exon 51 of the human dystrophin gene; (c2) an antisense oligomer containing a base sequence having 80% or more sequence identity with respect to the base sequence of any one of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and having an activity of inducing skipping of exon 51 of the human dystrophin gene; and (d2) an antisense oligomer that hybridizes under stringent conditions with an oligonucleotide consisting of a base sequence complementary to the base sequence of any one of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67, and 76, and having an activity of inducing skipping of exon 51 of the human dystrophin gene. The antisense oligomer according to claim 1, or a pharmaceutically acceptable salt thereof or a hydrate thereof.

5. The antisense oligomer according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof or a hydrate thereof, which is an oligonucleotide.

6. The antisense oligomer according to claim 5, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the sugar moiety and / or phosphate bond moiety of at least one nucleotide constituting the oligonucleotide is modified.

7. The sugar moiety of at least one nucleotide constituting the oligonucleotide has an -OH group at the 2'-position replaced by any group selected from the group consisting of OR, R, R'OR, SH, SR, NH ２ , NHR, NR ２ , N ３ , CN, F, Cl, Br and I (wherein R represents alkyl or aryl, and R' represents alkylene), which is ribose substituted with any of the groups selected from the group consisting of: The antisense oligomer according to claim 5 or 6, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.

8. The antisense oligomer according to any one of claims 5 to 7, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the phosphate bond moiety of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, and a boranophosphate bond.

9. The antisense oligomer according to any one of claims 1 to 4, which is a morpholino oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.

10. The antisense oligomer according to claim 9, which is a phosphorodiamidate morpholino oligomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.

11. The 5'-end is one of the following chemical formulas (1) to (3): The antisense oligomer according to claim 9 or 10, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the 5'-end is one of the following chemical formulas (1) to (3):

12. A pharmaceutical composition for treating muscular dystrophy, comprising the antisense oligomer according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.

13. The pharmaceutical composition according to claim 12, further comprising a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 12 or 13 for administration to a patient with muscular dystrophy, wherein the patient has a mutation targeted for exon 51 skipping in the dystrophin gene.

15. The pharmaceutical composition according to claim 14, wherein the patient has a dystrophin gene with a frameshift mutation caused by deletion of at least an exon near exon 51 and the amino acid reading frame is corrected by exon 51 skipping.

16. The pharmaceutical composition according to claim 14 or 15, wherein the patient has a frameshift mutation caused by deletion of exons 13 - 50, 29 - 50, 40 - 50, 43 - 50, 45 - 50, 47 - 50, 48 - 50, 49 - 50, 50, 52, 52 - 63 in the dystrophin gene.

17. The pharmaceutical composition according to any one of claims 14 to 16, wherein the patient is a human.

18. Use of the antisense oligomer according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, in the manufacture of a medicament for treating muscular dystrophy.

19. A method for treating muscular dystrophy, comprising the step of administering to a patient with muscular dystrophy an effective amount of the antisense oligomer according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or the pharmaceutical composition according to any one of claims 12 to 16.

20. The treatment method according to claim 19, wherein the patient is a human.

21. An antisense oligomer according to any one of claims 1 to 11, or a pharmaceutically acceptable salt or hydrate thereof, or a pharmaceutical composition according to any one of claims 12 to 16, for use in the treatment of muscular dystrophy.

22. The antisense oligomer according to claim 21, or a pharmaceutically acceptable salt or hydrate thereof, or a pharmaceutical composition, wherein the muscular dystrophy patient is a human in said treatment.