Antisense nucleic acid

Abstract

To provide an agent for highly efficiently skipping an exon.SOLUTION: The present invention provides an antisense oligomer in which two or more unit oligomers targeting sequences that are not contiguous or overlapping with each other in the same exon are linked.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a method for detecting a target exon comprising a nucleotide sequence complementary to two or more different sequences within the target exon. More specifically, the present invention relates to an antisense oligomer for exon skipping. Antisense oligonucleotides that allow skipping of the 44th exon of the strophin gene The present invention relates to oligomers and pharmaceutical compositions containing said oligomers. [Background technology]

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

[0003] DMD is known to be caused by mutations in the dystrophin gene. The gene is present on the X chromosome and is a huge gene consisting of 2.2 million bases of DNA. It is transcribed into a precursor, and then introns are removed by splicing to combine 79 exons. The resulting mRNA is 13,993 bases long. This mRNA is translated into 3,685 amino acids, which then become dystrophin. The dystrophin protein is involved in maintaining membrane stability in muscle cells. The dystrophin gene in DMD patients is essential for protecting muscle cells from damage. Because of the mutation, functional dystrophin protein is barely expressed in muscle cells. Therefore, in DMD patients, the structure of muscle cells cannot be maintained, and a large amount of calcium is released. As a result, an inflammation-like reaction occurs, and fibrosis progresses. Muscle cells become less able to regenerate.

[0004] Becker muscular dystrophy (BMD) is also caused by mutations in the dystrophin gene, Although the symptoms are muscle weakness, they are generally milder than DMD and the progression of muscle weakness is slower. In most cases, the onset occurs in adulthood. The clinical difference between DMD and BMD is the mutation that causes dystrophic The amino acid reading frame during translation of dystrophin mRNA into the dystrophin protein is disrupted. It is believed that this depends on whether the D In MD, mutations that shift the amino acid reading frame result in functional dystrophy. Although the protein is barely expressed, in BMD, part of the exon is deleted due to the mutation. However, the amino acid reading frame is maintained, so the dystrophy is functional, albeit incomplete. Fin proteins are produced.

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

[0006] Exon skipping can involve skipping either the 5' or 3' splice site or both, or This can be induced by binding of antisense nucleic acids that target the inside of the exon. The splice site is only recognized by the spliceosome complex when both splice sites are integrated into the mRNA. Therefore, targeting splice sites with antisense nucleic acids can This can induce exon skipping. The exon splicing enhancer (ESE) must have a serine and a nucleotide sequence to be recognized by the exon splicing enhancer (ESE). It is believed that binding of arginine-rich SR proteins is required to target ESEs. Exon skipping can also be induced by activating the nucleotide sequence.

[0007] The location and type of dystrophin gene mutations vary among DMD patients. Antisense nucleic acids corresponding to the single exon of the dystrophin gene are required. , antisense nucleic acids that target a single continuous sequence and induce exon skipping There have been several reports on this (Patent Documents 1 to 6 and Non-Patent Documents 1 and 2). Two types of antisense nucleic acids that target the same exon of the HIV gene are mixed and acted upon. (dual targeting) enhances skipping activity compared to when each antisense nucleic acid is used alone. It has been reported that this may occur (Patent Document 7).

[0008] However, linked single-stranded antisense nucleic acids targeting two or more sites within the same exon It has not yet been reported that an acid (linked type) exhibits skipping activity (Patent Document 1). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2004 / 048570 [Patent Document 2] International Publication No. 2009 / 139630 [Patent Document 3] International Publication No. 2010 / 048586 [Patent Document 4] U.S. Patent Publication No. 2010 / 0168212 [Patent Document 5] International Publication No. 2011 / 057350 [Patent Document 6] International Publication No. 2006 / 000057 [Patent Document 7] International Publication No. 2007 / 135105 [Non-patent literature]

[0010] [Non-Patent Document 1] Annemieke Aartsma-Rus et al., (2002) Neuromuscular Disorders 12: S71-S77 [Non-patent document 2] Wilton SD, et al., Molecular Therapy 2007: 15: p. 1288-96 Summary of the Invention [Problem to be solved by the invention]

[0011] In the above situation, the present invention provides a method for identifying a dystrophin gene having a different exon in the same exon. A novel linked antisense molecule that targets two base sequences to induce exon skipping The main objective of the present invention is to provide a muscular dystrophy treatment agent containing the oligomer. Let's say. [Means for solving the problem]

[0012] The present inventors have detailed the technical details and the structure of the dystrophin gene described in the above documents. As a result of this research, we have developed an opioid that targets two different regions of exon 44 of the human dystrophin gene. Antisense oligomers obtained by linking ligomers induce skipping of the same exon. Based on this finding, the present inventors have completed the present invention.

[0013] That is, the present invention is as follows. [1] (a) A base sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases in the target exon. a first unit oligomer comprising a string; and (b) bases complementary to a second nucleotide sequence of 7 to 15 consecutive bases within the target exon; a second unit oligomer comprising the sequence an antisense oligomer having a length of 15 to 30 bases, The first and second nucleotide sequences are contiguous or overlap each other. isn't it, an antisense oligomer or a pharmaceutical composition thereof that induces skipping of the target exon; A pharmaceutically acceptable salt or hydrate. [2] The first and / or second unit oligomers are introductory sequences adjacent to the target exon. The antisense molecule according to [1] above, which contains a base sequence complementary to a partial nucleotide sequence of the The oligomer, or a pharmaceutically acceptable salt or hydrate thereof. [3] [1] or [2], wherein the target exon is an exon of the human dystrophin gene. 1. An antisense oligomer according to claim 1, or a pharmaceutically acceptable salt or hydrate thereof. [4] The first nucleotide sequence is a contiguous sequence selected from the nucleotide sequence shown in SEQ ID NO:1. The antisense oligonucleotide according to [1] or [2] above, which is a nucleotide sequence of 7 to 15 bases. GOMER, or a pharmaceutically acceptable salt or hydrate thereof. [5] The second nucleotide sequence is a contiguous sequence selected from the nucleotide sequence shown in SEQ ID NO:2. The amplicon according to any one of [1] to [3] above, which is a nucleotide sequence of 7 to 15 bases. A thysene oligomer, or a pharmaceutically acceptable salt or hydrate thereof. [6] Two unit oligomers selected from the group consisting of (c) to (e) below are linked together. The antisense oligomer according to [1] or [2] above, which is: (c) a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence shown in SEQ ID NO: 3; a unit oligomer consisting of a base sequence complementary to the base sequence; (d) a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence shown in SEQ ID NO: 4; a unit oligomer consisting of a base sequence complementary to the base sequence; and (e) a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence shown in SEQ ID NO: 5; A unit oligomer consisting of a base sequence complementary to the base sequence, or a pharmaceutically acceptable salt thereof Salt or hydrate. [7] The above [1] or [2], which consists of any one of the base sequences selected from the group consisting of SEQ ID NOs: 6 to 9. The antisense oligomer according to [2], or a pharmaceutically acceptable salt or water thereof Japanese food. [8] The antisense oligonucleotide according to any one of [1] to [7] above, Ligomer, or a pharmaceutically acceptable salt or hydrate thereof. [9] The sugar moiety and / or linker of at least one nucleotide constituting the oligonucleotide The antisense oligomer according to [8] above, wherein the phosphate binding moiety is modified, or A pharmaceutically acceptable salt or hydrate of

[10] The sugar moiety of at least one nucleotide constituting the oligonucleotide , the -OH group at the 2' position is selected from OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br and I [8] or [9], wherein the ribose is substituted with any group selected from the group consisting of 1. An antisense oligomer according to claim 1, or a pharmaceutically acceptable salt or hydrate thereof. (The above R represents alkyl or aryl, and the above R' represents alkylene.)

[11] The phosphate bond of at least one nucleotide constituting the oligonucleotide The bond portion is a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, bond, a phosphoramidate bond, and a boranophosphate bond. The antisense oligonucleotide according to any one of [8] to

[10] above, Ligomer, or a pharmaceutically acceptable salt or hydrate thereof.

[12] The anion according to any one of [1] to [7], which is a morpholino oligomer. A thysene oligomer, or a pharmaceutically acceptable salt or hydrate thereof.

[13] The anthraquinone according to

[12] , which is a phosphorodiamidate morpholino oligomer. A thysene oligomer, or a pharmaceutically acceptable salt or hydrate thereof.

[14] The above

[12] or

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

[13] The antisense oligomer according to

[13] , or a pharmaceutically acceptable salt or hydrate thereof. thing. [ka]

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

[14] above, or A pharmaceutical composition for treating muscular dystrophy, comprising a pharmaceutically acceptable salt or hydrate thereof as an active ingredient. Pharmaceutical composition.

[16] The pharmaceutical composition according to

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

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

[12] above, or a pharmaceutical agent thereof or a physiologically acceptable salt or hydrate thereof, or the pharmaceutical composition according to [1] or

[16] . A method for treating muscular dystrophy, comprising administering the composition to a patient suffering from muscular dystrophy.

[18] The patient with muscular dystrophy had exon 44 skipping in the dystrophin gene. The method of treatment according to

[17] above, wherein the patient has the target mutation.

[19] The method of treatment according to

[17] or

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

[20]

[0033] In the manufacture of a pharmaceutical composition for treating muscular dystrophy, any one of [1] to

[14] above is used. Use of the antisense oligomer according to any one of claims 1 to 4, or a pharmaceutically acceptable salt or hydrate thereof For. [twenty one] The compound according to any one of [1] to

[14] above for use in treating muscular dystrophy. An antisense oligomer, or a pharmaceutically acceptable salt or hydrate thereof. [twenty two] In the treatment, a patient with muscular dystrophy has exon 44 skipping in the dystrophin gene. The antisense oligonucleotide according to

[21] above is for a patient having a mutation that is a target of the antisense oligonucleotide. or a pharmaceutically acceptable salt or hydrate thereof. [twenty three] The antisense oligomer according to

[21] or

[22] above, wherein the patient is a human; and is a pharmaceutically acceptable salt or hydrate thereof. [twenty four] (a) A base sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases in the target exon. a first unit oligomer comprising a string; and (b) bases complementary to a second nucleotide sequence of 7 to 15 consecutive bases within the target exon; a second unit oligomer comprising the sequence and preparing an antisense oligomer having a length of 15 to 30 bases by linking the wherein the first nucleotide sequence and the second nucleotide sequence are contiguous or overlap each other. It is not something that A method for producing the antisense oligomer described in [1] above. [twenty five] measuring the skipping efficiency of the antisense oligomer obtained in the above step; and selecting antisense oligomers having skipping efficiencies exceeding a reference value; The method according to

[24] , further comprising:

[26] (a)(i) a base sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases within the target exon; a first unit oligomer comprising a group sequence; and (ii) a second nucleotide sequence complementary to a contiguous sequence of 7 to 15 bases within the target exon; a second unit oligomer containing a base sequence a step of selecting a sequence of the first nucleotide sequence and the second nucleotide sequence, or are not overlapping with each other), (b) Linking the first and second unit oligomers to form an antisense oligonucleotide having a length of 15 to 30 bases. generating a sense oligomer; (c) measuring the skipping efficiency of the antisense oligomer obtained in step (b); , and (d) selecting antisense oligomers having skipping efficiencies exceeding a reference value; , A method for screening an antisense oligomer, comprising: [Effects of the Invention]

[0014] The antisense oligomer of the present invention targets exon 44 of the human dystrophin gene. It is possible to induce kipping with high efficiency. This can effectively alleviate the symptoms of Duchenne muscular dystrophy. . [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 2] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 3] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 4] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 5] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 6] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 7] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 8] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 9] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 10] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 11] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 12] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 13] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 14] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 15] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 16] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 17] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 18] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 19] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 20] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 21] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 22] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 23] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 24] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 25] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 26] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene (by oligomer concentration) in human rhabdomyosarcoma cells (RD cells). [Figure 27] This figure compares the efficiency of exon 44 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells) between a combination of two unit oligomers targeting different sites and a mixture of them. [Figure 28] This figure compares the efficiency of exon 44 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells) between a combination of two unit oligomers targeting different sites and a mixture of them. [Figure 29] This figure compares the efficiency of exon 44 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells) between a combination of two unit oligomers targeting different sites and a mixture of them. [Figure 30] This figure compares the efficiency of exon 44 skipping in the human dystrophin gene in human rhabdomyosarcoma cells (RD cells) when two unit oligomers targeting different sites are used alone, linked together, or mixed together. [Figure 31] This figure compares the efficiency of exon 44 skipping in the human dystrophin gene in human rhabdomyosarcoma cells (RD cells) when two unit oligomers targeting different sites are used alone, linked together, or mixed together. [Figure 32] FIG. 1 shows the efficiency of exon 44 skipping of the human dystrophin gene in exon 45-deficient DMD patient-derived cells. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below. The following embodiments are examples for explaining the present invention. The present invention is not intended to be limited to the embodiments. Unless otherwise specified, it can be implemented in various forms. All documents cited in this specification, as well as publications, patent publications and other patents, The documents are incorporated herein by reference. Details of the Japanese patent application (Patent Application No. 2014-124157) on which the present application claims priority This includes the contents described in the documents and drawings.

[0017] 1. Antisense oligomers The present invention provides a method for detecting a target exon comprising: (a) detecting a first nucleotide sequence of 7 to 15 consecutive bases in a target exon; a first unit oligomer containing a base sequence consistent with the present invention; and (b) bases complementary to a second nucleotide sequence of 7 to 15 consecutive bases within the target exon; a second unit oligomer comprising the sequence an antisense oligomer having a length of 15 to 30 bases, The first and second nucleotide sequences are contiguous or overlap each other. an antisense oligomer that induces skipping of the target exon, but is not provides pharmaceutically acceptable salts or hydrates thereof. Hereinafter, "antisense oligomer, or a pharmaceutically acceptable salt or hydrate thereof" will be referred to. are sometimes collectively referred to simply as "antisense oligomers."

[0018] The antisense oligomer is (a) A base sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases within the target exon. a first unit oligomer comprising: (b) bases complementary to a second nucleotide sequence of 7 to 15 consecutive bases within the target exon; a second unit oligomer comprising the sequence and preparing an antisense oligomer having a length of 15 to 30 bases by linking the wherein the first nucleotide sequence and the second nucleotide sequence are contiguous or overlap each other. It can be produced by a manufacturing method that is not intended to be used. The above-mentioned production method is carried out by measuring the skipping efficiency of the antisense oligomer obtained in the above-mentioned step. determining the The second step involves selecting antisense oligomers with skipping efficiencies exceeding the reference value. The course, It may further include:

[0019] In the second step of the production method, the skipping efficiency is evaluated by mRNA is collected from the test cells, and the polynucleotides of the band in which the target exon was skipped are extracted from the mRNA. Nucleotide content "A" and polynucleotides of bands where the target exon was not skipped Measure the quantity "B" and calculate it based on the measurements of "A" and "B" according to the following formula: It is possible. Skipping efficiency (%) = A / ( A + B ) x 100 For calculation of skipping efficiency, see WO 2012 / 029986 It is also possible.

[0020] In the second step, the skipping efficiency as a standard value is 10% or more, 20% or more, 30% or more. Above, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and 90% or more. By linking a plurality of unit oligomers in this way, the Even if each of the skipping activities is low (or non-existent), skipping This results in an antisense oligomer with improved binding activity.

[0021] Similarly, (a)(i) a base sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases within the target exon; a first unit oligomer comprising a group sequence; and (ii) a second nucleotide sequence complementary to a contiguous sequence of 7 to 15 bases within the target exon; a second unit oligomer containing a base sequence a step of selecting a sequence of the first nucleotide sequence and the second nucleotide sequence, or are not overlapping with each other), (b) Linking the first and second unit oligomers to form an antisense oligonucleotide having a length of 15 to 30 bases. generating a sense oligomer; (c) measuring the skipping efficiency of the antisense oligomer obtained in step (b); , and (d) selecting antisense oligomers having skipping efficiencies exceeding a reference value; , A method for screening an antisense oligomer is provided, comprising:

[0022] In the antisense oligomer, the first and second unit oligomers are both may be linked at the 5' or 3' end, but in one embodiment, the first unit The first unit oligomer is located on the 5' side, and the second unit oligomer is located on the 3' side and ligated. The antisense oligomer is a nucleotide sequence of the third of 7 to 15 consecutive bases in the target exon. The third unit oligomer may contain a base sequence complementary to the nucleotide sequence of good.

[0023] Here, "linkage" refers to whether two unit oligomers are directly linked or linked via an intermediate. When two unit oligomers are directly linked, the bond is 5. The 3' end of the unit oligomer located on the 3' end side and the unit oligomer located on the 3' end side The 5' end of the intermediate forms a phosphate bond or the following group: In addition to nucleic acids (chains) of 1 to 5 residues, ordinary nucleic acids and morpholino nucleic acid derivatives can be linked. Known compounds that are used can be used, for example, 3-aminopropyl, succinyl , 2,2'-diethanolsulfonyl, long chain alkylamino (LCAA) can be done. [ka] (Wherein, X is —OH, —CHR 1 , -O-CH2R 1 , -S-CH2R 1 , -NR 2 R 3 or represents F; R 1 represents H, alkyl; R 2 and R 3 are the same or different and represent H, alkyl, cycloalkyl, or aryl. death; Y1 is 0, S, CH2 or NR 1 represents; Y2 is 0, S or NR 1 represents; Z represents 0 or S.

[0024] The first and / or second unit oligomers are introductory sequences adjacent to the target exon. The nucleotide sequence may be complementary to a partial nucleotide sequence of the first The first unit oligomer is located on the 5' side, and the second unit oligomer is located on the 3' side. In this embodiment, the 5' side of the first unit oligomer is connected to the 5' side of the target exon. a base sequence complementary to the nucleotide sequence around the 3' end of the intron adjacent to and / or an introductory sequence adjacent to the 3' side of the target exon on the 3' side of the second unit oligomer. The nucleotide sequence may contain a base sequence complementary to the nucleotide sequence surrounding the 5' end of the gene.

[0025] The first and / or second unit oligomers are exon splices of the target exon. Complementary to a partial nucleotide sequence of the exonic splicing enhancer (ESE) The nucleic acid sequence may include a base sequence similar to that of the nucleic acid sequence of the present invention.

[0026] The target exon is not particularly limited, but in one embodiment is an exon of a human gene. and an exon of the human dystrophin gene. More specifically, it is exon 44 of the human dystrophin gene. Thus, in one embodiment, the present invention provides a method for the production of a dystrophin gene comprising the 44th exon of the human dystrophin gene. The antisense oligomer capable of skipping the target sequence (hereinafter referred to as "the oligomer of the present invention") The structure of the antisense oligomer of the present invention is described in detail below. Reveal.

[0027] [Exon 44 of the human dystrophin gene] In the present invention, the term "gene" includes not only genomic genes but also cDNA, mRNA precursors, and mRNA. Preferably, the gene is a precursor to mRNA, i.e., pre-mRNA. In the human genome, the human dystrophin gene is located at locus Xp21.2. The strophin gene is 3.0 Mbp in size, the largest known human gene. However, the coding region of the human dystrophin gene is only 14 kb, The coding region is distributed within the dystrophin gene as 79 exons (Roberts, RG., et al., Genomics, 16: 536-538 (1993)). A pre-mRNA is spliced ​​to generate a mature mRNA of 14 kb. The nucleotide sequence of the trophin gene is known (GenBank Accession No. NM_004006). The nucleotide sequence of exon 44 of the wild-type human dystrophin gene is shown in SEQ ID NO:10.

