Antisense nucleic acids

Abstract

This invention provides a drug that efficiently skips the 53rd exon of the human dystrophin gene. [Solution] The present invention provides an oligomer that enables skipping of the 53rd exon of the human dystrophin gene.

Description

[Technical Field]

[0001] This invention enables skipping of the 53rd exon of the human dystrophin gene. This invention relates to an antisense oligomer and a pharmaceutical composition containing the oligomer. [Background technology]

[0002] Duchenne muscular dystrophy (DMD) is the most common type of muscular dystrophy, affecting approximately 1 in 3,500 male births. It is a highly severe hereditary progressive muscular atrophy. In infancy, the symptoms are almost indistinguishable from those of a normal person. While motor function is present, muscle weakness is observed from around 4-5 years of age. This muscle weakness then progresses until around 12 years of age. It is a serious disease that can lead to inability to walk and death from heart failure or respiratory failure in one's 20s. Currently, there is no effective treatment for DMD, and the development of new therapeutic drugs is urgently needed.

[0003] DMD is known to be caused by mutations in the dystrophin gene. The gene is located on the X chromosome and is a huge gene consisting of 2.2 million base pairs of DNA. It is transcribed into a precursor, and then splicing removes the introns and binds to 79 exons. The resulting mRNA is synthesized. This mRNA is then translated into 3,685 amino acids, which are then converted into dystrophintan. Proteins are produced. Dystrophin protein is involved in maintaining the membrane stability of muscle cells. It is necessary to make muscle cells less susceptible to damage. The dystrophin gene in DMD patients is mutated. Because of this, the functional dystrophin protein is hardly expressed in muscle cells. Therefore, in DMD patients, the structure of muscle cells cannot be maintained, and large amounts of calcium are needed. Ions flow into muscle cells. As a result, an inflammation-like reaction occurs, and fibrosis progresses, causing muscle damage. Cells become less likely to regenerate.

[0004] Becker muscular dystrophy (BMD) is also caused by mutations in the dystrophin gene, but its symptoms show muscle weakness due to muscle atrophy, generally being mild compared to DMD, and the progression of muscle weakness is also slow, and in many cases, it develops in adulthood. The difference in clinical symptoms between DMD and BMD is thought to be due to whether the amino acid reading frame is disrupted or maintained when dystrophin mRNA is translated into dystrophin protein due to the mutation (Non-Patent Document 1 ). That is, in DMD, due to having a mutation that shifts the amino acid reading frame, almost no functional dystrophin protein is expressed, but in BMD, although a part of the exon is deleted due to the mutation, the amino acid reading frame is maintained, so an incomplete but functional dystrophin protein is produced.

[0005] As a treatment method for DMD, exon skipping is expected. This method repairs the amino acid reading frame of dystrophin mRNA by modifying splicing and induces the expression of a partially function-restored dystrophin protein (Non-Patent Document 2 ). The amino acid sequence part targeted for exon skipping will be lost. Therefore, the dystrophin protein expressed by this treatment will be shorter than normal, but because the amino acid reading frame is maintained, the function of stabilizing muscle cells is partially retained. Therefore , it is expected that by exon skipping, DMD will exhibit symptoms similar to those of milder BMD. The exon skipping method has undergone animal experiments using mice and dogs [Clinical trials are being conducted on human DMD patients.

[0006] Exon skipping can occur at either the 5' or 3' splice site, or both. This can be induced by the binding of antisense nucleic acids that target the inside of exons. The xon is mR only when both splice sites are recognized by the spliceosome complex. It is included in NA. Therefore, targeting the splice site with antisense nucleic acid is possible. This allows for the induction of exon skipping. Also, when exons are spliced... In order to be recognized by the splicing mechanism, the SR (Splicing Rate) to the Exons Splicing Enhancer (ESE) It is thought that protein binding is necessary, and by targeting ESE, This can induce Kuson to skip.

[0007] Because dystrophin gene mutations differ among DMD patients, the location and type of gene mutations are important. An antisense nucleic acid is needed accordingly. To date, Steve Wi of the University of Western Australia has... Rutton et al. developed an anti-sensor that induces exon skipping for all 79 exons. Nucleic acids have been produced (Non-Patent Document 3), by Annemieke Aartsma-Rus et al. in the Netherlands. Antisense nucleic acids that induce exon skipping for 39 types of exons have been created. (Non-patent document 4)

[0008] Approximately 8% of all DMD patients skip exon 53 (hereinafter referred to as "exon 53"). It is thought that it can be treated by pinging. In recent years, the exophen Several research institutions have reported on studies targeting exon skipping with n53. This has been done (Patent Documents 1-4; Non-Patent Document 5). However, to efficiently extract Exon 53 The technique for skipping flights has not yet been established. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Public Gazette WO 2006 / 000057 [Patent Document 2] International Public Gazette WO 2004 / 048570 [Patent Document 3] : U.S. Patent Publication US 2010 / 0168212 [Patent Document 4] International Public Gazette WO 2010 / 048586 [Non-patent literature]

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

[0011] In the situation described above, the skipping of exon 53 of the dystrophin gene is strongly Antisense oligomers that induce muscular dystrophy and muscular dystrophy treatments containing these oligomers are desired. It is being made. The inventors have conducted a detailed study of the structure of the dystrophin gene and found that the dystrophin gene Of the mRNA precursor of the gene (hereinafter referred to as "pre-mRNA"), the 32nd to 5th exon from the 5' end of exon 53 The sequence consisting of nucleotides around the 6th position is targeted with an antisense oligomer. We have found that by doing so, we can induce exon 53 skipping with high efficiency. Based on this knowledge, they completed the present invention.

[0012] In other words, the present invention is as follows: [1] An engineer that enables skipping of the 53rd exon of the human dystrophin gene. It is a chisense oligomer, and is the 5' end of exon 53 of the human dystrophin gene. From the end, the 31st to 53rd, the 31st to 54th, the 31st to 55th, the 31st to 56th, the 31st to 57th, the 31st ~58th, 32nd-53rd, 32nd-54th, 32nd-55th, 32nd-56th, 32nd-57th, Numbers 32-58, 33-53, 33-54, 33-55, 33-56, and 33-57 The 33rd to 58th, the 34th to 53rd, the 34th to 54th, the 34th to 55th, the 34th to 56th, the 34th 57th, 34th-58th, 35th-53rd, 35th-54th, 35th-55th, 35th-56th, 35th-57th, 35th-58th, 36th-53rd, 36th-54th, 36th-55th, 36th-56th , complementary to one of the sequences consisting of nucleotides 36-57 or 36-58 An antisense oligomer consisting of a nucleotide sequence. [2] The antisense oligomer described in [1], wherein the oligonucleotide is an oligonucleotide. [3] The sugar portion of at least one nucleotide constituting the oligonucleotide and / or The antisense oligomer described in [2] above, wherein the phosphate bond portion is modified. [4] The sugar portion of at least one nucleotide constituting the oligonucleotide is 2' The -OH group at the position consists of OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, and I. The antiseptic described in [3] above, which is ribose substituted with any group selected from the group. Nsu oligomer. (The above R represents alkyl or aryl, and the above R' represents alkylene.) [5] The phosphate bond portion of at least one nucleotide constituting the oligonucleotide However, phosphorothioate bond, phosphorodithioate bond, alkylphosphonate bond, Selected from the group consisting of phosphoramidate bonds and boranophosphate bonds. The antisense oligomer described in [3] or [4] above, wherein either one of the above is the antisense oligomer. [6] The antisense oligomer described in [1] above, which is a morpholino oligomer. [7] The antisense described in [6] above, which is a phosphorodiamidate morpholino oligomer. Oligomer. [8] Any of the above [1] to [7] whose 5' end is a group of any of the following chemical formulas (1) to (3). The antisense oligomer described in item 1. [ka] [9] From the 5' end of the 53rd exon of the human dystrophin gene, the 32nd to 56th or The sequence consisting of nucleotides 36 to 56 consists of a base sequence complementary to the sequence of [1] to [8] above. Either one of the antisense oligomers described in item 1.

[10] The above [1 An antisense oligomer as described in any one of items [8].

[11] Any one nucleotide sequence selected from the group consisting of sequence numbers 11, 17, 23, 29 and 35 An antisense oligomer according to any one of the above [1] to [8], comprising:

[12] Any one of the above [1] to [8] consisting of either the base sequence of SEQ ID NO: 11 or 35 The antisense oligomer described above.

[13] Antisense oligomers described in any one of the above paragraphs [1] to

[12] , the pharmaceutically acceptable ones. A pharmaceutical composition for the treatment of muscular dystrophy, comprising a possible salt or hydrate as an active ingredient.

[0013] The antisense oligomer of the present invention enables the suppression of exon 53 of the human dystrophin gene. It is possible to induce kipping with high efficiency. Furthermore, when the pharmaceutical composition of the present invention is administered... This can effectively alleviate the symptoms of Duchenne muscular dystrophy. . [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows the skipping efficiency of human dystrophin gene exon 53 in human rhabdomyosarcoma cell lines (RD cells). [Figure 2]This figure shows the skipping efficiency of exon 53 of the human dystrophin gene in cells differentiated into muscle cells by introducing the human myoD gene into human normal tissue-derived fibroblasts (TIG-119 cells). [Figure 3] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene in cells differentiated into muscle cells by introducing the human myoD gene into fibroblasts (5017 cells) derived from human DMD patients. [Figure 4] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene in fibroblasts derived from human DMD patients (exon 45-52 deletion) that were differentiated into muscle cells by introducing the human myoD gene. [Figure 5] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene in fibroblasts derived from human DMD patients (exon 48-52 deletion) that were differentiated into muscle cells by introducing the human myoD gene. [Figure 6] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene in fibroblasts derived from human DMD patients (exon 48-52 deletion) that were differentiated into muscle cells by introducing the human myoD gene. [Figure 7] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene in fibroblasts derived from human DMD patients (deletion of exons 45-52 or exon 48-52) that were differentiated into muscle cells by introducing the human myoD gene. [Figure 8] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene in fibroblasts derived from human DMD patients (exon 45-52 deletion) that were differentiated into muscle cells by introducing the human myoD gene. [Figure 9] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 10]This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 11] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 12] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 13] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 14] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 15] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 16] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 17] This figure shows the skipping efficiency of exon 53 of the human dystrophin gene (2'-OMe-S-RNA) in human rhabdomyosarcoma cells (RD cells). [Figure 18] This figure shows the skipping efficiency of human dystrophin gene exon 53 at different oligomer concentrations in human rhabdomyosarcoma cells (RD cells). [Figure 19] This figure shows the skipping efficiency of human dystrophin gene exon 53 at different oligomer concentrations in human rhabdomyosarcoma cells (RD cells). [Best Mode for Carrying Out the Invention]

[0015] The present invention will be described in detail below. The following embodiments are illustrative examples for illustrating the present invention. However, the present invention is not intended to be limited to these embodiments only. The present invention does not deviate from its gist. Unless otherwise specified, it can be implemented in various forms. Furthermore, all documents cited in this specification, as well as published gazettes, patent gazettes, and other patents References shall be incorporated herein by reference. This specification is also subject to change as of September 1, 2010. Specification of the Japanese patent application (Japanese Patent Application No. 2010-196032) on which the priority claim of this application is based. It also includes the contents described in the drawings.

