Antisense nucleic acid

Abstract

The present invention provides a novel linkage-type antisense oligomer that induces exon skipping by targeting base sequences at two different sites in the same exon of the dystrophin gene, and a therapeutic agent for muscular dystrophy comprising the oligomer.SOLUTION: It has been found that an antisense oligomer obtained by linking oligomers targeting two different sites of exon 45 of the human dystrophin gene can induce skipping of the exon.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention allows for skipping of the 45th exon of the human dystrophin gene. The present invention relates to antisense oligomers and pharmaceutical compositions containing the oligomers. [Background technology]

[0002] Duchenne muscular dystrophy (DMD) is the most common type of muscular dystrophy, occurring in approximately 1 in 3,500 male births. It is a hereditary progressive muscle disease with a high incidence of stroke. However, muscle weakness begins to decrease from around the age of 4 or 5. Muscle weakness then progresses and by the age of 12, the child is no longer able to walk. It is a serious disease that can lead to death due to heart or respiratory failure in people in their 20s. There is no effective treatment for D, and there is a strong demand for the development of new therapeutic drugs.

[0003] DMD is known to be caused by mutations in the dystrophin gene. The gene is located on the X chromosome and is a huge gene consisting of 2.2 million bases of DNA. It is transcribed into the genome and then spliced ​​to remove introns and combine 79 exons. The mRNA consists of 13,993 bases. This mRNA is translated into 3,685 amino acids, and the dystrophin protein The dystrophin protein is involved in maintaining membrane stability in muscle cells. The dystrophin gene in DMD patients is altered and is necessary for protecting muscle cells from damage. Because of this mutation, functional dystrophin protein is rarely expressed in muscle cells. Therefore, in DMD patients, the structure of muscle cells cannot be maintained, and a large amount of calcium Ions flow into muscle cells, causing an inflammation-like reaction and fibrosis, which leads to muscle damage. Cells become less able to regenerate.

[0004] Becker muscular dystrophy (BMD) is also caused by mutations in the dystrophin gene. Symptoms of DMD include muscle weakness, but 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 that the mutation causes dystrophy. The amino acid reading frame during translation of dystrophin mRNA into the dystrophin protein is disrupted. It is believed that the reason for this is whether the DMD is maintained or not (Non-Patent Document 1). In this study, a mutation that shifts the amino acid reading frame results in functional dystrophin. Although the protein is barely expressed, in BMD, a portion of the exon is deleted due to the mutation. However, the amino acid reading frame is maintained, so the dystrophin is functional, albeit incomplete. Inhibitory proteins are produced.

[0005] Exon skipping is a promising treatment for DMD. By modifying the ligation, the amino acid reading frame of dystrophin mRNA was restored, partially This is a method for inducing the expression of functionally restored dystrophin protein (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 contains The function of stabilizing muscle cells is partially maintained because the reading frame is maintained. Exon skipping causes 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 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. is incorporated into mRNA only when both splice sites are recognized by the spliceosome complex. Therefore, targeting splice sites with antisense nucleic acids can It can induce exon skipping. The exon splicing enhancer (ESE) must contain a serine and an alpha amino acid sequence for recognition 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 ligating the exon.

[0007] The dystrophin gene mutations vary among DMD patients, so the location and type of gene mutation 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 phenomenon (Patent Documents 1 to 6 and Non-Patent Documents 1 and 2). When two types of antisense nucleic acids targeting the same exon of the 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 (linked type) has not yet been reported to exhibit skipping activity. [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 two different genes in the same exon of the dystrophin gene. A novel linked antisense molecule that targets specific base sequences to induce exon skipping The main purpose of the present invention is to provide oligomers and therapeutic drugs for muscular dystrophy containing the same. do. [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 targeting two different sites in exon 45 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] 14, in which two unit oligomers selected from the group consisting of (a) to (e) below are linked together: An antisense oligomer having a length of about 32 bases, wherein the two unit oligomers are consecutive or alternate. antisense oligomers or pharmaceutically acceptable salts thereof, Hydrate: (a) Positions -5 to 15 from the 5' end of the 45th exon of the human dystrophin gene A nucleotide sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences Unit oligomers consisting of group sequences; (b) Positions 48-70 from the 5' end of the 45th exon of the human dystrophin gene A nucleotide sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences Unit oligomers consisting of group sequences; (c) From the 5' end of exon 45 of the human dystrophin gene to positions 128-150 Complementary to a contiguous nucleotide sequence of 7 to 16 bases selected from the nucleotide sequence A unit oligomer consisting of a specific base sequence; (d) 15th to 40th residues from the 5' end of the 45th exon of the human dystrophin gene A nucleotide sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences a unit oligomer consisting of a group sequence; and (e) From the 5' end of the 45th exon of the human dystrophin gene to positions 110-125 Complementary to a contiguous nucleotide sequence of 7 to 16 bases selected from the nucleotide sequence A unit oligomer consisting of a specific base sequence. [2] The anti-oxidant according to [1], wherein one of the two unit oligomers is (a). A sense oligomer or a pharmaceutically acceptable salt or hydrate thereof. [3] Any one selected from the group consisting of SEQ ID NOs: 7 to 12, 14 to 33, 40 to 52, 57, 64, 65, and 79 to 86 The antisense oligomer or its derivatives according to [1] or [2] above, which consists of one or more base sequences. A pharmaceutically acceptable salt or hydrate of [4] Any one of the base sequences selected from the group consisting of SEQ ID NOs: 8, 10, 25, 30, 33, 79, and 80 The antisense oligomer or its pharmaceutical use according to any one of [1] to [3] above, Acceptable salts or hydrates thereof. [5] The antisense oligonucleotide according to any one of [1] to [4] above, which is an oligonucleotide. or a pharmaceutically acceptable salt or hydrate thereof. [6] The sugar moiety and / or linker of at least one nucleotide constituting the oligonucleotide The antisense oligomer or its pharmaceutical composition according to [5] above, wherein the phosphate binding portion is modified. A pharmaceutically acceptable salt or hydrate. [7] The sugar moiety of at least one nucleotide constituting the oligonucleotide is 2 The -OH group at position 1 is selected from the group consisting of OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br and I.