[0028] In one embodiment, the oligomer of the invention is an oligomer that encodes an exon of the human dystrophin gene. Skipping of 44 allows the protein encoded by the DMD-type dystrophin gene to be expressed in BMD. It was created with the aim of modifying the dystrophin protein into a dystrophin-like protein. The exon of the dystrophin gene that is the target of exon skipping by the oligomer of the present invention is Son44 includes not only wild-type but also mutant types. Specifically, exon 44 of the mutant human dystrophin gene is (I) or (I It is a polynucleotide according to I). (I) A polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 10 and a string a polynucleotide that hybridizes under favorable conditions; (II) A polynucleotide having a base sequence having 90% or more identity to the base sequence of SEQ ID NO: 10. nucleotide As used herein, the term "polynucleotide" refers to DNA or RNA. As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to a polynucleotide that hybridizes under stringent conditions. For example, the entire polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 10 Alternatively, a portion of the DNA may be used as a probe for colony hybridization or plaque hybridization. Polypeptides obtained by using methods such as Southern hybridization or Southern hybridization. The hybridization method is described, for example, in "Sambrook & R ussell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, La. boratory Press 2001" and "Ausubel, Current Protocols in Molecular Biology, John W. The method described in "Billey & Sons 1987-1997" can be used. In the present specification, the term "complementary base sequence" refers to a base sequence that is complementary to the target base sequence and has a Watson-Crick pair structure. It is not limited to base sequences that form a wobble base pair. Watson-Crick pairs include adenine-thymine and adenine- The base pairs formed by hydrogen bonds between guanine and uracil and between guanine and cytosine are called fluctuating base pairs. The base pairs are guanine-uracil, inosine-uracil, inosine-adenine and inosine-cysteine. The term "complementary base sequence" refers to a base pair in which a hydrogen bond is formed between the nucleotides. It is not necessary to have 100% complementarity with the target base sequence. For example, The sequence may contain 1 to 3, 1 to 2, or 1 non-complementary base. As used herein, "stringent conditions" refers to low stringent conditions, medium stringent conditions, The conditions may be either low stringency or high stringency. "Optimal conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, 32 The "moderate stringent conditions" are, for example, conditions of 5x SSC, 5x denaturing acid, and 5x ethanol. soln, 0.5% SDS, 50% formamide, 42°C or 5x SSC, 1% SDS, 50 mM Tris-HCl ( The conditions are pH 7.5, 50% formamide, and 42°C. For example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, 50°C or 0.2x SSC, 0 The conditions were 0.1% SDS and 65°C. Under these conditions, the higher the temperature, the higher the identity. However, it is expected that polynucleotides that can be obtained efficiently will be hybridized. Factors that affect the stringency of the reaction include temperature, probe concentration, and probe length. Several factors, such as the temperature, ionic strength, time, and salt concentration, can be considered. Similar stringency can be achieved by appropriately selecting When using a commercially available kit for hybridization, for example, Alkphos Direct The Labelling and Detection System (GE Healthcare) can be used. Incubation with the labeled probe was performed according to the protocol provided with the kit. After overnight washing, the membrane was washed with the first washing buffer containing 0.1% (w / v) SDS at 55°C. After washing, the hybridized polynucleotides can be detected. When preparing a probe based on all or part of the base sequence complementary to the base sequence of No. 10 Using commercially available reagents (e.g., PCR Labeling Mix (Roche Diagnostics)), When the probe was labeled with digoxigenin (DIG), a DIG nucleic acid detection kit (Roche) was used. Hybridization can be detected using a ELISA kit (Diagnos). Polynucleotides other than the above hybridizable polynucleotides include homologous polynucleotides. The results were calculated using the BLAST software, which is a serologic search software, with default parameters. When the sequence of the polynucleotide of SEQ ID NO: 10 is detected, the sequence is 90% or more, 91% or more, 92% or more, or 93% or more. % 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 a polynucleotide having 99.9% or more identity thereto. The identity of the base sequences was evaluated using the BLAST algorithm by Carlin and Arthur (Bas ic Local Alignment Search Tool)(Proc. Natl. Acad. Sci. USA 872264-2268, 1990; P roc Natl Acad Sci USA 90: 5873, 1993). Programs called BLASTN and BLASTX based on this method have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing nucleotide sequences using BLASTN, use the parameter For example, the score is 100 and the word length is 12. When used, the default parameters of each program are used.

[0029] Specifically, the oligomer of the present invention comprises two units selected from the group consisting of the following (a) and (b): It is an antisense oligomer of 15 to 30 bases in length, consisting of a knit oligomer linked together. (a) a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence shown in SEQ ID NO: 1; a unit oligomer consisting of a base sequence complementary to the base sequence; and (b) a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence shown in SEQ ID NO: 2; A unit oligomer consisting of a base sequence complementary to the base sequence For example, the first nucleotide sequence is selected from the nucleotide sequence set forth in SEQ ID NO:1. and / or the second nucleotide may be a nucleotide sequence of 7 to 15 consecutive bases. The nucleotide sequence is a sequence of 7 to 15 consecutive nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 2. It may also be a nucleotide sequence. Preferably, the oligomer of the present invention comprises two selected from the group consisting of the following (c) to (e): It is an antisense oligomer of 15 to 30 bases in length, in which unit oligomers are linked. (c) a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence shown in SEQ ID NO: 3; a unit oligomer consisting of a sequence complementary to the nucleotide sequence; (d) a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence shown in SEQ ID NO: 4; a unit oligomer consisting of a sequence complementary to the sequence of the nucleotide; and (e) a nucleotide sequence of 7 to 15 consecutive bases selected from the nucleotide sequence shown in SEQ ID NO: 5; A unit oligomer consisting of a sequence complementary to the

[0030] Here, the nucleotide sequences shown in SEQ ID NOs: 1 and 2 correspond to the wild-type dystrophin gene of humans. The nucleotide sequence of exon 44 of the child (SEQ ID NO: 10) is: The sequence consisting of bases 1 to 44 and the sequence consisting of bases 58 to 115. SEQ ID NO: 3 The nucleotide sequence shown in Figure 1 is the nucleotide sequence of exon 44 of the human wild-type dystrophin gene. It is a sequence consisting of the 18th to 34th bases counting from the 5' end of the nucleotide sequence (SEQ ID NO: 10). Similarly, the nucleotide sequences shown in SEQ ID NOs: 4 and 5 respectively consist of the nucleotides 61 to 77 of and a sequence consisting of the 88th to 104th bases.

[0031] Each of the unit oligomers (a) to (e) above (hereinafter, sometimes simply referred to as "unit") The size of the nucleic acid fragment is 7 to 15 bases long, preferably 8 to 15 bases long, 9 to 15 bases long, 10 to 15 bases long, The lengths of the units (a) to (e) are 10-14 bases, 10-13 bases, and 11-13 bases. The sizes may be the same or different.

[0032] When selecting two unit oligomers from the group consisting of (a) and (b), The oligomers may be a combination of the same unit oligomers or may be a combination of different unit oligomers. That is, the two unit oligomers may be a combination of (a) and (a) or a combination of (a) and (b), or a combination of (a) and (b). stomach. In addition, when selecting two unit oligomers from the group consisting of (c) to (e), The oligomers may be combinations of the same unit oligomers or may be combinations of different units. Although it may be a combination of oligomers, preferably, one of each type of unit is selected. For example, if one unit chooses (c), the other unit can choose (d) or (e). ) is preferable. Similarly, if unit (d) is selected for one, the other unit is It is preferable that the unit be (c) or (e). If the unit (e) is selected for one of the two, the other Preferably, the unit is (c) or (d).

[0033] When units (a) and (b) are selected, which of the two selected units is the 5' end? However, if (a) and (b) are selected, unit (a) should be located at the 3' end. It is preferably connected to the side. If you select two units from (c) to (e), which of the two selected units is It may be located on the 5' end, but if (c) and (d) are selected, unit (c) If (d) and (e) are selected, unit (d) is linked to the 3' end. If (c) and (e) are selected, unit (c) is linked to the 3' end. It is preferable that

[0034] Here, "connection" means two units selected from (a) and (b), or (c) to (e). This means that the two units selected are directly connected. are linked, the 3' end of the unit located on the 5' end side and the This means that the 5' end of the unit forms a phosphate bond or the following group. [ka] (Wherein, X is —OH, —CHR 1 , -O-CH2R 1 , -S-CH2R 1 , -NR2 R 3 or represents F; R 1 represents H, alkyl; R 2 and R 3 are the same or different and represent H, alkyl, cycloalkyl, or aryl. death; Y1 is 0, S, CH2 or NR 1 represents; Y2 is 0, S or NR 1 represents; Z represents 0 or S.

[0035] "Enables skipping of exon 44 of the human dystrophin gene" , a region corresponding to exon 44 of the transcript (e.g., pre-mRNA) of the human dystrophin gene When the oligomer of the present invention binds to the transcript, For example, in a DMD patient with a deletion in exon 45, the nucleotide sequence corresponding to the 3' end of exon 43 The base sequence corresponding to the 5' end of exon 46 is linked to the nucleotide sequence, causing a codon frameshift. This means that untranslated mature mRNA is formed.

[0036] Here, the "binding" refers to the binding of the oligomer of the present invention to a transcript of the human dystrophin gene. When these two are mixed, they hybridize to form a double strand under physiological conditions. The above "physiological conditions" refers to conditions adjusted to pH, salt composition, and temperature similar to those in the body. For example, the temperature is 25 to 40°C, preferably 37°C, and the pH is 5 to 8, preferably 7.4. For example, a sodium chloride concentration of 150 mM is used.

[0037] Whether exon 44 skipping of the human dystrophin gene occurs or not is a dystrophin-related disorder. The oligomer of the present invention is introduced into a dysprotein-expressing cell (e.g., a human rhabdomyosarcoma cell), and the dysprotein is expressed in the dysprotein-expressing cell. From the total RNA of dystrophin-expressing cells, exon 44 of the mRNA of the human dystrophin gene was identified. The surrounding region is amplified by RT-PCR, and the PCR amplification product is subjected to nested PCR or sequence analysis. The skipping efficiency can be confirmed by The mRNA was collected from the test cells, and the polynucleotides in the band in which exon 44 was skipped were analyzed. The polynucleotide amount of the band in which exon 44 was not skipped is "A" and the polynucleotide amount of the band in which exon 44 was not skipped is "B". " and calculate the value of "A" and "B" according to the following formula based on the measured values ​​of "A" and "B". can. Skipping efficiency (%) = A / ( A + B ) x 100 For calculation of skipping efficiency, see WO 2012 / 029986 It is also possible.

[0038] Preferably, the antisense oligomer of the present invention has a nucleotide sequence of 10% or more, 20% or more, 30% or more, 40% or more, %, 50%, 60%, 70%, 80%, or 90% or more of the target exons (e.g., For example, skipping exon 44.

[0039] The antisense oligomer of the present invention may be, for example, an oligonucleotide having a length of 15 to 30 bases. oligonucleotides, morpholino oligomers, or peptide nucleic acids (PNA) ) oligomers. Preferably, the antisense oligomers of the present invention are 16-30 bases, 17-30 bases, 18-30 bases, 19-30 bases, 20-30 bases, 20-29 bases, 20-28 bases 20 to 27 bases, 20 to 26 bases, 21 to 26 bases, or 22 to 26 bases in length, Preferably it is a copolymer.

[0040] The oligonucleotide (hereinafter referred to as "the oligonucleotide of the present invention") is The oligomer of the present invention is composed of nucleotides as building blocks, and such nucleotides are ribonucleic acids. The nucleotides may be nucleotides, deoxyribonucleotides or modified nucleotides.

[0041] Modified nucleotides are nucleotides that are ribonucleotides or deoxyribonucleotides. It refers to a compound in which all or part of the acid-base, sugar moiety, and phosphate binding moiety are modified.

[0042] Examples of nucleic acid bases include adenine, guanine, hypoxanthine, cytosine, and thymine. Examples of such modified bases include uracil, uracil, and modified bases thereof. For example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g. 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halo Uracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), uracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydrogen (hydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil hydroxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-di Methylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyl Adenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylamino 5-methylmethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl 2-methylthiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetone Acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, Examples of suitable amines include, but are not limited to, benzophenone, imidazole, xanthine, etc. stomach.

[0043] Modifications of the sugar moiety include, for example, modification of the 2'-position of ribose and modifications of other parts of the sugar. Examples of modifications at the 2'-position of ribose include -O at the 2'-position of ribose. Modifications that replace H groups with OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, and I are listed. Here, R represents alkyl or aryl, and R' represents alkylene. Modifications of other sugar moieties include, for example, O at the 4' position of ribose or deoxyribose. The sugars are substituted with S, and the 2' and 4' positions of the sugars are linked together. For example, LNA (Locked Nucleic Acid Examples include ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acid) and ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acid). However, the present invention is not limited to the above.