[0016] 1. Antisense oligomers This invention enables skipping of the 53rd exon of the human dystrophin gene. It is an antisense oligomer, and is the 53rd exon of the human dystrophin gene. From the 5' end, positions 31-53, 31-54, 31-55, 31-56, and 31-57 , 31st-58th, 32nd-53rd, 32nd-54th, 32nd-55th, 32nd-56th, 32nd-57th 33rd, 32nd-58th, 33rd-53rd, 33rd-54th, 33rd-55th, 33rd-56th, 33rd ~57th, 33rd-58th, 34th-53rd, 34th-54th, 34th-55th, 34th-56th, Numbers 34-57, 34-58, 35-53, 35-54, 35-55, 35-56 The 35th to 57th, the 35th to 58th, the 36th to 53rd, the 36th to 54th, the 36th to 55th, the 36th A sequence consisting of the 56th, 36th-57th, or 36th-58th nucleotides (hereinafter referred to as "target sequence") It is also called an antisense oligomer consisting of a base sequence complementary to one of the following. The following invention provides the oligomer of the present invention.

[0017] [Exon 53 of the human dystrophin gene] In this invention, "gene" includes not only genomic genes but also cDNA, mRNA precursors, and mRNA. It is included. Preferably, the gene is an mRNA precursor, 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, making it the largest known human gene. It is the gene for [the disease]. However, the coding region of the human dystrophin gene is only 14kb, The coding region is distributed within the dystrophin gene as 79 exons (Roberts, RG., et al., Genomics, 16: 536-538 (1993). In the transcript of the human dystrophin gene... Certain pre-mRNAs undergo splicing to produce 14kb mature mRNA. Human wild-type dysplasmic sperm The nucleotide sequence of the trophin gene is publicly known (GenBank Accession No. NM_004006). The base sequence of exon 53 of the human wild-type dystrophin gene is shown in Sequence ID No. 1.

[0018] The oligomer of the present invention is obtained by skipping exon 53 of the human dystrophin gene. The protein encoded by the DMD-type dystrophin gene is the BMD-type dystrophin protein. It was manufactured with the purpose of modifying the material. Therefore, the oligomer of the present invention Exon 53 of the dystrophin gene, which is the target of exon skipping, is only available in the wild type. However, variant forms are also included. Specifically, exon 53 of the mutant human dystrophin gene is either (a) or (b) It is a polynucleotide as described in ). (a) Polynucleotides and stringers consisting of a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 1 Polynucleotides that hybridize under specific conditions; (b) A polynuclear sequence having 90% or more identity with the base sequence of Sequence ID No. 1 Cleotide

[0019] In this specification, "polynucleotide" means DNA or RNA. In this specification, "polynucleotides that hybridize under stringent conditions" and For example, all of the polynucleotides consisting of the base sequence complementary to the base sequence of SEQ ID NO: 1 Alternatively, using a portion as a probe, colony hybridization, plaque hybridization Poly This refers to nucleotides. Hybridization methods include, for example, "Sambrook & R ussell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, La. boratory Press 2001" and "Ausubel, Current Protocols in Molecular Biology, John W. You can use the methods described in "Iley & Sons 1987-1997," etc.

[0020] In this specification, “complementary nucleotide sequence” refers to the nucleotide sequence in question and the Watson-Crick pair. It is not limited to the base sequences that form a wobble base pair. This also includes the base sequence that makes up the pair. Here, the Watson-Crick pair is adenine-thymine, adenine This refers to a base pair in which a hydrogen bond is formed between n-uracil and guanine-cytosine, and is a fluctuating salt. The base pairs are guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cyan. This refers to base pairs that form hydrogen bonds between tosines. Furthermore, "complementary base sequences" means... The target base sequence does not need to have 100% complementarity; for example, the target salt The base sequence may contain 1 to 3, 1 to 2, or 1 non-complementary base.

[0021] In this specification, "stringent conditions" refers to low stringent conditions, medium stringent conditions, etc. Either stringent conditions or highly stringent conditions are acceptable. "Specific conditions" include, for example, 5x SSC, 5x Denhardt solution, 0.5% SDS, 50% formamide, 32 The condition is °C. Also, "medium stringent conditions" are, for example, 5 × SSC, 5 × Denha Luto solution, 0.5% SDS, 50% formamide, 42°C or 5×SSC, 1% SDS, 50 mM Tris-HCl ( The conditions are pH 7.5, 50% formamide, and 42°C. "High stringent conditions" are an example. For example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, 50°C or 0.2×SSC, 0 The conditions were 0.1% SDS and 65°C. Under these conditions, higher temperature resulted in higher identity. It is expected that polynucleotides can be efficiently obtained. However, hybridise Factors that affect the stringency of the probe include temperature, probe concentration, and probe length. Therefore, several factors such as ionic strength, time, and salt concentration can be considered, and a person skilled in the art would know these factors By selecting the appropriate options, it is possible to achieve similar stringency.

[0022] If you are using a commercially available kit for hybridization, for example, Alkphos Direct The Labelling and Detection System (GE Healthcare) can be used. Follow the protocol included in the kit to incubate the labeled probe. After the process is done overnight, the membrane is washed in a primary wash buffer containing 0.1% (w / v) SDS under conditions of 55°C. After washing, hybridized polynucleotides can be detected. Alternatively, the sequence When preparing a probe based on all or part of a nucleotide sequence complementary to the nucleotide sequence of number 1 Using commercially available reagents (for example, PCR labeling mix (Roche Diagnostics) etc.) If the probe is labeled with digoxigenin (DIG), then the DIG nucleic acid detection kit (Roche) Hybridization can be detected using (Diagnos, Inc.).

[0023] Other polynucleotides besides the hybridizable polynucleotides mentioned above include homologous polynucleotides. The sex search software BLAST calculated using default parameters. In addition, the polynucleotide of SEQ ID NO: 1 is 90% or more, 91% or more, 92% or more, 93% or more, and 94%. Above, 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 higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% Polynucleotides with more than 1% identity can be listed.

[0024] Furthermore, the identity of the base sequence is determined by the BLAST algorithm developed by Carlin and Arthur (Bas ic Local Alignment Search Tool)(Proc. Natl. Acad. Sci. USA 872264-2268, 1990; P This can be determined using the roc Natl Acad Sci USA 90: 5873, 1993). Programs called BLASTN and BLASTX have been developed based on this (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing the base sequence using BLASTN, the parameters —For example, let's set score = 100 and wordlength = 12. BLAST and Gapped BLAST programs When using them, use the default parameters for each program.

[0025] Exon 53, from the 5' end, 31st to 53rd, 31st to 54th, 31st to 55th, and 31st to 56th , 31st-57th, 31st-58th, 32nd-53rd, 32nd-54th, 32nd-55th, 32nd-56th 33rd, 32nd-57th, 32nd-58th, 33rd-53rd, 33rd-54th, 33rd-55th, 33rd ~56th, 33rd-57th, 33rd-58th, 34th-53rd, 34th-54th, 34th-55th, 34th-56th, 34th-57th, 34th-58th, 35th-53rd, 35th-54th, 35th-55th The 35th to 56th, the 35th to 57th, the 35th to 58th, the 36th to 53rd, the 36th to 54th, the 36th The sequence consisting of nucleotides 55, 36-56, 36-57, and 36-58 corresponds to Examples of complementary base sequences are shown in the table below.

[0026] [Table 1-A] [Table 1-B]

[0027] The oligomer of the present invention is preferably the 53rd exon of the human dystrophin gene. From the 5' end, the 32nd to 56th, 33rd to 56th, 34th to 56th, 35th to 56th, or 36th to 56th A base sequence complementary to any one of the sequences consisting of the nth nucleotide (for example, SEQ ID NO: 1) 1. Consists of sequence numbers 17, 23, 29, or 35. Preferably, the oligomer of the present invention is the 53rd exon of the human dystrophin gene. One of the sequences consisting of nucleotides 32-56 or 36-56 from the 5' end It consists of a complementary base sequence (for example, SEQ ID NO: 11 or SEQ ID NO: 35).

[0028] "Enabling the skipping of the 53rd exon of the human dystrophin gene" , the region corresponding to exon 53 of the transcript (e.g., pre-mRNA) of the human dystrophin gene. When the oligomer of the present invention is bound to the transcript, when the transcript undergoes splicing, For example, in a DMD patient with a deletion of exon 52, the base sequence corresponding to the 3' end of exon 51 A base sequence corresponding to the 5' end of exon 54 is ligated to the 3' side, causing a codon frameshift. This means that unprocessed mature mRNA is formed. Therefore, the oligomer of the present invention skips exon 53 of the human dystrophin gene. As long as it is possible, it does not have a base sequence that is 100% complementary to the target sequence. This is also fine. For example, the oligomer of the present invention may have 1 to 3, 1 to 2 or It may contain one non-complementary base. Here, the "binding" refers to the combination of the oligomer of the present invention and a transcript of the human dystrophin gene. When mixed, the two hybridize under physiological conditions to form a double helix. It tastes good. The above "physiological conditions" refers to conditions adjusted to have a pH, salt composition, and temperature similar to those found in living organisms. This means that, for example, at 25-40°C, preferably 37°C, and pH 5-8, preferably pH 7.4. One example is a condition where the sodium chloride concentration is 150 mM.

[0029] Whether or not exon 53 of the human dystrophin gene skipping occurred is determined by the dystrophin gene. The oligomer of the present invention is introduced into cells expressing the enzyme (for example, human rhabdomyosarcoma cells), and the aforementioned enzyme From the total RNA of trophin-expressing cells, exon 53 of the mRNA of the human dystrophin gene The surrounding region is amplified by RT-PCR, and the PCR amplification product is subjected to nested PCR or sequencing analysis. This can be confirmed by doing so. Skipping efficiency is measured by recovering the mRNA of the human dystrophin gene from the test cells, and the mRNA Of these, the polynucleotide amount "A" of the band skipped by exon 53, and exon 53 The polynucleotide amount "B" of the bands that were not skipped was measured, and these "A" and "B" Based on the measured values, the calculation can be performed according to the following formula. Skipping efficiency (%) = A / (A + B) x 100

[0030] The oligomer of the present invention is, for example, an oligonucleotide having a length of 18 to 28 bases. Tide, morpholino oligomer, or peptide nucleic acid (PNA) oligomer - can be listed. A length of 21-25 bases is preferred, and morpholino oligomers are preferred. stomach.

[0031] The aforementioned oligonucleotide (hereinafter referred to as "the oligonucleotide of the present invention") is a nucleus The present invention is an oligomer having ocide as a constituent unit, and such nucleotides are ribonucleotides. It may be any of ocide, deoxyribonucleotide, or modified nucleotide. Modified nucleotides are the nuclei that make up ribonucleotides or deoxyribonucleotides. This refers to substances in which all or part of the acid-base, sugar, and phosphate bond portions have been modified.