[0023] The ribose according to [5] or [6] above is ribose substituted with any group selected from the group consisting of The antisense oligomer described above, or a pharmaceutically acceptable salt or hydrate thereof. (The above R represents alkyl or aryl, and the above R' represents alkylene.) [8] A phosphate bond of at least one nucleotide constituting the oligonucleotide. The moiety is a phosphorothioate bond, a phosphorodithioate bond, or an alkylphosphonate bond. , phosphoramidate bond, and boranophosphate bond. The antisense oligomer or its derivatives according to [6] or [7] above, A pharmaceutically acceptable salt or hydrate. [9] The antisense oligomer according to any one of [1] to [4], which is a morpholino oligomer. A pharmaceutically acceptable salt or hydrate thereof.

[10] The anti-cancer agent according to [9], which is a phosphorodiamidate morpholino oligomer. A sense oligomer or a pharmaceutically acceptable salt or hydrate thereof.

[11] The anti-cancer agent according to [4], which is a phosphorodiamidate morpholino oligomer. A sense oligomer or a pharmaceutically acceptable salt or hydrate thereof.

[12] The 5'-end of any one of the above [9] to

[11] is a group represented by any one of the following chemical formulas (1) to (3): Any of the antisense oligomers or pharmaceutically acceptable salts or hydrates thereof . [ka]

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

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

[14] The pharmaceutical composition according to

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

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

[12] above or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to

[13] or

[14] above for the treatment of muscular dystrophy. A method for treating muscular dystrophy, comprising administering to a patient suffering from muscular dystrophy.

[16] The patient with muscular dystrophy had exon 45 skipping in the dystrophin gene. The method of treatment according to

[15] above, wherein the patient has a mutation that is a target of the treatment.

[17] The method of treatment according to

[15] or

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

[18]

[0023] In the production of a pharmaceutical composition for treating muscular dystrophy, the method according to any one of [1] to

[12] above is used. Use of the antisense oligomer or a pharmaceutically acceptable salt or hydrate thereof.

[19] The compound according to any one of [1] to

[12] above for use in treating muscular dystrophy. An antisense oligomer or a pharmaceutically acceptable salt or hydrate thereof.

[20] In the treatment, a patient with muscular dystrophy has exon 45 skipping in the dystrophin gene. The antisense oligonucleotide according to

[19] above is used for a patient having a mutation that is a target of the antisense oligonucleotide. or a pharmaceutically acceptable salt or hydrate thereof. [twenty one] The antisense oligomer or its derivative according to

[19] or

[20] above, wherein the patient is a human. A pharmaceutically acceptable salt or hydrate of [Effects of the Invention]

[0014] The antisense oligomer of the present invention can be used to target exon 45 of the human dystrophin gene. Furthermore, administration of the pharmaceutical composition of the present invention can effectively induce thrombosis. This can effectively alleviate the symptoms of Duchenne muscular dystrophy. The deleted exons in the target patients are 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, etc. Examples include: [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 2] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 3] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 4] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 5] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 6]FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 7] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 8] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 9] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 10] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 11] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 12] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 13] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 14] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 15] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 16] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 17] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 18]FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 19] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 20] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 21] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 22] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 23] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 24] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells). [Figure 25] FIG. 1 shows the efficiency of exon 45 skipping of the human dystrophin gene in human rhabdomyosarcoma cells (RD 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. Therefore, it is not intended that the present invention be limited to only this embodiment. 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. The Japanese patent application on which this application claims priority (Patent Application No. 2015-18) was filed on the 15th of this month. This patent includes the contents described in the specification and drawings of Patent No. 2145.