[0044] Modification of the phosphate bond moiety includes, for example, changing the phosphodiester bond to a phosphorothioate bond. bond, phosphorodithioate bond, alkylphosphonate bond, phosphoramidate bond , boranophosphate bond (Enya et al: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (for example, Patent Republished Publication No. 2006 (See Nos. 2006 / 129594 and 2006 / 038608).

[0045] The alkyl is preferably a linear or branched alkyl having 1 to 6 carbon atoms. is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- Butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl The alkyl may be substituted, and such substitution may be Examples of the group include halogen, alkoxy, cyano, and nitro. These may be substituted by 1 to 3 of these. The cycloalkyl is preferably a cycloalkyl having 5 to 12 carbon atoms. For example, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl Examples include cyclododecyl and cyclododecyl. Examples of halogen include fluorine, chlorine, bromine, and iodine. The alkoxy includes linear or branched alkoxy having 1 to 6 carbon atoms, for example, methoxy. ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy si, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, iso hexyloxy, etc. In particular, alkoxy having 1 to 3 carbon atoms is preferred. . The aryl is preferably an aryl having 6 to 10 carbon atoms. Examples of the alkyl group include phenyl, α-naphthyl, and β-naphthyl. Phenyl is particularly preferred. The aryl may be substituted, and such substituents include, for example, alkyl, halogen, and the like. Examples of the substituted alkyl include alkoxy, cyano, and nitro. good. The alkylene is preferably a linear or branched alkylene having 1 to 6 carbon atoms. Specifically, for example, methylene, ethylene, trimethylene, tetramethylene, pentamethylene , hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene. The acyl may be a straight-chain or branched-chain alkanoyl or aroyl. Examples of the alkanoyl include formyl, acetyl, 2-methyl acetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, penta Examples of aroyl include 2,2-dimethylpropionyl, hexanoyl, and the like. Examples of such aroyls include benzoyl, toluoyl, and naphthoyl. may be substituted at substitutable positions, and may be substituted with alkyl.

[0046] The oligonucleotide of the present invention preferably has a structure in which the -OH group at the 2'-position of ribose is substituted with methoxy. The phosphate bond moiety is a phosphorothioate bond. The oligomer of the present invention has the following structural units. [ka] (In the formula, Base represents a nucleic acid base.)

[0047] The oligonucleotides of the present invention can be synthesized using various automated synthesizers (e.g., AKTA oligopilot plus 1 0 / 100 (GE Healthcare)) or can be easily synthesized by a third party. Alternatively, the preparation can be outsourced to a research institution (for example, Promega or Takara).

[0048] The morpholino oligomer is an oligomer of the present invention having a group represented by the following general formula as a constituent unit. It's Gomer. [ka] (wherein Base has the same meaning as defined above; W represents a group represented by any of the following formulas: [ka] (Wherein, X is —CHR 1 , -O-CH2R 1 , -S-CH2R 1 , -NR 2 R 3 or represents F; R 1 represents H, alkyl; R 2 and R 3 are the same or different and represent H, alkyl, cycloalkyl, or aryl. death; Y1 is 0, S, CH2 or NR 1 represents; Y2 is 0, S or NR 1 represents; Z represents 0 or S.

[0049] The morpholino oligomer is preferably an oligomer having a group represented by the following formula as a constituent unit: phosphorodiamidate morpholino oligomer (hereinafter referred to as "PMO") . [ka] (In the formula, Base, R 2 , R 3 has the same meaning as above.)

[0050] Morpholino oligomers can be prepared, for example, from the compounds described in International Publication No. WO 1991 / 009033 or WO 2004 / 022664. In particular, PMOs can be prepared according to WO 2009 / 064471. 471, or the method described in WO 2013 / 100190. It can be produced according to the following:

[0051] [PMO manufacturing method] One embodiment of PMO is, for example, a compound represented by the following general formula (I) (hereinafter referred to as PMO(I)): ) can be mentioned. [ka] [In the formula, each Base, R 2 , R 3 has the same meaning as above; n is any integer in the range of 1 to 99, and preferably any integer in the range of 18 to 28. is an integer.]

[0052] PMO(I) can be produced according to known methods, for example, by carrying out the following steps: The film can be produced by carrying out the process. The compounds and reagents used in the following steps are those commonly used in the production of PMOs. If so, there is no particular limitation.

[0053] All of the following steps can be carried out using either liquid phase or solid phase synthesis (manual or commercially available solid phase automated synthesis). When PMO is produced by the solid phase method, the operation procedure can be simplified. In terms of accuracy of synthesis, a method using an automatic synthesizer is preferred.

[0054] (1) Process A: A compound represented by the following general formula (II) (hereinafter referred to as compound (II)) is reacted with an acid. By this, a compound represented by the following general formula (III) (hereinafter referred to as compound (III)) can be obtained. Manufacturing process. [ka] [where n, R 2 , R 3 has the same meaning as above; Each B P independently represent an optionally protected nucleobase; T represents a trityl group, a monomethoxytrityl group, or a dimethoxytrityl group; L is hydrogen, acyl, or a group represented by the following general formula (IV) (hereinafter referred to as group (IV)). represents.] [ka] B P The "nucleobase" in this case can be the same as the "base". , B P The amino group or hydroxyl group of the nucleic acid base may be protected. There are no particular restrictions on such amino group-protecting groups as long as they are used as protecting groups for nucleic acids. Specific examples include, but are not limited to, benzoyl, 4-methoxybenzoyl, acetyl, and propyl. Onyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butyl 4-Isopropylphenoxyacetyl, (dimethylamino)methyl Examples of the protecting group for the hydroxyl group include 2-cyanoethyl, 4-nitroethyl, and the like. phenylsulfonylethyl, methylsulfonylethyl, trimethylsilyl ethyl, phenyl optionally substituted with 1 to 5 electron-withdrawing groups at any substitutable position; , diphenylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, methylphenyl 1-pyrrolidinylcarbamoyl, morpholinocarbamoyl, 4-(tert- butylcarboxy)benzyl, 4-[(dimethylamino)carboxy]benzyl, 4-(phenyl carboxy)benzyl (see, for example, International Publication No. 2009 / 064471 reference). The "solid phase carrier" is not particularly limited as long as it can be used in a solid phase reaction of nucleic acid. For example, (i) a reagent (e.g., dichloromethane) that can be used in the synthesis of a morpholino nucleic acid derivative acetonitrile, tetrazole, N-methylimidazole, pyridine, acetic anhydride, (ii) It is used for the synthesis of morpholino nucleic acid derivatives. (iii) be chemically stable to available reagents; (iv) be chemically modifiable; and (v) have sufficient strength to withstand the high pressures applied during processing; (vi) It is desirable that the particle size range and distribution are constant. Specifically, swellable polystyrene (For example, aminomethyl polystyrene resin 1% dibenzylbenzene cross-linked (200-400 mesh) (2.4-3.0 mmol / g) (Tokyo Chemical Industry Co., Ltd.), Aminomethylated Polystyrene Resin·HCl [ Dibenzylbenzene 1%, 100-200 mesh (Peptide Institute Co., Ltd.), non-swelling polymer polyethylene (e.g., Primer Support (GE Healthcare)), PEG-linked polystyrene ( For example, NH2-PEG resin (manufactured by Watanabe Chemical Co., Ltd.), TentaGel resin, controlled pore glass (controlled pore glass (CPG) (e.g., manufactured by CPG), oxalyl-pore glass (e.g., manufactured by Alul et al., N (See Nucleic Acids Research, Vol. 19, 1527 (1991)), TentaGel support - aminopolyethylene ethylene glycol-derivatized supports (e.g., Wright et al., Tetrahedron Letters, Vol. 34, 3373 ( 1993), and examples thereof include Poros-polystyrene / divinylbenzene copolymers. do. The "linker" is a compound that is usually used to link nucleic acids or morpholino nucleic acid derivatives. Known alkyl groups can be used, such as 3-aminopropyl, succinyl, 2,2'-diaminopropyl, and the like. Examples include ethanol sulfonyl and long chain alkylamino (LCAA).

[0055] This step can be carried out by reacting compound (II) with an acid.

[0056] The "acid" that can be used in this step is, for example, trifluoroacetic acid, dichloroacetic acid, or trifluoroacetic acid. The amount of the acid used is, for example, 1 mol of Compound (II) The amount is suitably in the range of 0.1 to 1000 molar equivalents, preferably 1 to 100 It is within the range of molar equivalents. In addition, an organic amine can be used together with the acid. Although not limited to, for example, triethylamine can be mentioned. The amount of amine used is, for example, within the range of 0.01 to 10 molar equivalents per mole of acid. and preferably in the range of 0.1 molar equivalents to 2 molar equivalents. When a salt or a mixture of an acid and an organic amine is used in this step, for example, triflate Examples of the salt or mixture of fluoroacetic acid and triethylamine include triethylamine and more specifically, triethylamine and A mixture of 2 equivalents of trifluoroacetic acid and 1 equivalent of triethylamine can be given. do. The acid that can be used in this process is diluted with an appropriate solvent to a concentration within the range of 0.1% to 30%. The solvent is not particularly limited as long as it does not participate in the reaction. For example, dichloromethane, acetonitrile, alcohols (ethanol, isopropanol) , trifluoroethanol, etc.), water or a mixture thereof.

[0057] The reaction temperature in the above reaction is preferably within the range of, for example, 10°C to 50°C, more preferably The temperature is preferably in the range of 20°C to 40°C, and more preferably in the range of 25°C to 35°C. The reaction time varies depending on the type of acid used and the reaction temperature, but is usually in the range of 0.1 minutes to 24 hours. Preferably, it is within the range of 1 minute to 5 hours.

[0058] After this step is completed, if necessary, a base may be added to neutralize the acid present in the system. The "base" is not particularly limited, but for example, diisopropyl The base should be added at a concentration in the range of 0.1% (v / v) to 30% (v / v). It can also be used after diluting with an appropriate solvent. The solvent used in this step is not particularly limited as long as it does not participate in the reaction. ethane, acetonitrile, alcohols (ethanol, isopropanol, trifluoroethanol The reaction temperature can be, for example, 10 The temperature is preferably in the range of 20°C to 50°C, more preferably in the range of 20°C to 40°C, and even more preferably Preferably, it is in the range of 25°C to 35°C. The reaction time varies depending on the type of base used and the reaction temperature, but is usually between 0.1 minutes and 24 hours. The range is appropriate, and preferably, it is in the range of 1 minute to 5 hours.