[0032] Nucleic acid bases include, for example, adenine, guanine, hypoxanthine, cytosine, and thymine. Examples of such modified bases include uracil or modified bases thereof. For example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (example) For example, 5-methylcytosine), 5-alkyluracil (for example, 5-ethyluracil), 5-halo Uracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methylurethane) (Racil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydr Roxymethyl) Uracil, 5'-Carboxymethylaminomethyl-2-thiouracil, 5-Carbo Xymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-di Methylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyl Adenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyl Nomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl Thir-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyvinegar Acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, i Examples include ndol, imidazole, and xanthine, but are not limited to these. stomach.

[0033] Modifications of the sugar moiety include, for example, modifications at the 2' position of ribose and modifications of other parts of the sugar. One example of a modification at the 2' position of ribose is the -O at the 2' position of ribose. Modifications that replace the H group with OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, or I are listed below. This can be achieved. Here, R represents alkyl or aryl, and R' represents alkylene. Other modifications of sugars include, for example, the O at the 4' position of ribose or deoxyribose. Substitutions where S is substituted, or where the 2' and 4' positions of the sugar are bridged, for example, LNA (Locked Nucleic Acid). Examples include Acid) or ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids), but It is not limited to these.

[0034] Modifications of the phosphate bond include, for example, changing the phosphodiester bond to a phosphorothioate. Bonding, phosphorodithioate bond, alkylphosphonate bond, phosphoramidate bond , boranophosphate binding (Enya et al: Bioorganic & Medicinal Chemistry, 2008, Examples of modifications that can be substituted are listed (e.g., Patent Republication No. 2006) (See issues / 129594 and 2006 / 038608).

[0035] As the alkyl group, linear or branched alkyl groups having 1 to 6 carbon atoms are preferred. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, se c-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n- Examples include hexyl and isohexyl. The alkyl may be substituted, and such arrangement Examples of substitution groups include halogens, alkoxys, cyanos, and nitros. These may be substituted in groups of 1 to 3.

[0036] As for cycloalkyls, cycloalkyls having 5 to 12 carbon atoms are preferred. Specifically, for example, For example, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl Examples include cyclododecyl.

[0037] Examples of halogens include fluorine, chlorine, bromine, and iodine.

[0038] Examples of alkoxys include linear or branched alkoxys with 1 to 6 carbon atoms, such as methotrexate. Xy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy Xy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, i Examples include sohexyloxy. In particular, alkoxys with 1 to 3 carbon atoms are preferred. stomach.

[0039] As the aryl group, aryl groups with 6 to 10 carbon atoms are preferred. Specifically, for example, phenyl Examples include phenyl, α-naphthyl, and β-naphthyl. Phenyle is particularly preferred. The aryl may be substituted, and such substituents may be alkyl, halogen, etc. Examples include nucleotides, alkoxys, cyanos, and nitros, and even if 1 to 3 of these are substituted... good.

[0040] As the alkylene, linear or branched alkylenes having 1 to 6 carbon atoms are preferred. Physically, for example, methylene, ethylene, trimethylene, tetramethylene, pentamethylene Examples include hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene. It is possible.

[0041] Examples of acyls include linear or branched alkanoyls or aroyls. This can be done. Examples of alkanoyls include formyl, acetyl, 2-methylacetyl, 2,2-Dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2 Examples include 2-dimethylpropionyl and hexanoyl. Examples of aroyls include Examples include benzoyl, toluyl, and naphthoyl. Such aroyls are substituted. It may be substituted at any position, and may be substituted with alkyl.

[0042] The oligonucleotide of the present invention preferably has a methoxy group at the 2' position of the ribose--OH group. The group is converted, and the phosphate bond portion is a phosphorothioate bond, as represented by the following general formula. This is the oligomer of the present invention, which is used as a constituent unit. [ka] (In the formula, Base represents a nucleic acid base.)

[0043] The oligonucleotides of the present invention can be synthesized using various automated synthesis devices (e.g., AKTA oligopilot plus 1). It can be easily synthesized using 0 / 100 (GE Healthcare), or it can also be produced by commissioning a third-party institution (for example, Promega or Takara). The morpholino oligomer of the present invention is an oligomer of the present invention having a group represented by the following general formula as a constituent unit.

[0044] (In the formula, Base has the same meaning as described above; W represents a group represented by any of the following formulas. [Chemical formula] (In the formula, X represents -CH2R , -O-CH2R [Chemical formula] <确定 (In the formula, X represents -CH2R 1 , 1 , -O-CH2R 1 , -S-CH2R 1 , -NR 2 R 3 or F; R 1 represents H or alkyl; R 2 and R 3 are the same or different and represent H, alkyl, cycloalkyl, or aryl ; Y1 represents 0, S, CH2 or NR 1 ; Y2 represents 0, S or NR 1 ; Z represents 0 or S.))

[0045] The morpholino oligomer is preferably an oligomer (phosphorodiamidate morpholino oligomer (hereinafter referred to as "PMO")) having a group represented by the following formula as a constituent unit. . [Chemical formula] ​(In the formula, Base, R 2 , R 3 (This is synonymous with the above.)

[0046] Morpholino oligomers are described, for example, in International Publication No. 1991 / 009033, or International Publication It can be manufactured in accordance with International Publication No. 2009 / 064471. In particular, PMO can be manufactured in accordance with International Publication No. 2009 / 064471. It can be manufactured according to the method described in No. 471, or according to the method shown below. Cut.

[0047] [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)). One possible answer is (u). [ka] [In the formula, each Base, R 2 , R 3 This is synonymous with the above; n is any integer in the range of 1 to 99, preferably any integer in the range of 18 to 28. It is an integer.

[0048] PMO(I) can be manufactured according to known methods, for example, by performing the following steps. It can be manufactured by applying the following method. The compounds and reagents used in the following processes are those commonly used in the manufacture of PMO. Therefore, it is not particularly limited.

[0049] Furthermore, all of the following steps are performed using either a liquid-phase or solid-phase method (manual or commercially available solid-phase automated synthesis). This can be done using a machine. When manufacturing PMO using the solid-phase method, the operating procedure can be simplified. Furthermore, from the standpoint of accuracy in synthesis, a method using an automated synthesizer is preferable.

[0050] (1) Process A: When an acid is reacted with a compound represented by the following general formula (II) (hereinafter referred to as compound (II)), By doing so, a compound represented by the following general formula (III) (hereinafter referred to as compound (III)) is produced. The manufacturing process. [ka] [where n, R 2 , R 3 This is synonymous with the above; Each B P This independently represents a nucleic acid base that may be protected; 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)). [This represents...] [ka]

[0051] B P The "nucleic acid base" in this context is the same "nucleic acid base" as in "Base." However, B P The amino group or hydroxyl group of the nucleic acid base involved may be protected. As for protecting groups for such amino groups, any group used as a protecting group for nucleic acids is particularly suitable. Not limited to, specifically, for example, benzoyl, 4-methoxybenzoyl, acetyl, propyl Onyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-br Tylphenoxyacetyl, 4-isopropylphenoxyacetyl, (dimethylamino)methyl Examples of hydroxyl protecting groups include 2-cyanoethyl and 4-nitrate. Lophenethyl, phenylsulfonylethyl, methylsulfonylethyl, trimethylsilyl Ethyl, and phenyl which may be substituted with 1 to 5 electron-withdrawing groups at any substitutable position. , diphenylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, methylphenyl Nilcarbamoyl, 1-pyrrolidinylcarbamoyl, morpholinocarbamoyl, 4-(tert- Butylcarboxybenzyl, 4-[(dimethylamino)carboxybenzyl, 4-(phenyl Carboxy)benzyl can be cited (for example, International Publication No. 2009 / 064471). reference).

[0052] As for the "solid phase support," there are no particular restrictions as long as it is a support that can be used in solid phase reactions of nucleic acids. For example, (i) reagents that can be used in the synthesis of morpholino nucleic acid derivatives (e.g., dichlorometh N, acetonitrile, tetrazole, N-methylimidazole, pyridine, acetic anhydride, lutinous acid (ii) It is practically insoluble in din and trifluoroacetic acid, and is used in the synthesis of morpholino nucleic acid derivatives. (iii) chemically stable to usable reagents, (iv) can be chemically modified, and (iv) desirable (v) It can load ruforino nucleic acid derivatives and has sufficient strength to withstand the high pressure applied during processing. (vi) It is desirable that the particle size range and distribution be constant. Specifically, swellable polystyrene (For example, aminomethyl polystyrene resin 1% dibenzylbenzene crosslinked (200-400 mesh) (2.4~3.0 mmol / g) (manufactured by Tokyo Chemical Industry Co., Ltd.), Aminomethylated Polystyrene Resin·HCl [ Dibenzylbenzene 1%, 100-200 mesh (manufactured by Peptide Research Institute Co., Ltd.), non-swelling polyethylene Chilean (e.g., Primer Support (manufactured by GE Healthcare)), PEG chain-linked polystyrene ( For example, NH2-PEG resin (manufactured by Watanabe Chemical Co., Ltd.), TentaGel resin), controlled-porous glass (controlled Pore ​​glass (CPG) (e.g., manufactured by CPG), oxallylated porous glass (e.g., Alul et al., N See Ucleic Acids Research, Vol. 19, 1527 (1991), TentaGel support - aminopolyethylene Lenglycol-derived support (e.g., Wright et al., Tetrahedron Letters, Vol.34, 3373) (See 1993), Poros-polystyrene / divinylbenzene copolymers can be cited. ru.

[0053] As a "linker," it is typically used to link nucleic acids and morpholino nucleic acid derivatives. Known substances can be used, for example, 3-aminopropyl, succinyl, 2,2'-di Examples include ethanol sulfonyl and long-chain alkylamino (LCAA).

[0054] This process can be carried out by reacting compound (II) with an acid. Examples of "acids" that can be used in this process include trifluoroacetic acid, dichloroacetic acid, or Lichloroacetic acid can be cited as an example. The amount of acid used is, for example, 1 mole of compound (II). For this, a range of 0.1 molar equivalents to 1000 molar equivalents is appropriate, preferably 1 molar equivalent to 100 It is within the range of molar equivalents. In addition, an organic amine can be used together with the acid. While not limited to these, triethylamine can be cited as an example. The appropriate amount of min to use is, for example, within the range of 0.01 to 10 molar equivalents per mole of acid. The amount is preferably in the range of 0.1 molar equivalents to 2 molar equivalents.

[0055] When using a salt or mixture of an acid and an organic amine in this process, for example, truffle Examples include salts or mixtures of triethylamine and trioloacetic acid, more specifically, One example is a mixture of 2 equivalents of refluoroacetic acid and 1 equivalent of triethylamine. ru. The acid used in this process should be diluted with a suitable solvent to a concentration in the range of 0.1% to 30%. It can also be used as such. The solvent is not particularly limited as long as it does not participate in the reaction, but for example... For example, dichloromethane, acetonitrile, alcohols (ethanol, isopropanol) Examples include trifluoroethanol, water, or mixtures thereof.