[0017] 1. Antisense oligomers The present invention provides an enzyme capable of skipping the 45th exon of the human dystrophin gene. The present invention relates to a chemiluminescent oligomer or a pharmaceutically acceptable salt or hydrate thereof (hereinafter referred to as "the chemiluminescent oligomer of the present invention"). We provide a service called "Ligomar."

[0018] [Exon 45 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 45 of the human wild-type dystrophin gene is shown in SEQ ID NO: 13. , the nucleotide sequence of exon 45 of the human wild-type dystrophin gene (SEQ ID NO: 13) The sequence consisting of bases −5 to 15 counting from the 5′ end is shown in SEQ ID NO: 3. A sequence consisting of ~70 bases, a sequence consisting of 128-150 bases, a sequence consisting of 15-40 bases The sequence consisting of the above and the sequence consisting of bases 110 to 125 are shown in SEQ ID NOs: 4 to 6 and 143, respectively.

[0019] The oligomer of the present invention is capable of activating the dystrophin gene by skipping exon 45 of the human dystrophin gene. The protein encoded by the DMD-type dystrophin gene is called BMD-type dystrophin protein. Therefore, the oligomer of the present invention is Exon 45 of the dystrophin gene, which is the target of exon skipping, contains only the wild type. Not only that, but mutant forms are also included. Specifically, exon 45 of the mutant human dystrophin gene or a part thereof is The polynucleotide is a polynucleotide according to (I) or (II). (I) SEQ ID NO: 13, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143 a polynucleotide having a base sequence complementary to any of the base sequences selected from the group consisting of: a polynucleotide that hybridizes under stringent conditions; (II) SEQ ID NO: 13, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143 A base sequence having 90% or more identity to any base sequence selected from the group consisting of a polynucleotide consisting of

[0020] As used herein, the term "polynucleotide" refers to DNA or RNA. As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to a polynucleotide that hybridizes under stringent conditions. For example, SEQ ID NO: 13, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 14 A polynucleotide having a base sequence complementary to any of the base sequences selected from the group consisting of Using all or part of the octide as a probe, colony hybridization, plaque hybridization, By using hybridization or Southern hybridization, etc. The hybridization method is, for example, "S ambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Sprin g Harbor, Laboratory Press 2001" and "Ausubel, Current Protocols in Molecular Bio logy, John Wiley & Sons 1987-1997" can be used. do.

[0021] 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.

[0022] 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

[0023] 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. From the group consisting of SEQ ID NO: 13, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143 Any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143 A nucleic acid sequence based on all or part of a nucleic acid sequence complementary to any of the nucleic acid sequences selected from the group consisting of: When preparing probes, commercially available reagents (e.g., PCR labeling mix (Roche, DG)) are used. When the probe is labeled with digoxigenin (DIG) using a method such as IAGNOS, Hybridization was detected using an IG nucleic acid detection kit (Roche Diagnostics). It is possible.

[0024] 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. In this case, SEQ ID NO: 13, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143 90% or more, 91% or more of a sequence consisting of any polynucleotide selected from the group consisting of: 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more and polynucleotides having 99.8% or more, or 99.9% or more identity thereto. do. 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.

[0025] In one embodiment, the oligomer of the present invention comprises two selected from the group consisting of: An antisense oligomer having a length of 14 to 32 bases, in which the unit oligomers are linked, or a pharmaceutical composition thereof The compound is a physiologically acceptable salt or hydrate. (a) Positions -5 to 15 from the 5' end of the 45th exon of the human dystrophin gene A sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences oligomers consisting of strings of units; (b) Positions 48-70 from the 5' end of the 45th exon of the human dystrophin gene A sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences oligomers consisting of strings of units; (c) From the 5' end of exon 45 of the human dystrophin gene to positions 128-150 Complementary to a contiguous nucleotide sequence of 7 to 16 bases selected from the nucleotide sequence a unit oligomer consisting of a sequence of (d) 15th to 40th residues from the 5' end of the 45th exon of the human dystrophin gene A sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences an oligomer of units consisting of a series of units; and (e) From the 5' end of the 45th exon of the human dystrophin gene to positions 110-125 Complementary to a contiguous nucleotide sequence of 7 to 16 bases selected from the nucleotide sequence A unit oligomer consisting of a specific base sequence.

[0026] Each of the unit oligomers (hereinafter sometimes simply referred to as "unit") of (a) to (e) above The size of each unit is 7 to 16 bases long, preferably 8 to 16 bases long, or 9 to 16 bases long. The knits may be the same size or different sizes.