[0059] In addition, in the compound (II), n=1 and L is a group (IV), the following general formula (IIa) The compound represented by the formula (hereinafter referred to as compound (IIa)) can be produced by the following method. This can be done. [ka] [In the formula, B P , T, linker, and solid phase support are as defined above.]

[0060] Step 1: The compound represented by the following general formula (V) is reacted with an acylating agent to give the compound represented by the following general formula (V): A process for producing a compound represented by formula (VI) (hereinafter referred to as compound (VI)). [ka] [In the formula, B P , T, linker are as defined above; R 4 represents a hydroxyl group, a halogen, or an amino group.

[0061] This step is carried out by a known linker introduction reaction using compound (V) as a starting material. It is possible. In particular, the compound represented by the following general formula (VIa) can be prepared by reacting the compound (V) with succinic anhydride: It can be produced by carrying out a process known as an esterification reaction. [ka] [In the formula, B P , T has the same meaning as above.]

[0062] Step 2: Compound (VI) is reacted with a condensing agent or the like to react with a solid support to give compound (II a) A process for producing the product. [ka] [In the formula, B P , R 4 , T, linker, and solid phase support are as defined above.] This step is carried out by a method known as a condensation reaction using compound (VI) and a solid support. It is possible.

[0063] In the compound (II), n is 2 to 99, and L is a group (IV), and the compound is represented by the following general formula (IIa2): The compound (IIa) can be produced by the steps A and B of the method for producing PMO described herein, using compound (IIa) as a starting material. The process can be produced by repeating steps A and B a desired number of times. [ka] [In the formula, B P , R 2 , R 3 , T, linker, and solid phase support are as defined above; n' represents 1 to 98.

[0064] In addition, in the compound (II), n=1 and L is hydrogen, and the compound (II) is represented by the following general formula (IIb): The compound can be produced by the method described in, for example, WO 1991 / 009033. can. [ka] [In the formula, B P , T has the same meaning as above.]

[0065] In the compound (II), n is 2 to 99, and L is hydrogen, and the compound is represented by the following general formula (IIb2): The compound to be produced is a compound (IIb) starting from compound (IIb) and produced by the process for producing PMO described herein. The production can be carried out by repeating steps A and B a desired number of times. [ka] [In the formula, B P , n', R 2 , R 3 , T has the same meaning as above.]

[0066] In addition, in the compound (II), n=1 and L is acyl, the compound represented by the following general formula (IIc) The compound represented by the formula (IIb) is subjected to a process known as an acylation reaction. It can be produced by [ka] [In the formula, B P , T is as defined above; R 5 represents acyl.]

[0067] In the compound (II), n is 2 to 99, and L is acyl, and the compound is represented by the following general formula (IIc2): The compound to be produced is a compound (IIc) starting from compound (IIc) and produced by the process for producing PMO described herein. The production can be carried out by repeating steps A and B a desired number of times. [ka] [In the formula, B P , n', R 2 , R 3 , R 5 , T has the same meaning as above.]

[0068] (2) Process B: Compound (III) is reacted with a morpholino monomer compound in the presence of a base, A process for producing a compound represented by the following general formula (VII) (hereinafter referred to as compound (VII)): [ka] [In the formula, each B P , L, n, R 2 , R 3 , T has the same meaning as above.]

[0069] This step involves reacting compound (III) with a morpholino monomer compound in the presence of a base. This can be carried out by:

[0070] As the morpholino monomer compound, for example, a compound represented by the following general formula (VIII) Some examples include: [ka] [In the formula, B P , R 2 , R 3 , and T has the same meaning as above.] Examples of the "base" that can be used in this step include diisopropylamine, triethylamine, and the like. Examples of the base include N-ethylmorpholine and N-ethylmorpholine. The amount is preferably in the range of 1 to 1000 molar equivalents relative to 1 mole of compound (III). Alternatively, it is in the range of 10 molar equivalents to 100 molar equivalents. The morpholino monomer compound and base that can be used in this step have a concentration of 0.1% to 30%. The solvent may be diluted with a suitable solvent as long as it is not involved in the reaction. Examples of the methylimidazolidone include, but are not limited to, N,N-dimethylimidazolidone, N-methylpiperidone, DM F, dichloromethane, acetonitrile, tetrahydrofuran, or a mixture thereof. It is possible.

[0071] The reaction temperature is preferably within the range of, for example, 0°C to 100°C, more preferably 10°C to 50°C. It is within range. The reaction time varies depending on the type of base used and the reaction temperature, but is usually in the range of 1 minute to 48 hours. The suitable time is within this range, and preferably within the range of 30 minutes to 24 hours.

[0072] Furthermore, after the completion of this step, an acylating agent can be added as needed. Examples of the "acidifier" include acetic anhydride, acetic acid chloride, and phenoxyacetic anhydride. The acylating agent can be dissolved in a suitable solvent to give a concentration in the range of, for example, 0.1% to 30%. The solvent is not particularly limited as long as it is not involved in the reaction. However, for example, dichloromethane, acetonitrile, alcohols (ethanol, isopropyl alcohol, trifluoroethanol, etc.), water, or a mixture thereof. . If necessary, an acylating agent such as pyridine, lutidine, collidine, Use a base such as triethylamine, diisopropylethylamine, or N-ethylmorpholine. The amount of the acylating agent used is in the range of 0.1 to 10,000 molar equivalents. The amount of the base used is preferably in the range of 1 molar equivalent to 1000 molar equivalents, and more preferably in the range of 1 molar equivalent to 1000 molar equivalents. For example, the amount is suitably within the range of 0.1 molar equivalents to 100 molar equivalents relative to 1 mole of the acylating agent. The amount is preferably within the range of 1 molar equivalent to 10 molar equivalents. The reaction temperature of this reaction is preferably within the range of 10°C to 50°C, more preferably 10°C to 50°C. The temperature is preferably within the range of 20°C to 40°C, and more preferably within the range of 25°C to 40°C. The reaction time varies depending on, for example, the type of acylating agent used and the reaction temperature. Although it varies depending on the circumstances, a range of 0.1 minutes to 24 hours is usually appropriate, and preferably 1 minute to 5 hours. is within the range.

[0073] (3) Process C: In the compound (VII) produced in step B, the protecting group is removed using a deprotecting agent, A process for producing a compound represented by general formula (IX). [ka] [In the formula, Base, B P , L, n, R 2 , R 3 , T has the same meaning as above.]

[0074] This step can be carried out by reacting compound (VII) with a deprotecting agent.

[0075] Examples of the "deprotecting agent" include concentrated aqueous ammonia and methylamine. Examples of the deprotecting agent that can be used in this step include water, methanol, ethanol, isopropyl alcohol, and the like. Phenyl alcohol, acetonitrile, tetrahydrofuran, DMF, N,N-dimethylimidazoline It can also be used by diluting it with N-methylpiperidone, N-methylpiperidone, or a mixture of these solvents. However, ethanol is preferred. The amount of the deprotecting agent used is, for example, 1 mol of compound (VII). For example, the range of 1 to 100,000 molar equivalents is suitable, and preferably 10 molar equivalents. The range is 1 molar equivalent to 1000 molar equivalents.

[0076] The reaction temperature is, for example, suitably in the range of 15°C to 75°C, preferably in the range of 40°C to 70°C. The deprotection reaction time is within the range of 50°C to 60°C, more preferably within the range of 50°C to 60°C. Although it varies depending on the type of the compound, reaction temperature, etc., it is suitable to use a time period ranging from 10 minutes to 30 hours, preferably The time is within a range of 30 minutes to 24 hours, and more preferably within a range of 5 hours to 20 hours.

[0077] (4) Process D: PMO (I) is prepared by reacting compound (IX) prepared in step C with an acid. The manufacturing process. [ka] [where Base, n, R 2 , R 3 , T has the same meaning as above.]

[0078] This step can be carried out by adding an acid to compound (IX).

[0079] The "acid" that can be used in this step includes, for example, trichloroacetic acid, dichloroacetic acid, Examples of the acid include acetic acid, phosphoric acid, and hydrochloric acid. The amount of acid used depends on, for example, the pH of the solution. It is suitable to use it so that the value falls within the range of 0.1 to 4.0, and more preferably within the range of 1.0 to 3.0. The solvent is used so as to be within the range. There are no particular restrictions on the solvent as long as it does not participate in the reaction. For example, acetonitrile, water, or a mixed solvent thereof can be used.

[0080] The reaction temperature is preferably in the range of 10°C to 50°C, more preferably in the range of 20°C to 40°C. The deprotection reaction time is preferably within the range of 25° C. to 35° C. Although it varies depending on the type of compound, reaction temperature, etc., a range of 0.1 minutes to 5 hours is suitable, and preferably The time is within a range of 1 minute to 1 hour, and more preferably within a range of 1 minute to 30 minutes.

[0081] PMO(I) can be separated and purified from the reaction mixture obtained in this step by conventional separation and purification methods, such as extraction and concentration. , neutralization, filtration, centrifugation, recrystallization, C8 to C 18 reversed-phase column chromatography, positive ion Ion exchange column chromatography, anion exchange column chromatography, gel filtration column Simple methods such as column chromatography, high performance liquid chromatography, dialysis, and ultrafiltration are used. The desired PMO(I) can be isolated and purified by using these compounds alone or in combination. (See, for example, International Publication WO1991 / 09033). When PMO(I) is purified using reversed-phase chromatography, the elution solvent may be, for example, A mixture of 20 mM triethylamine / acetate buffer and acetonitrile can be used. do. When purifying PMO(I) using ion exchange chromatography, for example, 1M A mixed solution of 10 mM saline and 10 mM sodium hydroxide solution can be used.