[0056] The reaction temperature in the above reaction is preferably, for example, in the range of 10°C to 50°C, and more preferably The temperature is within the range of 20°C to 40°C, and more preferably within 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 within the range of 0.1 minutes to 24 hours. The range within the box is appropriate. Preferably, it is within the range of 1 minute to 5 hours.

[0057] Furthermore, after this process is completed, if necessary, a base is added to neutralize the acid present in the system. It can be added. The "base" is not particularly limited, but for example, diisopropyl Luamine is one example. The base should be concentrated within the range of 0.1% (v / v) to 30% (v / v). It can also be used after diluting with a suitable solvent. The solvent used in this process is not particularly limited as long as it does not participate in the reaction, but dichloromethyl Tan, acetonitrile, alcohols (ethanol, isopropanol, trifluoroethylene) Examples include tanol, water, or mixtures thereof. The reaction temperature is, for example, 10 A range of ℃ to 50℃ is preferred, more preferably a range of 20℃ to 40℃, and even more preferably Or, within the range of 25°C to 35°C. The reaction time varies depending on the type of base used and the reaction temperature, but is usually between 0.1 minutes and 24 hours. The range is appropriate, preferably between 1 minute and 5 hours.

[0058] Furthermore, in compound (II), n=1 and L is the group (IV), the following general formula (IIa) The compound represented by (hereinafter referred to as compound (IIa)) shall be manufactured according to the following method. It is possible. [ka] [In the formula, B P T, linker, and solid-phase support are synonymous with the above.

[0059] Step 1: By reacting a compound represented by the following general formula (V) with an acylating agent, the following general A process for producing a compound represented by formula (VI) (hereinafter referred to as compound (VI)). [ka] [In the formula, B P T and linker are synonymous with the above; R 4 [This represents a hydroxyl group, halogen, or amino acid.]

[0060] This process is carried out using compound (V) as a starting material and by a known linker introduction reaction. It is possible. In particular, the compound represented by the following general formula (VIa) can be obtained by using compound (V) and succinic anhydride. It can be produced by carrying out a method known as an esterification reaction. [ka] [In the formula, B P T is synonymous with the above.

[0061] Step 2: Compound (VI) is reacted with a condensing agent, etc., to form compound (II) The process for manufacturing (a). [ka] [In the formula, B P , R 4 T, linker, and solid-phase support are synonymous with the above.

[0062] This process involves manufacturing using compound (VI) and a solid support by a method known as a condensation reaction. It is possible.

[0063] In compound (II), n=2 to 99, and L is the group (IV), it is represented by the following general formula (IIa2): The compound is made using compound (IIa) as a starting material, and is produced in step A and step A of the method for producing PMO described herein. The product can be manufactured by repeatedly performing step B a desired number of times. [ka] [In the formula, B P , R 2 , R 3 T, linker, and solid support are synonymous with the above; n' represents the numbers 1 through 98.

[0064] Furthermore, in compound (II), n=1 and L is hydrogen, it can be represented by the following general formula (IIb). The compound can be produced, for example, by the method described in International Publication No. 1991 / 009033. can. [ka] [In the formula, B P T is synonymous with the above.

[0065] In compound (II), n=2 to 99, where L is hydrogen, it is represented by the following general formula (IIb2). The compound is formed using compound (IIb) as a starting material and following the process of the PMO manufacturing method described herein. The product can be manufactured by repeatedly performing steps A and B a desired number of times. [ka] [In the formula, B P ,n',R 2 , R 3 T is synonymous with the above.

[0066] Furthermore, in compound (II), n=1 and L is acyl, the following general formula (IIc) The represented compound undergoes a known acylation reaction with compound (IIb). It can be manufactured by doing so. [ka] [In the formula, B P T is synonymous with the above; R 5 This represents acyl.

[0067] In compound (II), n=2 to 99, where L is acyl, the following general formula (IIc2) is expressed The compound to be produced uses compound (IIc) as a starting material and the process according to the PMO production method described herein. The product can be manufactured by repeatedly performing steps A and B a desired number of times. [ka] [In the formula, B P ,n',R 2 , R 3 , R 5 T is synonymous with the above.

[0068] (2) Process B: By reacting compound (III) 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 is synonymous with the above.

[0069] This process involves reacting compound (III) with a morpholino monomer compound in the presence of a base. This can be done by [method].

[0070] Examples of morpholino monomer compounds include compounds represented by the following general formula (VIII): It can be listed. [ka] [In the formula, B P , R 2 , R 3 T is synonymous with the above.

[0071] Examples of "bases" that can be used in this process include diisopropylamine and triethylamine. Examples include mine or N-ethylmorpholine. The amount of base used is, for example, For 1 mole of compound (III), the appropriate and preferred amount is in the range of 1 molar equivalent to 1000 molar equivalents. Or it is within the range of 10 molar equivalents to 100 molar equivalents. The morpholino monomer compounds and bases that can be used in this process will be at concentrations of 0.1% to 30%. It can also be used after diluting with a suitable solvent. The solvent should not be involved in the reaction. While not particularly limited, examples include N,N-dimethylimidazolidone, N-methylpiperidone, DM Examples include F, dichloromethane, acetonitrile, telolahydrofuran, or mixtures thereof. It is possible.

[0072] The reaction temperature is preferably in the range of 0°C to 100°C, and more preferably in the range of 10°C to 50°C. It is within the range. The reaction time varies depending on the type of base used and the reaction temperature, but is usually within the range of 1 minute to 48 hours. The enclosed area should be appropriate, preferably within the range of 30 minutes to 24 hours.

[0073] Furthermore, after the completion of this process, an acylating agent may be added as needed. Examples of "chemical agents" include acetic anhydride, acetate chloride, and phenoxyacetic anhydride. This can be done. The acylating agent is used in a suitable solvent so that its concentration is, for example, within the range of 0.1% to 30%. It can also be used after dilution. The solvent is not particularly limited as long as it does not participate in the reaction. However, for example, dichloromethane, acetonitrile, alcohols (ethanol, isopropyl alcohol) Examples include panol (such as trifluoroethanol), water, or mixtures thereof. . Also, if necessary, along with acylating agents, for example, pyridine, lutidine, colidine, Use bases such as triethylamine, diisopropylethylamine, and N-ethylmorpholine. It can be carried out. The amount of the acylating agent used is preferably in the range of 0.1 molar equivalent to 10,000 molar equivalents, and more preferably in the range of 1 molar equivalent to 1,000 molar equivalents. The amount of the base used is, for example, appropriate in the range of 0.1 molar equivalent to 100 molar equivalents with respect to 1 mole of the acylating agent, and preferably in the range of 1 molar equivalent to 10 molar equivalents.

[0074] The reaction temperature of this reaction is preferably in the range of 10°C to 50°C, more preferably in the range of 10°C to 50°C, still more preferably in the range of 20°C to 40°C, and even more preferably in the range of 25°C to 35°C. The reaction time varies depending on, for example, the type of the acylating agent used and the reaction temperature, but is usually appropriate in the range of 0.1 minute to 24 hours, and preferably in the range of 1 minute to 5 hours. <\

[0075] (3) Step C: In the compound (VII) produced in Step B, a protecting group is removed using a deprotecting agent to produce a compound represented by the general formula (IX).

Chemical formula

[0076] This step can be carried out by allowing a deprotecting agent to act on the compound (VII).

[0077] Examples of the "deprotecting agent" include concentrated aqueous ammonia and methylamine. The "deprotecting agent" that can be used in this step includes, for example, water, methanol, ethanol, and isopropanol. ​​​​​​​Pyral alcohol, acetonitrile, tetrahydrofuran, DMF, N,N-dimethylimidazoli It can also be used diluted with dohn, N-methylpiperidone, or a mixture thereof. However, ethanol is preferred. The amount of 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 appropriate, preferably 10 molar equivalents. It is within the range of 1000 molar equivalents.

[0078] The reaction temperature is suitable, for example, in the range of 15°C to 75°C, preferably in the range of 40°C to 70°C. The temperature is within the range of 50°C to 60°C. The deprotection reaction time is within the range of compound (VII The appropriate and preferred time range is between 10 minutes and 30 hours, although this varies depending on the type of reaction, reaction temperature, etc. This is within the range of 30 minutes to 24 hours, and more preferably within the range of 5 hours to 20 hours.

[0079] (4) Process D: PMO(I) is produced by reacting compound (IX), manufactured in step C, with an acid. The manufacturing process. [ka] [where Base, n, R 2 , R 3 T is synonymous with the above.

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

[0081] Examples of acids that can be used in this process include trichloroacetic acid, dichloroacetic acid, Examples include acetic acid, phosphoric acid, and hydrochloric acid. The amount of acid used depends, for example, on the pH of the solution. It is appropriate to use it so that the value is within the range of 0.1 to 4.0, and more preferably within the range of 1.0 to 3.0. Use it so that it is within the enclosed area. The solvent is not particularly limited as long as it does not participate in the reaction, Examples include acetonitrile, water, or a mixture thereof.

[0082] The reaction temperature is preferably in the range of 10°C to 50°C, and more preferably in the range of 20°C to 40°C. Yes, and more preferably, within the range of 25°C to 35°C. The deprotection reaction time is for compound (IX) The appropriate time range varies depending on the type of reaction, reaction temperature, etc., but is generally between 0.1 minutes and 5 hours, preferably. The range is between 1 minute and 1 hour, and more preferably between 1 minute and 30 minutes.

[0083] PMO(I) is obtained from the reaction mixture obtained in this process by conventional separation and purification methods, such as extraction and concentration. Neutralization, filtration, centrifugation, recrystallization, C8 to C 18 Reverse-phase column chromatography, cation io Anion exchange column chromatography, gel filtration chromatography Methods such as Lamb chromatography, high-performance liquid chromatography, dialysis, and limit filtration are used. It can be obtained by using either alone or in combination, and the desired PMO(I) can be isolated and purified. This is possible (see, for example, International Publication WO1991 / 09033). When purifying PMO(I) using reversed-phase chromatography, the elution solvent is, for example, A mixed solution of 20 mM triethylamine / acetate buffer and acetonitrile can be used. ru. Furthermore, when purifying PMO(I) using ion exchange chromatography, for example, 1M A mixed solution of saline solution and a 10 mM sodium hydroxide solution can be used.

[0084] Peptide nucleic acid is an oligomer of the present invention having a group represented by the following general formula as a constituent unit . [Chemical formula] (In the formula, Base has the same meaning as described above.)