[0027] In addition, when selecting two unit oligomers from the group consisting of (a) to (e), Ligomers are the same combinations of (a) to (e) (i.e., (a) and (a), (b) and (b), (c) and (c), (d) and (d), (e) and (e)) may be used, or different combinations may be used, but preferably different combinations. For example, if you select (a) as one unit, the other unit will be It is preferable that the unit be one of (b) to (e). Similarly, if the unit (b) is selected as one of the units, In this case, the other unit is preferably (a), (c), (d) or (e), and If one person chooses unit (c), the other unit will be (a), (b), (d) or (e). It is preferable that

[0028] If you select two units from (a) to (e), which of the two selected units is It may be located on the 5' end, but if (a) and (b) are selected, unit (a) If (b) and (c) are selected, unit (b) is linked to the 3' end. If (a) or (c) is selected, unit (a) is linked to the 3' end. If (a) and (d) are selected, unit (a) is linked to the 3' end, and (a) and If option (e) is selected, it is preferable that unit (a) is linked to the 3' end.

[0029] Here, "connected" means that two units selected from (a) to (e) are directly connected. In other words, when two units are linked, the unit located at the 5' end The 3' end of the unit and the 5' end of the unit located on the 3' end side are connected by a phosphate bond or the following group: It means to form. [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.

[0030] "Enables skipping of the 45th exon of the human dystrophin gene" , a region corresponding to exon 45 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 44, the base corresponding to the 3' end of exon 43 The base corresponding to the 5' end of exon 46 is linked, and no codon frameshift occurs. This means that mature mRNA is formed.

[0031] 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.

[0032] Whether exon 45 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 45 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 45 was skipped were analyzed. The polynucleotide amount of the band in which exon 45 was not skipped is "A" and the polynucleotide amount of the band in which exon 45 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

[0033] Preferably, the oligomer of the present invention is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more Exon 45 skipping efficiency of ≥ 60%, ≥ 70%, ≥ 80%, and ≥ 90% . For the calculation of skipping efficiency, see WO 2012 / 029986. can.

[0034] The oligomer of the present invention may be, for example, an oligonucleotide having a length of 14 to 32 bases. amides, morpholino oligomers, or peptide nucleic acid (PNA) oligomers Preferably, the oligomer of the present invention has a length of 16 to 30 bases, 17 to 30 bases, 18-30 bases, 19-30 bases, 20-30 bases, 20-29 bases, 20-28 bases, 20-27 bases, 20-26 bases It is preferable that the length of the oligonucleotide is 21 to 26 bases and that the oligonucleotide is a morpholino oligomer.

[0035] 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.

[0036] Modified nucleotides are nucleotides that are ribonucleotides or deoxyribonucleotides. It refers to a molecule in which all or part of the acid-base, sugar moiety, and phosphate binding moiety are modified.

[0037] 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.

[0038] 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.

[0039] 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).

[0040] 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.

[0041] 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. .

[0042] 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. This can be done.

[0043] Acyl may include straight or branched chain alkanoyl or aroyl. Examples of alkanoyl include formyl, acetyl, 2-methylacetyl, 2, 2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2- Examples of the aroyl include benzoyl, ... Examples of the aroyl include zoyl, toluoyl, and naphthoyl. It may be substituted at position 1 or may be substituted with alkyl.

[0044] 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.)

[0045] 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).

[0046] 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.

[0047] 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.)

[0048] 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:

[0049] [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 an integer ranging from 1 to 99, and preferably an integer ranging from 13 to 31. is an integer.]

[0050] PMO(I) can be produced according to a known method, for example, by carrying out the following steps: It can be produced by 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.

[0051] All of the following steps can be carried out using either the liquid phase method or the solid phase method (manual or commercially available solid phase automated synthesizers). When PMOs are produced by the solid phase method, the procedure is simplified and From the viewpoint of synthesis accuracy and the like, a method using an automatic synthesizer is preferable.

[0052] (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).

[0053] 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 to synthesize a morpholino nucleic acid derivative , acetonitrile, tetrazole, N-methylimidazole, pyridine, acetic anhydride, lutidine (ii) It is used in the synthesis of morpholino nucleic acid derivatives. (iii) chemically stable to potential reagents; (iv) capable of chemical modification; (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% divinylbenzene cross-linked (200-400 mesh) (2.4-3.0 mmol / g) (Tokyo Chemical Industry Co., Ltd.), Aminomethylated Polystyrene Resin·HCl [Divinyl 1% phenylbenzene, 100-200 mesh (Peptide Institute), non-swelling polystyrene (e.g., Primer Support (GE Healthcare)), PEG-linked polystyrene (e.g., , NH2-PEG resin (Watanabe Chemical Co., Ltd.), TentaGel resin, controlled pore glass glass (CPG) (e.g., manufactured by CPG), oxalyl-controlled hole glass (e.g., Alul et al., Nucleic Acids Research, Vol. 19, 1527 (1991)), TentaGel support - amino polyethylene Ricoh derivatized supports (e.g., Wright et al., Tetrahedron Letters, Vol. 34, 3373 (1993) ), and Poros-polystyrene / divinylbenzene copolymers.

[0054] The "linker" is a publicly known compound that is usually used to link nucleic acids or morpholino nucleic acid derivatives. Known amines can be used, for example, 3-aminopropyl, succinyl, 2,2'-diaminopropyl, 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 mole of compound (II). The range of 0.1 to 1000 molar equivalents is suitable, and preferably 1 to 100 molar equivalents. The range is within the range of 1 / 2 equivalent. 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.