[0082] The peptide nucleic acid oligomer of the present invention has a group represented by the following general formula as a constituent unit: It's Ligomar. [ka] (In the formula, Base has the same meaning as defined above.) Peptide nucleic acids can be produced, for example, according to the following literature: 1)PE Nielsen, M. Egholm, RH Berg, O. Buchardt,Science, 254, 1497 (1991) 2) M. Egholm, O. Buchardt, PE Nielsen, RH Berg, Jacs., 114, 1895 (1992) 3) KL Dueholm, M. Egholm, C. Behrens, L. Christensen, HF Hansen, T. Vulpiu s, KH Petersen, RH Berg, PE Nielsen, O. Buchardt, J. Org. Chem., 59, 57 67 (1994) 4)L. Christensen, R. Fitzpatrick, B. Gildea, KH Petersen, HF Hansen, T.K och, M. Egholm, O. Buchardt, PE Nielsen, J. Coull, RH Berg, J. Pept. Sci., 1, 175 (1995) 5)T. Koch, HF Hansen, P. Andersen, T. Larsen, HG Batz, K. Otteson, H. Oru m, J. Pept. Res., 49, 80 (1997)

[0083] The oligomer of the present invention has a 5'-end which is a group represented by any one of the following chemical formulas (1) to (3): Preferably, it is (3)-OH. [ka] Hereinafter, the groups represented by (1), (2), and (3) above will be referred to as "group (1)," "group (2)," and "group (3)," respectively. 3)"

[0084] 2. Pharmaceutical Compositions The oligomer of the present invention enables skipping of exon 44 of the dystrophin gene. Therefore, a pharmaceutical composition containing the oligomer of the present invention is administered to the dystrophin gene containing exon 44. DM with a mutation that is subject to skipping (a mutation that is in-frame with exon 44 skipping) It is predicted that administering this drug to patients with muscular dystrophy will alleviate the symptoms of the disease. Furthermore, the oligomer of the present invention, which has a short chain length, can be produced by a simple process. This has the advantage of reducing costs. Thus, in another embodiment, the oligomer of the present invention, its pharmaceutically acceptable salt or water a pharmaceutical composition for treating muscular dystrophy containing a compound thereof as an active ingredient (hereinafter referred to as "the composition of the present invention"); (hereinafter referred to as "the Company")

[0085] Examples of pharmaceutically acceptable salts of the oligomers of the invention that can be included in the compositions of the invention include: Alkali metal salts such as sodium salts, potassium salts, and lithium salts, calcium salts, magnesium salts, Alkaline earth metal salts such as sodium salts; aluminum salts, iron salts, zinc salts, copper salts, nickel salts salts, metal salts such as cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine salts Salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine Amine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroproca amine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, pipet ammonium salt, tetramethylammonium salt, tris(hydroxymethyl)aminomethane salt, etc. organic amine salts such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides; Hydrochlorides; inorganic acid salts such as nitrates, perchlorates, sulfates, and phosphates; methanesulfonates lower alkanesulfonates such as ammonium salts, trifluoromethanesulfonates, and ethanesulfonates; sulfonates; aryl sulfonates such as benzenesulfonates and p-toluenesulfonates Salts: acetate, malate, fumarate, succinate, citrate, tartrate, oxalate Salts, organic acid salts such as maleates; glycine salts, lysine salts, arginine salts, ornithine salts , and amino acid salts such as glutamate and aspartate. can be produced by known methods. Alternatively, the present invention can be The oligomer may be in the form of its hydrate.

[0086] The dosage form of the composition of the present invention is not particularly limited as long as it is a pharmaceutically acceptable dosage form. It can be selected depending on the treatment method, but from the viewpoint of ease of delivery to muscle tissue, intravenous administration is recommended. Intra-arterial administration, intramuscular administration, subcutaneous administration, oral administration, intratissue administration, transdermal administration, etc. are preferred. The dosage form that the composition of the present invention can take is not particularly limited, but may be, for example, various Examples include injections, oral preparations, drip infusions, inhalants, ointments, lotions, and the like.

[0087] When the oligomer of the present invention is administered to a patient with muscular dystrophy, the composition of the present invention Preferably, the ligomer comprises a carrier that facilitates delivery to muscle tissue. There are no particular limitations as long as it is pharmaceutically acceptable, and examples thereof include cationic liposomes, Examples of carriers include cationic carriers such as cationic polymers, and carriers that utilize viral envelopes. Examples of cationic liposomes include 2-O-(2-diethylamino) It consists of (ethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids as essential components. Liposomes formed by the above procedure (hereinafter referred to as "Liposome A"), Oligofectamine (registered trademark) (registered trademark) (manufactured by Invitrogen), Lipofectin (registered trademark) (manufactured by Invitrogen), Lipofectin (registered trademark) (manufactured by Invitrogen), Lipofectamine (registered trademark) (Invitrogen), Lipofectamine 2000 (registered trademark) (Invi Invitrogen), DMRIE-C (registered trademark) (Invitrogen), GeneSilencer (registered trademark) ene Therapy Systems), TransMessenger (registered trademark) (QIAGEN), TransIT TKO (registered trademark) (Mirus), and Nucleofector II (Lonza). Among these, Liposome A is preferred. Examples of cationic polymers include JetSI (registered trademark). (registered trademark) (manufactured by Qbiogene), Jet-PEI (registered trademark) (polyethyleneimine, manufactured by Qbiogene) Examples of carriers using viral envelopes include GenomeOn e (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Kaisha). Pharmaceutical devices described in Patent Publication No. 2924179, Patent Publication No. 2006 / 129594 and Patent Publication No. The cationic carriers described in 2008 / 096690 can also be used.

[0088] The concentration of the oligomer of the present invention contained in the composition of the present invention varies depending on the type of carrier, etc. The range of 0.1 nM to 100 μM is appropriate, the range of 1 nM to 10 μM is preferable, and the range of 10 nM to 1 The oligomer of the present invention contained in the composition of the present invention and the carrier are more preferably in the range of 1 μM. The weight ratio (carrier / oligomer of the present invention) of the carrier to the oligomer of the present invention depends on the properties of the oligomer, the type of the carrier, etc. Although it varies depending on the type, a range of 0.1 to 100 is appropriate, a range of 1 to 50 is preferable, and a range of 10 to 20 is preferable. It is more preferable that the range is within the range of .

[0089] The composition of the present invention may contain, in addition to the oligomer of the present invention and the carrier described above, any pharmaceutically acceptable carrier. Such additives include, for example, an emulsifying aid (e.g., For example, fatty acids having 6 to 22 carbon atoms and pharmaceutically acceptable salts thereof, albumin, dextran, stabilizers (e.g., cholesterol, phosphatidic acid), tonicity agents (e.g., sodium chloride), sodium, glucose, maltose, lactose, sucrose, trehalose), pH adjuster (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine) These may be used alone or in combination of two or more. The content of the additive in the composition of the present invention is suitably 90% by weight or less, and preferably 70% by weight or less. It is preferable that the content is 50% by weight or less, and more preferable that the content is 50% by weight or less.

[0090] The composition of the present invention can be prepared by adding the oligomer of the present invention to a dispersion of a carrier and stirring appropriately. The additive may be added either before or after the addition of the oligomer of the present invention. Water that can be used when adding the oligomer of the present invention can also be added at an appropriate stage. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water for injection, Examples include electrolyte solutions such as distilled water for injection, physiological saline solution, and sugar solutions such as glucose solution and maltose solution. In such cases, conditions such as pH and temperature can be appropriately selected by those skilled in the art. Cut.

[0091] The composition of the present invention can be, for example, a liquid formulation or a freeze-dried formulation thereof. The dry preparation can be prepared by freeze-drying the composition of the present invention in the form of a liquid preparation in a conventional manner. For example, the composition of the present invention in the form of a liquid can be prepared by After sterilization, dispense the specified amount into vials and pre-freeze at approximately -40 to -20°C for 2 hours. The drying is carried out for about 1 hour, followed by primary drying under reduced pressure at about 0 to 10°C, and then secondary drying under reduced pressure at about 15 to 25°C. It can be dried and lyophilized, and the inside of the vial is typically flushed with nitrogen gas. The container is then sealed to obtain a freeze-dried preparation of the composition of the present invention.

[0092] The lyophilized formulation of the composition of the present invention can generally be reconstituted by adding any suitable solution (reconstitution solution). Such a reconstitution liquid can be used by reconstitution with water for injection, physiological saline, etc. The amount of the reconstituted solution varies depending on the intended use. Although there are no particular limitations, an amount of 0.5 to 2 times the liquid volume before freeze-drying, or 500 mL or less, is appropriate.

[0093] The dosage when administering the composition of the present invention depends on the type of the oligomer of the present invention contained therein, Prepare the medicine taking into consideration the dosage form, the patient's condition such as age and weight, the route of administration, and the nature and severity of the disease. However, the amount of the oligomer of the present invention for adults is preferably 0.1 mg to 10 g / day. Generally, the dose is within the human range, preferably within the range of 1 mg to 1 g / human. It may also vary depending on the type of disease, the administration method, and the target molecule. In some cases, a dose less than this may be sufficient, but conversely, a dose greater than this may be necessary. The administration can be once or several times a day, or at intervals of one day to several days.

[0094] Another embodiment of the composition of the present invention is a vector capable of expressing the oligonucleotide of the present invention. and the above-mentioned carrier. The composition may be capable of expressing the oligonucleotide of the present invention. As with the compositions of the present invention containing the oligomers of interest, pharmaceutically acceptable additives may be added. The concentration of the expression vector contained in the composition can be adjusted depending on the type of carrier, etc. The concentration varies depending on the individual, but is suitably in the range of 0.1 nM to 100 μM, preferably in the range of 1 nM to 10 μM, and The range of 0 nM to 1 μM is more preferable. The weight ratio (carrier / expression vector) varies depending on the properties of the expression vector, the type of carrier, etc. The range of 0.1 to 100 is suitable, the range of 1 to 50 is preferable, and the range of 10 to 20 is more preferable. The content of the carrier contained in the composition is preferably 100% or more, and more preferably 100% or more, including the oligomer of the present invention. The same applies to the composition of the present invention, and the preparation method thereof is also the same as that of the composition of the present invention. is the same as:

[0095] The present invention will be described in more detail below with reference to examples and test examples. The range is not limited to the range shown in the table. [Example]

[0096] [Reference example 1] 4-{[(2S,6R)-6-(4-benzamido-2-one]- Aminopolystyrene Resin-Supported 4-{[(2S,6R)-6-(4-benzamido-2-one]- xopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxo Butanoic acid Step 1: 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl] Preparation of {(4-trityl)morpholin-2-yl]methoxy}-4-oxobutanoic acid Under an argon atmosphere, N-{1-[(2R,6S)-6-(hydroxymethyl)-4-trimethylmo [[ ...(())])((((())])))))) 0.44g and 1.1g of 4-dimethylaminopyridine (4-DMAP) were suspended in 50mL of dichloromethane and 0.90 g of succinic acid was added, and the mixture was stirred at room temperature for 3 hours. 10 mL of methanol was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was extracted with ethyl acetate and 0.5M aqueous potassium dihydrogen phosphate solution. The resulting organic layer was washed with 0.5 M aqueous potassium dihydrogen phosphate solution, water, and saturated saline in this order. The resulting organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain 4.0 g of the desired product.

[0097] Step 2: Aminopolystyrene Resin-Supported 4-{[(2S,6R)-6-(4-benzamide] -2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4 -Oxobutanoic acid production 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl) 4.0 g of [4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid was dissolved in pyridine ( Dissolve 0.73 g of 4-DMAP and 1-ethyl-3-(3-dimethylaminopropyl) carboxymethylcellulose in 200 mL of dehydrated water. 11.5 g of rubodiimide hydrochloride was added. Then, aminopolystyrene resin Primer support 25.0 g of 200 amino acid (GE Healthcare Japan, 17-5214-97) and 8.5 mL of triethylamine The mixture was added and shaken at room temperature for 4 days. After the reaction, the resin was filtered off. The resulting resin was dissolved in pyridine, methyl The resin was washed with ethanol and dichloromethane in that order and dried under reduced pressure. 200 mL of ran (dehydrated), 15 mL of acetic anhydride, and 15 mL of 2,6-lutidine were added and shaken at room temperature for 2 hours. The resin was collected by filtration, washed with pyridine, methanol, and dichloromethane in that order, and dried under reduced pressure. 0.7g of the desired product was obtained. The loading amount of the target compound can be determined by a known method using the moles of trityl per gram of resin. The amount of resin was determined by measuring the UV absorbance at 409 nm. The concentration was 29.2 μmol / g.