[0085] Peptide nucleic acid can be produced, for example, according to the following documents. 1) P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science, 254, 1497 (1991) 2) M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, Jacs., 114, 1895 (1992) 3) K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen, T. Vulpi us, K. H. Petersen, R. H. Berg, P. E. Nielsen, O. Buchardt, J. Org. Chem., 59, 5 767 (1994) 4) L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. F. Hansen, T. Koch, M. Egholm, O. Buchardt, P. E. Nielsen, J. Coull, R. H. Berg, J. Pept. Sci., 1, 175 (1995) 5) T. Koch, H. F. Hansen, P. Andersen, T. Larsen, H. G. Batz, K. Otteson, H. Or um, J. Pept. Res., 49, 80 (1997)

[0086] Furthermore, the oligomer of the present invention has a 5' end that is one of the following chemical formulas (1) to (3) It may be present. Preferably it is (3)-OH. [ka] Hereinafter, the groups shown in (1), (2) and (3) above will be referred to as "group (1)", "group (2)", and "group ( 3) is called "."

[0087] 2. Pharmaceutical Compositions The oligomer of the present invention is more efficient than the antisense oligomer of the prior art. This enables the skipping of exon 53. Therefore, pharmaceutical compositions comprising the oligomer of the present invention By administering this to DMD patients, it is possible to alleviate the symptoms of muscular dystrophy with high efficiency. It is predicted that this will be possible. For example, when using a pharmaceutical composition containing the oligomer of the present invention, conventional techniques Because it can achieve the same level of therapeutic effect with a smaller dose compared to the oligomers used in the procedure, side effects It can reduce costs and is also economical. Therefore, as another embodiment, the oligomer of the present invention, its pharmaceutically acceptable salt or water A pharmaceutical composition for the treatment of muscular dystrophy, comprising a Japanese compound as an active ingredient (hereinafter referred to as "the composition of the present invention"). It provides (that).

[0088] Examples of pharmaceutically acceptable salts of the oligomers of the present invention contained in the composition of the present invention include: Alkali metal salts such as sodium salts, potassium salts, lithium salts, calcium salts, magnesium Alkaline earth metal salts such as zinc salts; aluminum salts, iron salts, zinc salts, copper salts, nickel Salts, metal salts such as cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine Salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenedi Amine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine Salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroproca Ino salt, procaine salt, diethanolamine salt, N-benzylphenethylamine salt, pipette radin salt, tetramethylammonium salt, tris(hydroxymethyl)aminomethane salt Organic amine salts; such as hydrofluoric acid, hydrochloride, hydrobromide, and hydroiodide. Hydrochlorides; inorganic salts such as nitrates, perchlorates, sulfates, and phosphates; methanesulfone Lower alkansulfonates such as salts, trifluoromethanesulfonates, and ethanesulfonates Aryl sulfonic acids such as benzenesulfonates and p-toluenesulfonates. Salts; acetate, malate, fumarate, succinate, citrate, tartrate, oxalic acid Salts, organic acid salts such as maleates; glycine salts, lysine salts, arginine salts, ornithine salts Examples include amino acid salts such as glutamate and aspartate. It can be manufactured by known methods. Alternatively, the present invention can be incorporated into the composition of the present invention. The oligomer may also exist in the form of its hydrate.

[0089] The dosage form of the composition of the present invention is not particularly limited as long as it is a pharmaceutically acceptable dosage form. The choice of treatment method can vary, but intravenous administration is preferred due to its ease of delivery to muscle tissue. Administration by intra-arterial, intramuscular, subcutaneous, oral, intratissue, or transdermal means is preferable. Furthermore, the dosage form of the composition of the present invention is not particularly limited, but for example, various... Examples include injectable preparations, oral preparations, intravenous preparations, inhaled preparations, ointments, lotions, etc.

[0090] When administering the oligomer of the present invention to a patient with muscular dystrophy, the composition of the present invention is the O It is preferable to include a carrier that facilitates the delivery of ligomers to muscle tissue. There are no particular restrictions as long as it is pharmacodynamically acceptable, for example, cationic liposomes. Cationic carriers such as cationic polymers, or carriers utilizing viral envelopes Examples include cationic liposomes such as 2-O-(2-diethylamino Ethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids are essential components. Liposomes formed by this process (hereinafter referred to as "liposome A"), oligofectamine ( (Registered Trademark) (Manufactured by Invitrogen), Lipofectin (Registered Trademark) (Manufactured by Invitrogen), Lipo Fectamine® (manufactured by Invitrogen), Lipofectamine 2000® (manufactured by Invitrogen) (Manufactured by Invitrogen, Inc.), DMRIE-C (Registered Trademark) (Manufactured by Invitrogen, Inc.), GeneSilencer (Registered Trademark) (Manufactured by Gene Therapy Systems), TransMessenger® (manufactured by QIAGEN), TransIT Examples include TKO (registered trademark) (manufactured by Mirus Corporation) and Nucleofector II (Lonza). Among these, liposome A is preferred. As a cationic polymer, for example, JetSI (registered Trademark (manufactured by Qbiogene), Jet-PEI (registered trademark) (polyethyleneimine, manufactured by Qbiogene) Examples of carriers utilizing the viral envelope include, for example, Genome One example is One(registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Co., Ltd.). Alternatively, Pharmaceutical device described in Patent No. 2924179, Patent Republication Publication No. 2006 / 129594 and Patent Republication Publication A cationic carrier as described in Report No. 2008 / 096690 can also be used.

[0091] 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, a range of 0.1 nM to 100 μM is appropriate, and a range of 1 nM to 10 μM is preferred, and 10 nM to A range of 1 μM is more preferable. Also, the oligomer of the present invention contained in the composition of the present invention and The weight ratio of the carrier (carrier / oligomer of the present invention) depends on the properties of the oligomer and the type of carrier. While it varies depending on the factors, a range of 0.1 to 100 is appropriate, a range of 1 to 50 is preferable, and 10 to 2 A range of 0 is preferable.

[0092] In addition to the oligomer of the present invention and the carrier described above, the composition of the present invention may optionally contain other pharmaceutically acceptable materials. Possible additives can be incorporated. Such additives include, for example, emulsifying agents (for example) For example, fatty acids with 6 to 22 carbon atoms and their pharmaceutically acceptable salts, albumin, dextran, Stabilizers (e.g., cholesterol, phosphatidic acid), isotonic agents (e.g., sodium chloride) (Lium, glucose, maltose, lactose, sucrose, trehalose), pH adjuster (For example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethano Examples include sylamines. One or more of these can be used. The content of the additive in the composition of the present invention is preferably 90% by weight or less, and preferably 70% by weight or less. Preferably, 50% by weight or less is preferred.

[0093] The composition of the present invention is obtained by adding the oligomer of the present invention to a dispersion of a carrier and stirring appropriately. It can be further prepared. Also, the additive can be added before or after the addition of the oligomer of the present invention. Water can also be added in an appropriate process. The solvent is not particularly limited as long as it is pharmaceutically acceptable, for example, water for injection, Examples include distilled water for injection, electrolyte solutions such as physiological saline, glucose solution, and sugar solutions such as maltose solution. This can be done. Furthermore, the conditions such as pH and temperature in such cases can be appropriately selected by a person skilled in the art. Cut.

[0094] The composition of the present invention can be, for example, a liquid formulation or a freeze-dried formulation thereof. The dried formulation is obtained by freeze-drying the composition of the present invention, which has a liquid form, by a conventional method. It can be prepared by, for example, the composition of the present invention having the form of a liquid After sterilization, dispense the specified amount into vials and pre-freeze at approximately -40 to -20°C for 2 hours. This process is carried out for a period of time, followed by primary drying under reduced pressure at approximately 0-10°C, and then secondary drying under reduced pressure at approximately 15-25°C. It can be dried and freeze-dried. Generally, the inside of the vial is then replaced with nitrogen gas. Then, by capping the container, a freeze-dried formulation of the composition of the present invention can be obtained.

[0095] The lyophilized formulation of the composition of the present invention can generally be obtained by adding any suitable solution (re-dissolving solution). It can then be redissolved and used. Examples of such redissolving solutions include sterile water for injection and physiological saline. Examples include water and other general intravenous fluids. The volume of this redissolving solution varies depending on the application, etc. While there are no particular restrictions, an amount equal to 0.5 to 2 times the volume of liquid before freeze-drying, or 500 mL or less, is appropriate.

[0096] The dosage when administering the composition of the present invention depends on the type of oligomer contained in the present invention. The medication is prepared considering the dosage form, the patient's condition (age, weight, etc.), the route of administration, and the nature and severity of the disease. While this is preferable, the amount of the oligomer of the present invention for adults is 0.1 mg to 10 g per day. Within the human range, preferably 1 mg to 1 g / human is common. This value is the target and It may also vary depending on the type of disease, the form of administration, and the target molecule. Therefore, depending on the case... Therefore, a lower dose may be sufficient, and conversely, there may be times when a higher dose is needed. It can be administered once or several times a day, or at intervals of one to several days.

[0097] Another embodiment of the composition of the present invention is a vector capable of expressing the oligonucleotide of the present invention. Examples of pharmaceutical compositions include the carrier described above. Such expression vectors may include multiple The composition may be capable of expressing the oligonucleotide of the present invention. Similar to the composition of the present invention containing the oligomer, pharmaceutically acceptable additives may be added. The concentration of the expression vector contained in the composition can be determined by the type of carrier, etc. Although different, a range of 0.1 nM to 100 μM is appropriate, and a range of 1 nM to 10 μM is preferred. A range of 0 nM to 1 μM is more preferable. The expression vector contained in the composition and the support The weight ratio (carrier / expression vector) varies depending on the properties of the expression vector, the type of carrier, etc. A range of 0.1 to 100 is appropriate, a range of 1 to 50 is preferred, and a range of 10 to 20 is more preferred. Furthermore, the amount of carrier contained in the composition is such that it contains the oligomer of the present invention. The same applies to the composition of the present invention, and the method of preparation, etc., is also the same as in the case of the composition of the present invention. It is similar to that.

[0098] The present invention will be described in more detail below with reference to examples and test examples, but the present invention is not applicable. This is not limited to the scope shown in the example. [Examples]

[0099] [Reference example 1] 4-{[(2S,6R)-6-(4-benzamide) supported on aminomethyl polystyrene resin -2-Oxopyrimidine-1-yl)-4-Tritylmorpholin-2-yl]methoxy}-4 -Oxobutanoic acid Step 1: 4-{[(2S,6R)-6-(4-benzamide-2-oxopyrimidine-1(2H)- Preparation of (L)-4-tritylmorpholin-2-yl]methoxy-4-oxobutanoic acid Under an argon atmosphere, N-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmethyl Ruforin-2-yl]-2-oxo-1,2-dihydropyrimidine-4-yl}benzamide 2 2.0 g and 7.04 g of 4-dimethylaminopyridine (4-DMAP) were suspended in 269 mL of dichloromethane, and Add 5.76 g of aqueous succinic acid and stir at room temperature for 3 hours. Add 40 mL of methanol to the reaction mixture and concentrate under reduced pressure. It shrunk. The residue was extracted using ethyl acetate and a 0.5 M potassium dihydrogen phosphate aqueous solution. The obtained organic layer was washed in the following order: 0.5 M potassium dihydrogen phosphate aqueous solution, water, and saturated saline solution. The resulting organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain 25.9 g of the target product.