[0057] When a salt or a mixture of an acid and an organic amine is used in this step, for example, triflate Examples include a salt or mixture of diethyl amine and diethyl amine, more particularly triethyl amine. A mixture of 2 equivalents of fluoroacetic acid and 1 equivalent of triethylamine can be given. . 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.

[0058] The reaction temperature in the above reaction is preferably within the range of, for example, 10°C to 50°C, and 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.

[0059] 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 examples thereof include diisopropyl The base should be in a concentration range of 0.1% (v / v) to 30% (v / v). It can also be used after diluting with a suitable solvent as shown below. 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.

[0060] In the compound (II), n=1 and L is a group (IV), and the compound represented by the following general formula (IIa) The compound represented by the formula (IIa) can be prepared by the following method. can be done. [ka] [In the formula, B P , T, linker, and solid phase support are as defined above.]

[0061] 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.

[0062] This step is carried out by a known linker introduction reaction using compound (V) as a starting material. This can be done. 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.]

[0063] Step 2: Compound (VI) is reacted with a solid support by reacting with a condensing agent or the like to give compound (II a) A process for producing the product. [ka] [In the formula, BP , 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.

[0064] 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) is a compound obtained by the steps A and B of the method for producing PMO described herein, starting from compound (IIa). The production can be carried out by repeating step B a desired number of times. [ka] [In the formula, B P , R 2 , R 3 , T, linker, and solid support are as defined above; n' represents 1 to 98.

[0065] 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 prepared, for example, by the method described in WO 1991 / 009033. Cut. [ka] [In the formula, B P , T has the same meaning as above.]

[0066] 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 (IIb) can be prepared by the method for producing PMO described in this specification using compound (IIb) as a starting material. The step A can be produced by repeating the steps A and B as many times as desired. [ka] [In the formula, B P , n', R 2 , R 3 , T has the same meaning as above.]

[0067] In addition, in the compound (II), n=1 and L is acyl, and the compound (II) is represented by the following general formula (IIc): The compound to be synthesized can be obtained by subjecting compound (IIb) to a method known as an acylation reaction. It can be produced by the following. [ka] [In the formula, B P , T is as defined above; R 5 represents acyl.]

[0068] 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.]

[0069] (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.]

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

[0071] Examples of the morpholino monomer compound include compounds represented by the following general formula (VIII): It can be done. [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 diisopropylethylamine, triethylamine, and the like. Examples of the base include N-ethylamine and N-ethylmorpholine. For example, the amount is suitably within the range of 1 to 1000 molar equivalents relative to 1 mole of compound (III). The amount is preferably within the range of 10 to 100 molar equivalents.

[0072] The morpholino monomer compound and base that can be used in this step should be diluted to a concentration of 0.1% to 30%. The solvent may be any solvent that is not involved in the reaction. For example, but not limited to, N,N-dimethylimidazolidone, N-methylpiperidone, DMF, Examples include dichloromethane, acetonitrile, tetrahydrofuran, or mixtures thereof. This can be done.

[0073] The reaction temperature is, for example, preferably in the range of 0°C to 100°C, more preferably in the range of 10°C to 50°C. It is within the enclosure. 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.

[0074] Furthermore, after the completion of this step, an acylating agent can be added, if necessary. Examples of the "agent" 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 may be diluted before use. There is no particular limitation on the solvent as long as it does not participate in the reaction. However, for example, dichloromethane, acetonitrile, alcohols (ethanol, isopropanol, Examples of suitable solvents include alcohol, trifluoroethanol, water, and mixtures 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.

[0075] (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.]

[0076] This step can be carried out by reacting compound (VII) with a deprotecting agent.

[0077] Examples of the "deprotecting agent" include concentrated aqueous ammonia and methylamine. The "deprotecting agent" that can be used in this step is, for example, water, methanol, ethanol, isopropyl alcohol, or the like. Alcohol, acetonitrile, tetrahydrofuran, DMF, N,N-dimethylimidazolide It can also be used by diluting it with ethanol, N-methylpiperidone or a mixture of these solvents. Ethanol is also preferred. The amount of the deprotecting agent used is, for example, 1 mole of compound (VII). For example, a range of 1 molar equivalent to 100,000 molar equivalents is suitable, and preferably 10 molar equivalents. The range is from 1 molar equivalent to 1000 molar equivalents.

[0078] The reaction temperature is, for example, suitably within the range of 15°C to 75°C, preferably within the range of 40°C to 70°C. The deprotection reaction time is preferably within a range of 50°C to 60°C. Although it varies depending on the type of compound, reaction temperature, etc., a range of 10 minutes to 30 hours is suitable, and 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.

[0079] (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.]

[0080] This step can be carried out by adding an acid to compound (IX).