[0098] UV measurement conditions Equipment: U-2910 (Hitachi) Solvent: methanesulfonic acid Wavelength: 409 nm ε value: 45000

[0099] [Reference example 2] Aminopolystyrene Resin-Supported 4-{[(2S,6R)-6-(5-methyl-2,4-dioxa Sopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanol tannic acid The title compound was produced in the same manner as in Reference Example 1. However, the N -{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]- Instead of 2-oxo-1,2-dihydropyrimidin-4-yl}benzamide, in this process , 1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-5 -methylpyrimidine-2,4(1H,3H)-dione was used. The loading amount of the target compound can be determined by a known method using the molar amount of trityl per 1 g of resin. The loading of the resin was determined by measuring the UV absorbance at 409 nm. .0 μmol / g.

[0100] [Reference example 3] 4-{[(2S,6R)-6-(6-benzamidopurine] supported on aminopolystyrene resin -9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid The title compound was produced in the same manner as in Reference Example 1. However, the N -{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]- Instead of 2-oxo-1,2-dihydropyrimidin-4-yl}benzamide, in this process , N-{9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]purine-6-yl {Il}benzamide was used. The loading amount of the target compound can be determined by a known method using the molar amount of trityl per 1 g of resin. The loading of the resin was determined by measuring the UV absorbance at 409 nm. 0.7μmol / g.

[0101] [Reference example 4] 4-{{(2S,6R)-6-{6-(2-cyanoethoxy) )-2-[(2-phenoxyacetyl)amino]purin-9-yl}-4-tritylmorpholine -2-yl}methoxy}-4-oxobutanoic acid The title compound was produced in the same manner as in Reference Example 1. However, the N -{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]- Instead of 2-oxo-1,2-dihydropyrimidin-4-yl}benzamide, in this process , N-{6-(2-cyanoethoxy)-9-[(2R,6S)-6-(hydroxymethyl)-4-thiazolinone ritylmorpholin-2-yl]purin-2-yl}-2-phenoxyacetamide . The loading amount of the target compound can be determined by a known method using the molar amount of trityl per 1 g of resin. The loading of the resin was determined by measuring the UV absorbance at 409 nm. 0.8μmol / g.

[0102] PMOs shown in Table 1 as PMO Nos. 1 to 118 were synthesized according to the description in Example 1 below. PMO No. 119 and 120 were purchased from Gene Tools. The synthesized PMO was dissolved in water for injection (Otsuka Pharmaceutical Factory). Dissolved.

[0103] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0104] [Example 1] 4-{[(2S,6R)-6 -(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholine -2-yl]methoxy}-4-oxobutanoic acid (Reference Example 1), or aminopolystyrene 4-{[(2S,6R)-6-(5-methyl-2,4-dioxopyrimidin-1- {4-tritylmorpholin-2-yl}methoxy}-4-oxobutanoic acid (Reference Example 2) , or 4-{[(2S,6R)-6-(6-benzyl)-2-methyl-2-benzo[4- ... Amidopurin-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanol acetic acid (Reference Example 3) or 4-{{(2S,6R) -6-{6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9 -yl}-4-tritylmorpholin-2-yl}methoxy}-4-oxobutanoic acid (Reference Example 4 0.2 g of the sample was packed into a column with a filter and synthesized using a nucleic acid synthesizer (AKTA Oligopilot 10 plus). The following synthesis cycle was started using the nucleotide sequences of the compounds listed in Table 1. The desired morpholino monomer compound was added in the coupling cycle (see Table 2 below). see).

[0105] [Table 2]

[0106] The deblocking solution was dichloromethane containing 3% (w / v) trifluoroacetic acid. The neutralization and washing solution used was N,N-diisopropylethylamine at 10% (v / v). v) and tetrahydrofuran to 5% (v / v), Coupling solution A was dissolved in dichloromethane containing acetonitrile. The morpholino monomer compound was dissolved in tetrahydrofuran to a concentration of 0.10 M. Coupling solution B was prepared by dissolving N,N-diisopropylethylamine in 2 mL of HCl. acetone to 0% (v / v) and tetrahydrofuran to 10% (v / v). The capping solution was prepared by dissolving the compound in acetonitrile. A solution of 20% (v / v) acetic anhydride and 30% (v / v) 2,6-lutidine was used.

[0107] The aminopolystyrene resin carrying the synthesized PMO was recovered from the reaction vessel and left for 2 hours or more. The dried aminopolystyrene resin-supported PMO was placed in a reaction vessel. 5 mL of 28% aqueous ammonia-ethanol (1 / 4) was added and the mixture was stirred at 55°C for 15 hours. The polystyrene resin was filtered off and washed with 1 mL of water-ethanol (1 / 4). The resulting residue was diluted with 20 mM acetic acid-triethylamine buffer (TEAA buffer) and acetone. The resulting solution was dissolved in 10 mL of a mixed solvent of nitriles (4 / 1) and filtered through a membrane filter. The filtrate was purified by reverse phase HPLC under the conditions shown in Table 3 below.

[0108] [Table 3]

[0109] Each fraction was analyzed, and the target substance was collected and concentrated under reduced pressure. 0.5 mL of the solution was added and stirred for 15 minutes. Then, 2 mL of 2M aqueous sodium hydroxide solution was added to the solution. The solution was diluted with potassium and filtered through a membrane filter (0.45 μm). The resulting aqueous solution containing the target product was purified using an anion exchange resin column. is as shown in Table 4 below.

[0110] [Table 4]

[0111] Each fraction was analyzed (HPLC) to obtain the target substance as an aqueous solution. The mixture was neutralized with phosphate buffer (pH 6.0). Then, the mixture was subjected to reverse phase HPLC under the conditions shown in Table 5 below. Desalted.

[0112] [Table 5]

[0113] The target product was collected and concentrated under reduced pressure. The resulting residue was dissolved in water and freeze-dried to give a white, fluffy solid. The target compound was obtained as a solid. The calculated and measured values ​​of ESI-TOF-MS are shown in Table 6 below. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0114] [Test Example 1] In vitro assays RD cells (human rhabdomyosarcoma cell line) 3.5 x 10 5 0.1 of the antisense oligomer in Table 1 ~30 μM was transfected using the Amaxa Cell Line Nucleofector Kit L and Nucleofector II (Lonza). The program used was T-030. After transfection, the cells were cultured in Eagle's medium containing 10% fetal calf serum (FCS) (Invitrogen). In 2 mL of EMEM (Sigma), the culture medium was incubated at 37°C and 5% CO2. The cells were cultured under these conditions for three nights. The cells were washed once with PBS (manufactured by Nissui Co., Ltd., the same applies hereinafter), and then incubated with 1% 2-mercaptoethanol ( Add 350 μL of Buffer RLT (Qiagen) containing HCl (Nacalai Tesque) to the cells and incubate for a few minutes. The cells were lysed by leaving the mixture at room temperature for 1 hour, and then collected in a QIAshredder homogenizer (Qiagen). The homogenate was prepared by centrifugation at 15,000 rpm for 2 minutes. Total RNA was extracted according to the protocol attached to the kit (manufactured by Ion Biosciences). The concentration of the extracted total RNA was was measured using a NanoDrop ND-1000 (manufactured by LMS).

[0115] The QIAGEN OneStep RT-PCR Kit (Qiagen) was used for 400 ng of extracted total RNA. One-Step RT-PCR was performed using the same DNA. The reaction mixture was prepared according to the protocol provided with the kit. The thermal cycler was PTC-100 (MJ Research) or TaKaRa PCR Thermal Cycler. Dice Touch (Takara Bio Inc.) was used. The RT-PCR program used was as follows: be. 50℃, 30 minutes: reverse transcription 95℃, 15 minutes: Polymerase activation, reverse transcriptase inactivation, cDNA denaturation PCR amplification: [94°C, 30 seconds; 60°C, 30 seconds; 72°C, 1 minute] x 35 cycles 72℃, 10 minutes: Final extension reaction

[0116] The nucleotide sequences of the forward and reverse primers used for RT-PCR are as follows: is. Forward primer: 5'-GCTCAGGTCGGATTGACATT-3' (SEQ ID NO: 125) Reverse primer: 5'-GGGCAACTCTTCCACCAGTA -3' (SEQ ID NO: 126)

[0117] 1 μL of the PCR reaction product was analyzed using a Bioanalyzer (Agilent). The polynucleotide amount of the band in which exon 44 was skipped, "A", and the polynucleotide amount of the band in which exon 44 was skipped, "B", are shown. The amount of polynucleotides in the unpaired bands, "B," was measured. Based on the constant value (unit: nmol / L), the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / ( A + B ) x 100

[0118] The experimental results are shown in Figures 1 to 26. This experiment demonstrated that the wild-type dystrophin gene in humans In the nucleotide sequence of exon 44 (SEQ ID NO: 10), the -1st to 44th positions from the 5' end ( a short unit oligomer selected from positions 58 to 115 (SEQ ID NO: 2) The oligomer of the present invention obtained by ligating the above can effectively skip exon 44. It was revealed.

[0119] [Test Example 2] In vitro assays The experiment was carried out in the same manner as in Test Example 1, except that 3.5 × 10 RD cells (human rhabdomyosarcoma cell line) were used. 5 The oligomers of the present invention, PMO Nos. 34, 100, 45, 73, 49, and 47, were used alone or in combination with each other. The two unit oligomers that make up each of these are used alone or in combination to produce 1 and 3 or at a concentration of 10 μM using the Amaxa Cell Line Nucleofector Kit L and Nucleofector II (L The sequence was introduced using the T-030 program. The following sequence combinations were introduced: As stated above. [Table 7]

[0120] Experimental results The results are shown in Figures 27 to 31. In this experiment, PMO Nos. 110 to 115, which target exon 44, , PMO No. 117 and PMO No. 118 do not cause exon 44 skipping by themselves. In addition, the mixture of two antisense nucleic acids targeting different sites within exon 44 was found. Compounds (PMO No. 114 and PMO No. 115, PMO No. 109 and PMO No. 114, PMO No. 110 and PMO No. 111, PMO No. 112 and PMO No. 113, PMO No. 117 and PMO No. 118, and PMO No. 119 and PMO No. 120) In comparison, PMO No. 34, PMO No. 100, PMO No. 45, PMO No. 73, and PMO The oligomers of the present invention, No. 49 and PMO No. 47, induce exon 44 skipping with high efficiency. It was discovered that...