[0100] Step 2: 4-{[(2S,6R)-6-(4-benzyl acetate) supported on aminomethyl polystyrene resin [amide-2-oxopyrimidine-1-yl)-4-tritylmorpholin-2-yl]methoxy Production of}-4-oxobutanoic acid 4-{[(2S,6R)-6-(4-benzamide-2-oxopyrimidine-1(2H)-yl) 23.5g of pyridine (-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid Dissolve in 336 mL (dehydrated), and add 4.28 g of 4-DMAP and 1-ethyl-3-(3-dimethylaminopropyl) 40.3g of rubodiimide hydrochloride was added. Then, aminomethyl polystyrene resin 1% DVB frame Add 25.0g of bridge compound (manufactured by Tokyo Chemical Industry Co., Ltd., A1543) and 24mL of triethylamine, and shake at room temperature for 4 days. The reaction was completed. After the reaction, the resin was filtered off. The obtained resin was then mixed with pyridine, methanol, and dichloromethic acid. The resin was washed in the following order and dried under reduced pressure. 150 mL of tetrahydrofuran (dehydrated) was added to the resulting resin. Add 15 mL of hydroxide and 15 mL of 2,6-lutidine, and shake at room temperature for 2 hours. Filter out the resin and pyle The sample was washed with din, methanol, and dichloromethane in that order, then dried under reduced pressure to obtain 33.7 g of the target product. The loading amount of the target substance is determined by a known method, using the molars of trityl per gram of resin. The amount was determined by measuring the UV absorbance at 409 nm. The resin loading amount was 3 The concentration was 97.4 μmol / g.

[0101] UV measurement conditions Equipment: U-2910 (Hitachi, Ltd.) Solvent: Methanesulfonic acid Wavelength: 265 nm ε value: 45000

[0102] [Reference example 2] 4-oxo-4-{[(2S,6R)-6-(6-O Xo-2-[2-phenoxyacetamide]-1H-purine-9-yl)-4-tritylmorphoyl [-2-yl]methoxybutanoic acid Process 1:N 2 - Production of (phenoxyacetyl)guanosine 100g of guanosine was dried at 80°C under reduced pressure for 24 hours. 500mL of pyridine (dehydrated), dichloro Add 500 mL of methane (dehydrated) and, under an argon atmosphere at 0°C, add 401 mL of chlorotrimethylsilane. The mixture was added dropwise and stirred at room temperature for 3 hours. It was then cooled again on ice, and 66.3 g of phenoxyacetyl chloride was added dropwise. The mixture was then stirred for a further 3 hours under ice cooling. 500 ml of methanol was added to the reaction mixture, and after stirring overnight at room temperature, The solvent was removed under reduced pressure. 500 mL of methanol was added to the residue, and the mixture was concentrated under reduced pressure three times. Add 4 L of water to the residue and stir under ice cooling for 1 hour, then filter out the precipitate. This was then mixed with water, and then... Washed with cold methanol and dried, 150.2 g of the target compound was obtained (yield: 102%) (Reference: Org. Lett. (2004), Vol. 6, No. 15, 2555-2557).

[0103] Step 2: N-{9-[(2R,6S)-6-(hydroxymethyl)-4-morpholin-2-yl]- 6-Oxo-6,9-dihydro-1H-purine-2-yl}-2-phenoxyacetamide p- Luen sulfonate 30 g of the compound obtained in step 1 was suspended in 480 mL of methanol, and 130 mL of 2N hydrochloric acid was added under ice cooling. Next, 56.8g of ammonium tetraborate tetrahydrate and 16.2g of sodium periodate were added in that order. In addition, the mixture was stirred at room temperature for 3 hours. The reaction mixture was cooled on ice, and insoluble matter was filtered out. This mixture was then converted to methanol. Washed with 100 mL of water. Combined the filtrate and washing solution, cooled with ice, and added 11.52 g of 2-picolinborane. After stirring for 20 minutes, slowly add 54.6g of p-toluenesulfonic acid monohydrate and stir overnight at 4°C. The precipitate was filtered off, washed with 500 mL of cold methanol, and dried to obtain 17.7 g of the target compound. Yield: 43.3%. 1H NMR(δ,DMSO-d6):9.9-9.2(2H,br), 8.35(1H,s), 7.55(2H,m), 7.35( 2H,m), 7.10(2H,d, J=7.82Hz), 7.00(3H,m), 5.95(1H,dd, J=10.64,2.42H z), 4.85(2H,s), 4.00(1H,m), 3.90-3.60(2H,m), 3.50-3.20(5H,m), 2.9 0 (1 H, m), 2.25 (3 H, s)

[0104] Step 3: N-{9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholine-2- Il]-6-oxo-6,9-dihydro-1H-purine-2-yl}-2-phenoxyacetami Manufacturing 2.0 g of the compound obtained in step 2 is suspended in 30 mL of dichloromethane and, under ice cooling, triethylamine is added. Add 13.9g of sodium bicarbonate and 18.3g of trityl chloride, and stir at room temperature for 1 hour. The reaction mixture was then mixed with saturated sodium bicarbonate solution. Next, the mixture was washed with water, dried, and the organic layer was concentrated under reduced pressure. The residue was buffered with 0.2 M sodium citrate. Add 40 mL of solution (pH 3) / methanol (1:4 (v / v)) and stir, then add 40 mL of water and cool with ice. The mixture was stirred for 1 hour. This was filtered, washed with cold methanol, and dried to obtain 1.84 g of the target compound. (Yield: 82.0%).

[0105] Step 4: 4-oxo-4-{[(2S,6R)-6- (6-oxo-2-[2-phenoxyacetamide]-1H-purine-9-yl)-4-trityl Production of Morpholin-2-yl]methoxybutanoic acid The indicated compound was prepared in the same manner as in Reference Example 1. However, the N-{1} used in step 1 of Reference Example 1 was replaced. -[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-ol Instead of xo-1,2-dihydropyrimidine-4-ylbenzamide, in this process, N- {9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-6 Using Oxo-6,9-dihydro-1H-purine-2-yl}-2-phenoxyacetamide Ta.

[0106] [Reference example 3] 4-{[(2S,6R)-6-(5-methyl-2,4- [Dioxo-3,4-dihydropyrimidine-1-yl)-4-tritylmorpholine-2-yl] Toxy-4-oxobutanoic acid The indicated compound was prepared in the same manner as in Reference Example 1. However, the N-{1} used in step 1 of Reference Example 1 was replaced. -[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-ol Instead of xo-1,2-dihydropyrimidine-4-ylbenzamide, in this step, 1- [(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-5-methyl Lupyrimidine-2,4(1H,3H)-dione was used.

[0107] [Reference example 4] 1,12-dioxo-1-(4-tritylpiper) supported on aminomethyl polystyrene resin Zin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoic acid The indicated compound was prepared in the same manner as in Reference Example 1. However, the N-{1} used in step 1 of Reference Example 1 was replaced. -[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-ol Instead of xo-1,2-dihydropyrimidine-4-ylbenzamide, in this process, 2- [2-(2-hydroxyethoxy)ethoxy]ethyl 4-tritylpiperazine-1-carb A compound called nic acid (described in International Publication No. 2009 / 064471) was used.

[0108] According to the descriptions in Examples 1-12 and Comparative Examples 1-3 below, various PMOs are used as shown in Table 2 PMO No. 1-11, 13-16. PMO was synthesized. The synthesized PMO was dissolved in sterile water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.). Note: PMO No. 12 I purchased it from GeneTools. [Table 2]

[0109] [Example 1] PMO No. 8 4-{[(2S,6R)-6-(4-benzamine) supported on aminomethyl polystyrene resin [do-2-oxopyrimidine-1(2H)-yl)-4-tritylmorpholine-2-yl]methoxymethyl Transfer 2g (800μmol) of cy-4-oxobutanoic acid (Reference Example 1) to the reaction vessel, and add dichlorometh 30 mL of [amount missing] was added and allowed to stand for 30 minutes. Furthermore, after washing twice with 30 mL of dichloromethane, the following steps were taken: The synthesis cycle was initiated. In each cycle, the desired sequence was obtained to achieve the base sequence of the indicated compound. A morpholino monomer compound was added. [Table 3]

[0110] The deblocking solution consists of trifluoroacetic acid (2 equivalents) and triethylamine (1 equivalent) Mix the following quantities to make a 3% (w / v) mixture: 1% (v / v) ethanol and 10% (v / v) 2,2 A solution of ,2-trifluoroethanol-containing dichloromethane was used. As the neutralizing solution, use 25% N,N-diisopropylethylamine to make a 5% (v / v) solution. A solution containing (v / v) 2-propanol in dichloromethane was used. As the pulling solution A, a morpholino monomer compound is prepared to a concentration of 0.15 M, at a concentration of 10% (v / v). 1,3-dimethyl-2-imidazolidinone containing N,N-diisopropylethylamine A solution dissolved in [a certain substance] was used. As coupling solution B, N,N-diisopropylethyl [another substance] was used. Min dissolved in 1,3-dimethyl-2-imidazolidinone to a concentration of 10% (v / v). The following was used: As the capping solution, 20% (v / v) acetic anhydride relative to dichloromethane. A solution of 30% (v / v) 2,6-lutidine was used.

[0111] The aminomethyl polystyrene resin on which the PMO synthesized above is supported is recovered from the reaction vessel. The P was dried under reduced pressure at room temperature for more than 2 hours. Place the MO in a reaction vessel, add 200 mL of 28% aqueous ammonia-ethanol (1 / 4), and incubate at 55°C for 15 hours. The mixture was stirred. The aminomethyl polystyrene resin was filtered off and washed with 50 mL of water-ethanol (1 / 4). The obtained filtrate was concentrated under reduced pressure. The resulting residue was buffered with 20 mM triethylamine acetate. Dissolve in 100 mL of a mixed solvent (4 / 1) of solution (TEAA buffer) and acetonitrile, and then fill the membrane with The filtrate was filtered using a filter. The resulting filtrate was purified by reverse-phase HPLC. The conditions used were as follows: be. [Table 4] Each fraction was analyzed, and the target substance was recovered with 100 mL of acetonitrile-water (1 / 1), and ethanol was added. 200 mL of Nol was added and the mixture was concentrated under reduced pressure. Further, it was dried under reduced pressure to obtain a white solid. 300 mL of 10 mM phosphoric acid solution was added to the solid and suspended. 10 mL of 2 M phosphoric acid solution was added, and 15 Stirring for minutes. Then, 15 mL of 2 M sodium hydroxide solution was added to neutralize. Add 15 mL of sodium hydroxide solution to make it alkaline, and then put a membrane filter (0.45 μm) into it. The solution was filtered using m). It was washed with 100 mL of 10 mM sodium hydroxide solution, and the target substance was obtained as an aqueous solution. I got it. The aqueous solution containing the obtained target product was purified using an anion exchange resin column. Conditions used: The details are as follows: [Table 5] Each fraction was analyzed (HPLC) to obtain the target product as an aqueous solution. A 0.1M solution was added to the obtained aqueous solution. 225 mL of phosphate buffer (pH 6.0) was added to neutralize the solution. The solution was then filtered through a membrane filter (0.45 μm). The sample was filtered. Then, ultrafiltration was performed under the following conditions to remove the salt. [Table 6]