[0081] The "acid" that can be used in this step includes, for example, trichloroacetic acid, dichloroacetic acid, acetic acid, Examples of the acid include phosphoric acid and hydrochloric acid. The amount of acid used is determined, for example, by the pH of the solution. It is suitable to use it so that it falls within the range of 0.1 to 4.0, and more preferably within the range of 1.0 to 3.0. The solvent is not particularly limited as long as it does not participate in the reaction. For example, acetonitrile, water, or a mixed solvent thereof can be mentioned.

[0082] 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, reaction temperature, etc., a range of 0.1 minutes to 5 hours is appropriate, and preferably 1 The time is in the range of 1 minute to 1 hour, and more preferably in the range of 1 minute to 30 minutes.

[0083] PMO(I) can be separated and purified from the reaction mixture obtained in this step by conventional separation and purification methods, such as extraction, concentration, Neutralization, filtration, centrifugation, recrystallization, C8 to C 18 Reversed-phase column chromatography of cations exchange column chromatography, anion exchange column chromatography, gel filtration column methods such as column chromatography, high performance liquid chromatography, dialysis, and ultrafiltration Alternatively, the desired PMO(I) can be obtained by using them in combination, and the desired PMO(I) can be isolated and purified. (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.

[0084] 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)

[0085] 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)"

[0086] 2. Pharmaceutical Compositions The oligomer of the present invention enables skipping of exon 45 of the dystrophin gene. Therefore, a pharmaceutical composition containing the oligomer of the present invention is administered to a dystrophin gene containing exon 45. DM with a mutation that is eligible for skipping (a mutation that is in-frame with exon 45 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")

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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. However, 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 The oligomer of the present invention contained in the composition of the present invention is more preferably in the range of 1 μM. The weight ratio of the carrier (carrier / oligomer of the present invention) depends on the nature of the oligomer and the type of the carrier. Although it varies depending on factors such as the amount of water, a range of 0.1 to 100 is appropriate, a range of 1 to 50 is preferable, and a range of 10 to 2 It is more preferable that it is in the range of 0.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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:

[0097] 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]

[0098] [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

[0099] 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.

[0100] Step 2: Aminopolystyrene Resin-Supported 4-{[(2S,6R)-6-(4-benzamide] -2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4 -Production of oxobutanoic acid 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl) 4.0 g of [4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid was dissolved in pyridine ( Dissolve 0.73 g of 4-DMAP and 1-ethyl-3-(3-dimethylaminopropyl) carboxymethylcellulose in 200 mL of dehydrated water. 11.5 g of rubodiimide hydrochloride was added. Then, aminopolystyrene resin Primer support 25.0 g of 200 amino acid (GE Healthcare Japan, 17-5214-97) and 8.5 mL of triethylamine The mixture was added and shaken at room temperature for 4 days. After the reaction, the resin was filtered off. The resulting resin was dissolved in pyridine, methyl The resin was washed with ethanol and dichloromethane in that order and dried under reduced pressure. 200 mL of ran (dehydrated), 15 mL of acetic anhydride, and 15 mL of 2,6-lutidine were added and shaken at room temperature for 2 hours. The resin was collected by filtration, washed with pyridine, methanol, and dichloromethane in that order, and dried under reduced pressure. 0.7g of the desired product was obtained.

[0101] 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. 0.2μmol / g. UV measurement conditions Equipment: U-2910 (Hitachi) Solvent: methanesulfonic acid Wavelength: 409 nm ε value: 45000

[0102] [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.

[0103] [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.

[0104] [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.

[0105] According to the description of Example 1 below, PMOs (R 2 , R 3 Hamechi The synthesized PMO was dissolved in water for injection (Otsuka Pharmaceutical Factory, It was dissolved in HCl (manufactured by Sigma-Aldrich).

[0106] [Table 1-1]

[0107] [Table 1-2]

[0108] [Table 1-3]

[0109] [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).

[0110] [Table 2]

[0111] 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.

[0112] 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.

[0113] [Table 3]

[0114] 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.

[0115] [Table 4]

[0116] 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.

[0117] [Table 5]

[0118] 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.

[0119] [Table 6-1]

[0120] [Table 6-2]

[0121] [Table 6-3]

[0122] [Test Example 1] In vitro assays RD cells (human rhabdomyosarcoma cell line) 3.5 x 10 5 For each individual, antisense oligomers 1 to 3 in Table 1 were used. 10 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 bovine serum (FBS) (Invitrogen). In 2 mL of EMEM (Sigma), the culture 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).

[0123] 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

[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: 1) Reverse primer: 5'-GGGCAACTCTTCCACCAGTA -3' (SEQ ID NO: 2)

[0125] 1 μL of the PCR reaction product was analyzed using a Bioanalyzer (Agilent) and a MultiNA (Shimadzu). Analysis was performed using a fluororesin (manufactured by Sigma-Aldrich). The polynucleotide amount of the band in which exon 45 was skipped, "A", and the polynucleotide amount of the band in which exon 45 was skipped, "B", are shown. The amount of polynucleotides in the unpaired bands, "B," was measured. Based on the fixed values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / ( A + B ) x 100

[0126] The experimental results are shown in Figures 1 to 5, 8, 10, 11 and 16 to 24. was found to effectively skip exon 45.