[0121] [Test Example 3] In vitro assay using human fibroblasts GM05112 cells (fibroblasts derived from a DMD patient with exon 45 deletion, Coriell Institute for Medical The exon 44 skipping activity of the oligomers of the present invention was examined using a PCR-based PCR product. The growth medium consisted of 10% FCS (Hyclone Laboratories) and 1% Penicillin / Streptomycin (P / S ) (Sigma-Aldrich) containing Dulbecco's Modified Eagle Medium: Nutrient Mixture e F-12 (DMEM / F-12) (Life Technologies) was used and cultured at 37°C in the presence of 5% CO2. Ta.

[0122] The cells were cultured in a T225 flask, and 2.5 mL of human myoD (SEQ ID NO: 1) was added to 30 mL of growth medium. 27) Expression retrovirus (co-expressing ZsGreen1) and polybrene (Sigma) at a final concentration of 8 μg / mL After culturing at 32°C for 2 days, the medium was replaced with fresh growth medium. The cells were further cultured at 37°C for 3 days. ZsGreen was analyzed using a BD FACSAria Cell Sorter (BD Biosciences). MyoD-transformed fibroblasts were collected by selecting 1-positive cells. The collected cells were then cultured in differentiation medium. (2% horse serum (Life Technologies), 1% P / S and ITS Liquid Media Supplement ( The cells were suspended in DMEM / F-12 containing guanidine-Aldrich (Gamma-Aldrich), and placed on a collagen-coated 24-well plate. 9.4 x 10 4 The cells were seeded at 1000 cells / well. The medium was changed every 2-3 days. They were induced to differentiate into ductal cells.

[0123] On the 7th day after seeding on a 24-well plate, PMO Nos. 34, 45, and 4 were added to a final concentration of 10 μM. After 2 days of incubation, the differentiation medium was replaced with a PMO-free differentiation medium containing 9 and 73. The medium was replaced with new medium and incubated for another 5 days. The cells were harvested and purified using the RNeasy Mini Kit (Kia). Total RNA was extracted using QIAGEN OneS. 50 ng of extracted total RNA was used. RT-PCR was performed using the Step RT-PCR Kit. The reaction mixture was prepared according to the attached protocol. The thermal cycler used was an iCycler (Bio-Rad Laboratories). The program is as follows: 50℃, 30 minutes: reverse transcription 95℃, 15 minutes: Polymerase activation, reverse transcriptase inactivation, cDNA denaturation PCR amplification: [94°C, 1 min; 60°C, 1 min; 72°C, 1 min] x 35 cycles 72℃, 7 minutes: final extension reaction

[0124] The nucleotide sequences of the forward and reverse primers used for RT-PCR are as follows: is. Forward primer: 5'-GCTCAGGTCGGATTGACATT-3' (SEQ ID NO: 125) Reverse primer: 5'-GGGCAACTCTTCCACCAGTA-3' (SEQ ID NO: 126)

[0125] 1 μL of PCR product was analyzed using Experion DNA 1K Analysis Kits (Bio-Rad Laboratories). Analysis was performed using an Experion Electrophoresis Station (Bio-Rad Laboratories). Select the DNA 1K assay on the BioRad Laboratories software version 3.2 and measure. The bands around 317 bp (A) and 465 bp (B) were quantified using Experion Software. (Unit: nmol / L) was calculated using Excel 2007 SP3 (Microsoft) using the following formula: The ping efficiency (%) was calculated. Skipping efficiency (%) = A / ( A + B ) x 100

[0126] Experimental results The results are shown in Figure 32. This experiment confirmed that the oligomers of the present invention, PMO Nos. 34, 45, 49 and 73 showed high efficiency of exon 44 skipping in exon 45-deficient DMD patient-derived cells. It was found that this can be done. [Industrial Applicability]

[0127] From the experimental results shown in the test examples, it was found that the oligomer of the present invention, which is a combination of short oligomers, inhibits the growth of RD cells. Therefore, the oligonucleotides of the present invention were found to induce exon 44 skipping in the These drugs are highly useful in the treatment of DMD.

[0128] [Sequence List Free Text]

[0129]

Claims

1. (a) A base sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases in the target exon. a first unit oligomer comprising a string; and (b) bases complementary to a second nucleotide sequence of 7 to 15 consecutive bases within the target exon; a second unit oligomer comprising the sequence An antisense oligomer having a length of 15 to 30 bases, The first and second nucleotide sequences are contiguous or overlap each other. isn't it, an antisense oligomer or a pharmaceutical composition thereof that induces skipping of the target exon; A pharmaceutically acceptable salt or hydrate.

2. The first and / or second unit oligomers are introductory sequences adjacent to the target exon. The antisense oligonucleotide according to claim 1, comprising a base sequence complementary to a partial nucleotide sequence of the oligonucleotide. Ligomer, or a pharmaceutically acceptable salt or hydrate thereof.

3. 3. The method of claim 1, wherein the target exon is an exon of the human dystrophin gene. or a pharmaceutically acceptable salt or hydrate thereof.

4. The first nucleotide sequence is a contiguous sequence selected from the nucleotide sequence shown in SEQ ID NO:

1.

3. The antisense oligomer according to claim 1 or 2, which is a nucleotide sequence of 7 to 15 bases. or a pharmaceutically acceptable salt or hydrate thereof.

5. The second nucleotide sequence is a contiguous sequence selected from the nucleotide sequence shown in SEQ ID NO:

2. The antisense oligonucleotide according to any one of claims 1 to 3, which is a nucleotide sequence of 7 to 15 bases. A oligomer, or a pharmaceutically acceptable salt or hydrate thereof.

6. Two unit oligomers selected from the group consisting of (c) to (e) below are linked together. The antisense oligomer of claim 1 or 2, which is: (c) a sequence of 7 to 15 consecutive nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 3; a unit oligomer consisting of a base sequence complementary to the base sequence; (d) a sequence of 7 to 15 consecutive nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 4; a unit oligomer consisting of a base sequence complementary to the base sequence; and (e) a sequence of 7 to 15 consecutive nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 5; A unit oligomer consisting of a base sequence complementary to the base sequence, or a pharmaceutically acceptable salt thereof Salt or hydrate.

7. Claim 1 or 2, which consists of any one base sequence selected from the group consisting of SEQ ID NOs: 6 to 9 1. An antisense oligomer according to claim 1, or a pharmaceutically acceptable salt or hydrate thereof.

8. The antisense oligomer according to any one of claims 1 to 7, which is an oligonucleotide. or a pharmaceutically acceptable salt or hydrate thereof.

9. The sugar moiety and / or linker of at least one nucleotide constituting the oligonucleotide 9. The antisense oligomer of claim 8, wherein the phosphate binding moiety is modified, or A pharmaceutically acceptable salt or hydrate.

10. The sugar moiety of at least one nucleotide constituting the oligonucleotide has a -O H group is OR, R, R'OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F, Cl, Br and I 10. The antisense oligonucleotide according to claim 8 or 9, wherein the ribose is substituted with any of the selected groups. A oligomer, or a pharmaceutically acceptable salt or hydrate thereof. (The above R represents alkyl or aryl, and the above R' represents alkylene.)

11. The phosphate binding moiety of at least one nucleotide constituting the oligonucleotide is Phosphorothioate bond, phosphorodithioate bond, alkylphosphonate bond, phospho Any bond selected from the group consisting of a boranoamidate bond and a boranophosphate bond An antisense oligomer according to any one of claims 8 to 10, which is one of A pharmaceutically acceptable salt or hydrate thereof.

12. The antisense oligomer of any one of claims 1 to 7, which is a morpholino oligomer. or a pharmaceutically acceptable salt or hydrate thereof.

13. 13. The antisense oligo of claim 12, which is a phosphorodiamidate morpholino oligomer. GOMER, or a pharmaceutically acceptable salt or hydrate thereof.

14. The compound according to claim 12 or 13, wherein the 5'-end is a group represented by any one of the following chemical formulas (1) to (3): An antisense oligomer, or a pharmaceutically acceptable salt or hydrate thereof. 【Chemistry 26】

15. The antisense oligomer according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for treating muscular dystrophy, comprising a possible salt or hydrate thereof as an active ingredient.

16. 16. The pharmaceutical composition of claim 15, further comprising a pharmaceutically acceptable carrier.

17. The antisense oligomer according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof. or the pharmaceutical composition of claim 1 or 16. A method for treating muscular dystrophy, comprising administering to a patient suffering from the strophy.

18. The muscular dystrophy patient is subject to exon 44 skipping in the dystrophin gene. The method of claim 17, wherein the patient has a mutation.

19. 19. The method of claim 17 or 18, wherein the patient is a human.

20. A method according to any one of claims 1 to 14 for the manufacture of a pharmaceutical composition for treating muscular dystrophy. or a pharmaceutically acceptable salt or hydrate thereof.

21. The antisense oligonucleotide according to any one of claims 1 to 14 for use in treating muscular dystrophy. or a pharmaceutically acceptable salt or hydrate thereof.

22. In the treatment, a patient with muscular dystrophy has exon 44 skipping in the dystrophin gene. The antisense oligomer of claim 21, or a pharmaceutically acceptable salt or hydrate thereof.

23. 23. The antisense oligomer of claim 21 or 22, wherein the patient is a human. A pharmaceutically acceptable salt or hydrate.

24. (a) A base sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases in the target exon. a first unit oligomer comprising a string; and (b) bases complementary to a second nucleotide sequence of 7 to 15 consecutive bases within the target exon; a second unit oligomer comprising the sequence a step of producing an antisense oligomer of 15 to 30 bases in length by linking wherein the first nucleotide sequence and the second nucleotide sequence are contiguous or overlap each other. It is not something that A method for producing the antisense oligomer according to claim 1.

25. measuring the skipping efficiency of the antisense oligomer obtained in the above step; and selecting antisense oligomers having skipping efficiencies exceeding a reference value; 25. The method of claim 24, further comprising:

26. (a) (i) a base sequence complementary to a first nucleotide sequence of 7 to 15 consecutive bases within the target exon; a first unit oligomer comprising a group sequence; and (ii) a second nucleotide sequence complementary to a contiguous sequence of 7 to 15 bases within the target exon; a second unit oligomer containing a base sequence a step of selecting a sequence of the first nucleotide sequence and the second nucleotide sequence, or are not overlapping with each other), (b) Linking the first and second unit oligomers to form an antisense oligonucleotide having a length of 15 to 30 bases. generating a sense oligomer; (c) measuring the skipping efficiency of the antisense oligomer obtained in step (b); , and (d) selecting antisense oligomers having skipping efficiencies exceeding a reference value; 、 A method for screening an antisense oligomer, comprising:

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