[0112] The filtrate was concentrated to obtain approximately 250 mL of aqueous solution. The obtained aqueous solution was filtered through a membrane filter (0.4 The solution was filtered through a 5 μm filter. The resulting aqueous solution was freeze-dried to obtain 1.5 g of the target compound as a white, cotton-like solid. I obtained something. ESI-TOF-MS calculated value: 6924.82 Measured value: 6923.54

[0113] [Example 2] PMO.No.1 The indicated compound was prepared according to the same method as in Example 1. MALDI-TOF-MS calculated value: 8291.96 Measured value: 8296.24

[0114] [Example 3] PMO.No.2 The indicated compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 7310.13 Measurement value: 7309.23

[0115] [Example 4] PMO.No.3 The indicated compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 8270.94 Measurement value: 8270.55

[0116] [Example 5] PMO.No.4 The indicated compound was prepared according to the same method as in Example 1. However, as a starting material, 4-(((2S,6R)-6-(5-methyl-2,4-diodeon supported on methyl polystyrene resin Xo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl) Methoxy)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 7310.13 Measured value: 7310.17

[0117] [Example 6] PMO.No.5 The indicated compound was prepared according to the same method as in Example 1. However, as a starting material, 4-(((2S,6R)-6-(5-methyl-2,4-diode polystyrene resin supported Xo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorpholin-2-yl) Methoxy)-4-oxobutanoic acid (Reference Example 3) was used. ESI-TOF-MS calculated value: 8270.94 Measured value: 8270.20

[0118] [Example 7] PMO.No.6 The indicated compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 5964.01 Measured value: 5963.68

[0119] [Example 8] PMO.No.7 The indicated compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 6609.55 Measured value: 6608.85

[0120] [Example 9] PMO.No.9 The indicated compound was prepared according to the same method as in Example 1. However, as a starting material, 4-oxo-4-(((2S,6R)-6-(6-oxo supported on methyl polystyrene resin -2-(2-phenoxyacetamide)-1H-purine-9(6H)-yl)-4-tritylmorphone Phosphate-2-yl(methoxy)butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 7280.11 Measured value: 7279.42

[0121] [Example 10] PMO.No.10 The indicated compound was prepared according to the same method as in Example 1. However, as a starting material, 4-oxo-4-(((2S,6R)-6-(6-oxo supported on methyl polystyrene resin -2-(2-phenoxyacetamide)-1H-purine-9(6H)-yl)-4-tritylmorphone Phosphate-2-yl(methoxy)butanoic acid (Reference Example 2) was used. ESI-TOF-MS calculated value: 8295.95 Measured value: 8295.91

[0122] [Example 11] PMO.No.13 The indicated compound was prepared according to the same method as in Example 1, except that amino acids were used as the starting material. 1,12-dioxo-1-(4-tritylpiperazine-1) supported on methyl polystyrene resin -yl)-2,5,8,11-tetraoxa-15-pentadecanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 7276.15 Measurement value: 7276.69

[0123] [Example 12] PMO.No.14 The indicated compound was prepared according to the same method as in Example 1, except that amino acids were used as the starting material. 1,12-dioxo-1-(4-tritylpiperazine-1) supported on methyl polystyrene resin -yl)-2,5,8,11-tetraoxa-15-pentadecanoic acid (Reference Example 4) was used. ESI-TOF-MS calculated value: 8622.27 Measured value: 8622.29

[0124] [Comparative Example 1] PMO.No.11 The indicated compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 10274.63 Measured value: 10273.71

[0125] [Comparative Example 2] PMO.No.15 The indicated compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 9941.33 Measured value: 9940.77

[0126] [Comparative Example 3] PMO.No.16 The indicated compound was prepared according to the same method as in Example 1. ESI-TOF-MS calculated value: 8238.94 Measured value: 8238.69

[0127] [Test Example 1] In vitro assay RD cells (human rhabdomyosarcoma cell line) 4 × 10 5 For each individual, the oligosaccharides of the present invention, PMO No. 1 to 8. - and 10 μM of PMO No. 11 antisense oligomer in the Amaxa Cell Line Nucleofector Kit It was installed using L via Nucleofector II (Lonza). The program used was T-030.

[0128] After introduction, the cells were placed in Eagle's m with 10% fetal bovine serum (FCS) (Invitrogen). Initial essential medium (EMEM) (Sigma-Ace, same applies below) 2 mL in a 5% CO2 strip at 37°C. The cells were cultured overnight under the specified conditions. After washing the cells twice with PBS (Nissui Co., Ltd., the same applies below), ISOGEN was applied. Add 500 μl (manufactured by Nippon Gene Co., Ltd.) to the cells and leave at room temperature for several minutes to lyse the cells. The dissolved product was collected in an Eppendorf tube. According to the protocol attached to ISOGEN, total RNA was extracted. The concentration of the extracted total RNA was measured using NanoDrop ND-1000 (manufactured by LMS Co., Ltd.). ) was used for measurement.

[0129] For 400 ng of extracted total RNA, the Titan One Tube RT-PCR Kit (Roche) was used. One-Step RT-PCR was performed. The reaction mixture was prepared according to the protocol provided with the kit. A PTC-100 thermal cycler (manufactured by MJ Research) was used. The RT-PCR program used was... The following is the case for Mu: 50°C, 30 minutes: Reverse transcription reaction 94°C, 2 minutes: Thermal denaturation [94°C, 10 seconds; 58°C, 30 seconds; 68°C, 45 seconds] x 30 cycles: PCR amplification 68°C, 7 minutes: Thermal inactivation of polymerase

[0130] The nucleotide sequences of the forward and reverse primers used in RT-PCR are as follows: That is the case. Forward primer: 5'-AGGATTTGGAACAGAGGCGTC-3' (SEQ ID NO: 40) Reverse primer: 5'-GTCTGCCACTGGCGGAGGTC-3' (SEQ ID NO: 41)

[0131] Next, the amplification product of the above RT-PCR was nested using Taq DNA Polymerase (Roche). ed PCR was performed. The PCR program used is as follows: 94°C, 2 minutes: Thermal denaturation [94°C, 15 seconds; 58°C, 30 seconds; 68°C, 45 seconds] x 30 cycles: PCR amplification 68°C, 7 minutes: Thermal inactivation of polymerase

[0132] The base sequences of the forward and reverse primers used in the above nested PCR are as follows: It is as follows: Forward primer: 5'-CATCAAGCAGAAGGCAACAA-3' (SEQ ID NO: 42) Reverse primer: 5'-GAAGTTTCAGGGCCAAGTCA-3' (SEQ ID NO: 43)

[0133] One μl of the reaction product from the above nested PCR was analyzed using a Bioanalyzer (Agilent). The amount of polynucleotides "A" in the band skipped by exon 53, and the amount of polynucleotides "A" in the band skipped by exon 53. The polynucleotide amount "B" of the bands that did not show up was measured. Based on constant values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / (A + B) x 100

[0134] Experimental results The results are shown in Figure 1. This experiment showed that the oligomers of the present invention, PMO No. 1 to 8, are all PMO Compared to antisense oligomer No. 11, it skips exon 53 with significantly higher efficiency. It was found that this causes [something]. In particular, the oligomers of the present invention of PMO No. 3 and 8 are [something] of PMO No. 11. It exhibits more than four times higher exon skipping efficiency compared to antisense oligomers.

[0135] [Test Example 2] In vitro assay using human fibroblasts TIG-119 cells (human normal tissue-derived fibroblasts) were induced by the ZsGreen1 co-expression retroviral vector. Cells, National Institute of Biomedical Innovation) or 5017 cells (human DMD patient-derived fibroblasts, Coriell Institute) The human myoD gene (SEQ ID NO: 44) was introduced into the (for Medical Research) model. After incubation for 4 to 5 days, FACS was used to identify ZsGreen-positive MyoD-converted fibroblasts. Collect, 5 x 10 4 pieces / cm 2 Seeds were sown in 12-well plates to achieve the following result. The growth medium was 10% FCS and 1% Dulbecco's Modified Penicillin / Streptomycin (P / S) (Sigma-Aldrich) Use 1 mL of Eagle Medium: Nutrient Mixture F-12 (DMEM-F-12) (Invitrogen). Ta. After 24 hours, differentiation medium (2% horse serum (Invitrogen), 1% P / S and ITS Liquid Medi) was used. The culture medium was replaced with DMEM / F-12 containing a Supplement (Sigma-A). The culture medium was changed every 2-3 days. The cells were incubated for 14 days to differentiate them into myotubes. Subsequently, the differentiation medium was replaced with a differentiation medium containing 6 μM Endo-Porter (GeneTool Co., Ltd.), and the process was completed. Morpholino oligomers were added to a concentration of 10 μM. After incubation for 48 hours, T Total RNA was extracted from cells using RIzol (Invitrogen). RT-PCR was performed on 50 ng of RNA using the QIAGEN OneStep RT-PCR Kit. The reaction solution was prepared according to the instructions. A thermal cycler (iCycler, manufactured by Bio-Rad) was used. The RT-PCR program used was as follows: 50°C, 30 minutes: Reverse transcription reaction 95°C, 15 minutes: Thermal denaturation [94°C, 1 minute; 60°C, 1 minute; 72°C, 1 minute] x 35 cycles: PCR amplification 72°C, 7 minutes: Thermal inactivation of polymerase

[0136] hEX51F and hEX55R were used as primers. hEX51F:5'-CGGGCTTGGACAGAACTTAC-3' (SEQ ID NO: 45) hEx55R:5'-TCCTTACGGGTAGCATCCTG-3' (Sequence ID 46)

[0137] The reaction products of the above RT-PCR reaction were separated by 2% agarose gel electrophoresis and then GeneFlash was used. Gel photographs were taken using Syngene. Image J (manufactured by the National Institutes of Health, USA) The amount of polynucleotides "A" in the band skipped by exon 53, and the amount of polynucleotides "A" in the band skipped by exon 53. The polynucleotide amount "B" of the bands that did not show up was measured. Based on constant values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / (A + B) x 100

[0138] Experimental results The results are shown in Figures 2 and 3. This experiment confirmed that the oligomers of the present invention of PMO No. 3, 8 and 9 ( Figure 2) shows the results in TIG-119 cells, compared to antisense oligomers of PMO No. 12. It was found that exon 53 could be skipped with high efficiency (Figure 2). In particular, PMO No. 3 The oligomers of the present invention and 8 are more than twice as effective as the antisense oligomer of PMO No. 12. It exhibits high exon skipping efficiency (Figure 2). Furthermore, this experiment showed that the oligomers of PMO No. 3 and 8-10 of the present invention (Figure 3) were found in 5017 cells. In both cases, compared to the antisense oligomer of PMO No. 12, exophthalmos were produced with higher efficiency. It was found that 53 was skipped (Figure 3). In particular, the original of the present invention of PMO No. 3 and 8 Gomar has more than 7 times higher exon skimming compared to PMO No. 12 antisense oligomer. Ping efficiency is shown (Figure 3).