[0127] [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 oligomer of the present invention alone (PMO No. 11 or PMO No. 9) Two individual unit oligomers or a mixture thereof were incubated at 3 μM each in Amaxa Cell Line N The cells were transfected using Nucleofector Kit L with Nucleofector II (Lonza). 30 was used. The combinations of introduced sequences are as follows:

[0128] [Table 7]

[0129] Experimental results The results are shown in Figures 6 and 25. This experiment identified two enzymes that target different sites within exon 45. PMO No. 11 (SEQ ID NO: 10), PMO No. 9 (SEQ ID NO: 8), or The oligomer of the present invention, PMO No. 72 (SEQ ID NO: 79), is composed of individual antisense oligonucleotides. oligomers (PMO No. 27 (SEQ ID NO: 36), PMO No. 28 (SEQ ID NO: 37), PMO No. 25 (SEQ ID NO: 38) No. 34), PMO No. 26 (SEQ ID NO: 35), PMO No. 82 (SEQ ID NO: 144), or PMO No. 83 (SEQ ID NO: No. 145)) or mixtures thereof (PMO No. 27 and PMO No. 28, PMO No. 25 and PMO No. 26, or PMO No. 82 and PMO No. 83), skipping exon 45 with high efficiency. It was discovered that...

[0130] [Test Example 3] In vitro assays 2'-O-methoxy-phosphoro Experiments were performed using antisense oligomers of thioate (2'-OMe-S-RNA). The various antisense oligomers used in the assay were purchased from Japan Bioservices. The sequences of the various antisense oligomers are shown below.

[0131] [Table 8-1]

[0132] [Table 8-2]

[0133] 5 x 10 RD cells (human rhabdomyosarcoma cell line) in a 24-well plate 4 100 cells / well and cultured in 10% fetal bovine serum ( Eagle's minimal essential medium (EMEM) containing FCS (Invitrogen) The cells were cultured overnight in 0.5 mL of Sigma-Aldrich (Sigma, hereafter the same) at 37°C under 5% CO2 conditions. Various antisense oligomers for Son45 skipping (Japan Bioservices) (1 μM or 300 nM) and Lipofectamine 2000 (Invitrogen) were complexed and 0.45 mL 50 μL per well of the solution was added to the RD cells whose medium had been replaced in step 1, to give a final concentration of 100 nM or 30 nM. After the addition, the cells were cultured overnight. After washing once with PBS (manufactured by Nissui Co., Ltd., the same applies below), the cells were Buffer RLT (Qiagen) containing 2-mercaptoethanol (Nacalai Tesque) was added for 35 min. Add 0 μL of the solution to the cells, leave it at room temperature for a few minutes to lyse the cells, and then homogenize using a QIAshredder homogenizer. The cells were collected in a Qiagen tube and centrifuged at 15,000 rpm for 2 minutes to prepare a homogenate. Total RNA was extracted according to the protocol attached to the Neasy Mini Kit (Qiagen). The concentration of the extracted total RNA was measured using a NanoDrop ND-1000 (LMS). Ta.

[0134] 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

[0135] 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: 1) Reverse primer: 5'-GGGCAACTCTTCCACCAGTA -3' (SEQ ID NO: 2)

[0136] 1 μL of the PCR reaction product was analyzed using a Bioanalyzer (Agilent) and a MultiNA (Shimadzu). Analysis was performed using a chromatograph (manufactured by the company). The polynucleotide amount of the band in which exon 45 was skipped, "A", and the polynucleotide amount of the band in which exon 45 was skipped, "B", are shown. The amount of polynucleotides in the unpaired bands, "B," was measured. Based on the fixed values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / ( A + B ) x 100

[0137] Experimental results The results are shown in Figures 7 and 12 to 15. This experiment demonstrated that the antisense oligomer of the present invention is effective It was found to effectively skip exon 45.

[0138] [Test Example 4] 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 For each of these, the oligomer of the present invention alone (PMO No. 2, PMO No. 31 or PMO No. 32) The two individual unit oligomers constituting the oligomers were incubated at a concentration of 3 μM or 10 μM in Amaxa Cell Lin e The transfection was performed using Nucleofector Kit L with Nucleofector II (Lonza). The T-030 was used. The combination of introduced sequences is as follows:

[0139] [Table 9]

[0140] Experimental results The results are shown in Figure 9. This experiment identified two amplicons targeting different sites within exon 45. PMO No. 2 (SEQ ID NO: 7), PMO No. 31 (SEQ ID NO: 11), or PMO No. The oligomer of the present invention of 32 (SEQ ID NO: 12) is composed of the individual antisense nucleic acids (PM PMO No. 66, PMO No. 63, PMO No. 64, or PMO No. 65) It was discovered that skipping was the solution. [Industrial Applicability]

[0141] 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. It has been shown that this gene induces exon 45 skipping in the present invention. Gomers are very useful in the treatment of DMD.

Claims

1. 14, in which two unit oligomers selected from the group consisting of (a) to (e) below are linked together: An antisense oligomer having a length of up to 32 bases, wherein the two unit oligomers are consecutive or alternate. antisense oligomers or pharmaceutically acceptable salts thereof, or hydrate: (a) Positions −5 to 15 from the 5' end of the 45th exon of the human dystrophin gene A nucleotide sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences A unit oligomer consisting of a group sequence; (b) 48th to 70th residues from the 5' end of the 45th exon of the human dystrophin gene A nucleotide sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences A unit oligomer consisting of a group sequence; (c) positions 128-150 from the 5' end of the 45th exon of the human dystrophin gene complementary to a contiguous nucleotide sequence of 7 to 16 bases selected from the nucleotide sequence a unit oligomer consisting of a base sequence; (d) 15th to 40th residues from the 5' end of the 45th exon of the human dystrophin gene A nucleotide sequence complementary to a nucleotide sequence of 7 to 16 consecutive bases selected from the nucleotide sequences a unit oligomer consisting of a group sequence; and (e) positions 110 to 125 from the 5' end of the 45th exon of the human dystrophin gene complementary to a contiguous nucleotide sequence of 7 to 16 bases selected from the nucleotide sequence A unit oligomer consisting of a specific base sequence.

2. The antisense oligomer according to claim 1, wherein one of the two unit oligomers is (a).

1. An oligomer or a pharmaceutically acceptable salt or hydrate thereof.

3. Any of the sequences selected from the group consisting of SEQ ID NOs: 7 to 12, 14 to 33, 40 to 52, 57, 64, 65, and 79 to 86 The antisense oligomer or the pharmaceutical composition thereof according to claim 1 or 2, which comprises one or more base sequences. A commercially acceptable salt or hydrate thereof.

4. Any one of the base sequences selected from the group consisting of SEQ ID NOs: 8, 10, 25, 30, 33, 79, and 80 The antisense oligomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, Possible salts or hydrates.

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

6. The sugar moiety and / or linker of at least one nucleotide constituting the oligonucleotide 6. The antisense oligomer or pharmaceutical composition thereof according to claim 5, wherein the phosphate binding moiety is modified. A commercially acceptable salt or hydrate thereof.

7. 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 7. The antisense oligonucleotide according to claim 5 or 6, wherein the ribose is substituted with any of the selected groups. A pharmaceutically acceptable salt or hydrate thereof. (The above R represents alkyl or aryl, and the above R' represents alkylene.)

8. 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 The antisense oligomer or its pharmaceutically acceptable salt thereof according to claim 6 or 7, Possible salts or hydrates.

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

10. 10. The antisense oligo of claim 9, which is a phosphorodiamidate morpholino oligomer.

5. A medicament for the treatment of rhesus malabsorption, or a pharmaceutically acceptable salt or hydrate thereof.

11. The antisense oligonucleotide of claim 4, which is a phosphorodiamidate morpholino oligomer. Ligomer or a pharmaceutically acceptable salt or hydrate thereof.

12. The 5'-end of any one of the following chemical formulas (1) to (3) is a group according to any one of claims 9 to 11. The antisense oligomer described above, or a pharmaceutically acceptable salt or hydrate thereof. 【Chemistry 25】

13. The antisense oligomer according to any one of claims 1 to 12, its pharmaceutically acceptable salt A pharmaceutical composition for treating muscular dystrophy, comprising a salt or hydrate thereof as an active ingredient.

14. 14. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable carrier.

15. The antisense oligomer according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 13 or 14, - A method for treating muscular dystrophy, comprising administering to a patient

16. The muscular dystrophy patient is subject to exon 45 skipping in the dystrophin gene. The method of claim 15, wherein the patient has a mutation.

17. 17. The method of claim 15 or 16, wherein the patient is a human.

18. A method according to any one of claims 1 to 12 for the manufacture of a pharmaceutical composition for treating muscular dystrophy. Use of an antisense oligomer of the formula (I) or a pharmaceutically acceptable salt or hydrate thereof.

19. The antisense oligonucleotide according to any one of claims 1 to 12 for use in treating muscular dystrophy.

1. An oligomer or a pharmaceutically acceptable salt or hydrate thereof.

20. In the treatment, a patient with muscular dystrophy has exon 45 skipping in the dystrophin gene. The antisense oligomer or antibody of claim 19 is used in a patient having a mutation targeted for the treatment of cancer. is a pharmaceutically acceptable salt or hydrate thereof.

21. The antisense oligomer or the pharmaceutical composition thereof according to claim 19 or 20, wherein the patient is a human. A commercially acceptable salt or hydrate thereof.

Images