[0139] [Test Example 3] In vitro assay using human fibroblasts Upper left of a DMD patient with deletion of exons 45-52 or DMD patient with deletion of exons 48-52. A biopsy was taken from the inner arm to collect dermal fibroblast cells (from human DMD patients (exons 45-52 or exons 48-52)). 2) Derived fibroblast cells were established. Fibroblast cells were induced using a ZsGreen1 co-expression retroviral vector. The human myoD gene (SEQ ID NO: 44) was introduced into the cells. After incubation for 4 to 5 days, FACS was used to identify ZsGreen-positive MyoD-converted fibroblasts. Collect, 5 x 10 4 pieces / cm 2 Seeds were sown in 12-well plates to achieve the following result. The growth medium was 10% FCS and 1% Dulbecco's Modified Penicillin / Streptomycin (P / S) (Sigma-Aldrich) Use 1 mL of Eagle Medium: Nutrient Mixture F-12 (DMEM / F-12) (Invitrogen). Ta. After 24 hours, differentiation medium (2% horse serum (Invitrogen), 1% P / S and ITS Liquid Medi) was used. The culture medium was replaced with DMEM / F-12 containing a Supplement (Sigma-A). The culture medium was changed every 2-3 days. They were incubated for 14 or 20 days to differentiate into myotubes. Subsequently, the differentiation medium was replaced with a differentiation medium containing 6 μM Endo-Porter (GeneTool Co., Ltd.), and the process was completed. Morpholino oligomers were added to a concentration of 10 μM. After incubation for 48 hours, T Total RNA was extracted from cells using RIzol (Invitrogen). RT-PCR was performed on 50 ng of RNA using the QIAGEN OneStep RT-PCR Kit. The reaction solution was prepared according to the instructions. A thermal cycler (iCycler, manufactured by Bio-Rad) was used. The RT-PCR program used was as follows: 50°C, 30 minutes: Reverse transcription reaction 95°C, 15 minutes: Thermal denaturation [94°C, 1 minute; 60°C, 1 minute; 72°C, 1 minute] x 35 cycles: PCR amplification 72°C, 7 minutes: Thermal inactivation of polymerase

[0140] hEx44F and h55R were used as primers. hEx44F:5'- TGTTGAGAAATGGCGGCGT-3' (Sequence ID 48) hEx55R:5'- TCCTTACGGGTAGCATCCTG-3' (Sequence ID 46)

[0141] The reaction products of the above RT-PCR reaction were separated by 2% agarose gel electrophoresis and then GeneFlash was used. Gel photographs were taken using Syngene. Image J (manufactured by the National Institutes of Health, USA) The amount of polynucleotides "A" in the band skipped by exon 53, and the amount of polynucleotides "A" in the band skipped by exon 53. The polynucleotide amount "B" of the bands that did not show up was measured. Based on constant values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / (A + B) x 100

[0142] Experimental results The results are shown in Figures 4 and 5. This experiment showed that the oligomers of PMO No. 3 and 8 of the present invention are... In cells derived from DMD patients with exon 45-52 deletion (Figure 4) or exon 48-52 deletion (Figure 5), 80% It was found that Exxon 53 could be skipped with the above high efficiency. Also, PMO No. 3 and The oligomer of the present invention, in cells derived from exon 45-52 deletion DMD patients, is PMO No. 15 It was found to skip exon 53 with higher efficiency compared to antisense oligomers. Revealed (Figure 4).

[0143] [Test Example 4] Western blotting The oligomer of the present invention, PMO No. 8, was added to cells at a concentration of 10 μM, and after 72 hours, Com was obtained from the cells. RIPA buffer containing plete Mini (manufactured by Roche Applied Science) (Thermo Fisher Scientific) Proteins are extracted using (manufactured by Thermo Fisher Scientific), and then the BCA protein assay kit (manufactured by Thermo Fisher Scientific) is used. Protein quantification was performed using NuPAGE Novex Tris-Acetate Gel 3-8% (Invitrogen). Electrophoresis was performed at 0 V for 75 minutes, and the samples were transferred to a PVDF membrane (Millipore) using a semi-dry rotter. After blocking the F membrane with 5% ECL Blocking agent (GE Healthcare), anti-dystrophin The membrane was incubated in antibody (NCL-Dys1, Novocastra) solution. Furthermore, peroxidas After incubation in e-conjugated goat-antimouse IgG (model number, Bio-Rad) solution, EC The color was developed using the L Plus Western blotting system (manufactured by GE Healthcare).

[0144] immunostaining Add the oligomer of the present invention, PMO No. 3 or No. 8, to cells, and after 72 hours, the cells are treated with 3% parabens. Ormaldehyde was immobilized in 10 minutes. Incubated in 10% Triton-X for 10 minutes. 10% goat Blocked with serum-containing PBS, the membrane was in an anti-dystrophin antibody (NCL-Dys1, Novocastra) solution. The film was incubated, and then incubated in a solution of anti-mouse IgG antibody (Invitrogen). The sample was mounted using Pro Long Gold Antifade reagent (Invitrogen) and then scanned under a fluorescence microscope. Observed using [this method].

[0145] Experimental results The results are shown in Figures 6 and 7. This experiment showed that the oligomers of the present invention for PMO No. 3 and 8 are dis Inducing trophin protein expression is possible in Western blotting (Figure 6) and immunotherapy. This was confirmed by epidemiological staining (Figure 7).

[0146] [Test Example 5] In vitro assay using human fibroblasts The experiment was conducted using the same method as in Test Example 3.

[0147] Experimental results The results are shown in Figure 8. This experiment shows that the oligomers of the present invention, PMO No. 3 and 8, are exon 4 In cells derived from 5-52 deletion DMD patients, the oligomers of the present invention with PMO No. 13 and 14 showed higher levels. It was found that skipping Exon 53 was efficient (Figure 8).

[0148] [Test Example 6] In vitro assay The 2'-O-methoxyphosphorothioate (2'-OMe-S-RNA) described in SEQ ID NOs. 49-123 Experiments were conducted using antisense oligomers. Various antisense oligomers were used in the assay. The gomers were purchased from Japan Bioservices Co., Ltd. The sequences of various antisense oligomers are as follows: This will be shown. [Table 7-A] [Table 7-B] [Table 7-C]

[0149] RD cells (human rhabdomyosarcoma cell line) 3 × 10 5 Seeds were planted in a 6-well plate, and 10% fetal bovine serum was added. Eagle's minimal essential medium (EMEM) medium containing (FCS) (manufactured by Invitrogen). (Sigma-Ace, same below) 2 mL was incubated overnight at 37°C under 5% CO2 conditions. Exon 5 as described above. 3. Various antisense oligomers for skipping (manufactured by Japan Bioservices Co., Ltd.) (1 μM) and A complex of Lipofectamine 2000 (manufactured by Invitrogen) was prepared, and the culture medium was changed with 1.8 mL of R 200 μl was added to D cells to achieve a final concentration of 100 nM. After addition, the cells were cultured overnight. The cells were washed twice with PBS (Nissui Co., Ltd., the same applies below) and then subjected to ISOG. Add 500 μl of EN (manufactured by Nippon Gene Co., Ltd.) to the cells and leave at room temperature for several minutes to lyse the cells. The dissolved substance was then collected in an Eppendorf tube. Following the protocol provided with ISOGEN, Total RNA was extracted. The concentration of the extracted total RNA was measured using NanoDrop ND-1000 (LMS). Measurements were taken using a device manufactured by the company. For 400 ng of extracted total RNA, the Titan One Tube RT-PCR Kit (Roche) was used. One-Step RT-PCR was performed. The reaction mixture was prepared according to the protocol provided with the kit. A PTC-100 thermal cycler (manufactured by MJ Research) was used. The RT-PCR program used was... The following is the case for Mu: 50°C, 30 minutes: Reverse transcription reaction 94°C, 2 minutes: Thermal denaturation [94°C, 10 seconds; 58°C, 30 seconds; 68°C, 45 seconds] x 30 cycles: PCR amplification 68°C, 7 minutes: Thermal inactivation of polymerase

[0150] The nucleotide sequences of the forward and reverse primers used in RT-PCR are as follows: That is the case. Forward primer: 5'-CATCAAGCAGAAGGCAACAA-3' (SEQ ID NO: 42) Reverse primer: 5'-GAAGTTTCAGGGCCAAGTCA-3' (SEQ ID NO: 43) Next, the amplification product of the above RT-PCR was nested using Taq DNA Polymerase (Roche). ed PCR was performed. The PCR program used is as follows: 94°C, 2 minutes: Thermal denaturation [94°C, 15 seconds; 58°C, 30 seconds; 68°C, 45 seconds] x 30 cycles: PCR amplification 68°C, 7 minutes: Thermal inactivation of polymerase

[0151] The base sequences of the forward and reverse primers used in the above nested PCR are as follows: It is as follows: Forward primer: 5'-AGGATTTGGAACAGAGGCGTC-3' (SEQ ID NO: 40) Reverse primer: 5'-GTCTGCCACTGGCGGAGGTC-3' (SEQ ID NO: 41)

[0152] One μl of the reaction product from the above nested PCR was analyzed using a Bioanalyzer (Agilent). The amount of polynucleotides "A" in the band skipped by exon 53, and the amount of polynucleotides "A" in the band skipped by exon 53. The polynucleotide amount "B" of the bands that did not show up was measured. Based on constant values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / (A + B) x 100

[0153] Experimental results The results are shown in Figures 9 to 17. This experiment revealed that the 53rd e of the human dystrophin gene If antisense oligomers are designed from the 5' end of xon to positions 31-61, high efficiency It was discovered that the Exxon 53 was being skipped.

[0154] [Test Example 7] RD cells (human rhabdomyosarcoma cell line) 3.5 × 10⁻⁶ 5 For each individual, antisense oligomer 0.3~3 0 μM was processed using the Amaxa Cell Line Nucleofector Kit L with Nucleofector II (Lonza) It was implemented. The program used was T-030. After introduction, the cells were placed in Eagle's m with 10% fetal bovine serum (FCS) (Invitrogen). Initial essential medium (EMEM) (Sigma-Ace, same applies below) 2 mL in a 5% CO2 strip at 37°C. The cells were cultured overnight under the specified conditions. After washing the cells twice with PBS (Nissui Co., Ltd., the same applies below), ISOGEN was applied. Add 500 μl (manufactured by Nippon Gene Co., Ltd.) to the cells and leave at room temperature for several minutes to lyse the cells. The dissolved product was collected in an Eppendorf tube. According to the protocol attached to ISOGEN, total RNA was extracted. The concentration of the extracted total RNA was measured using NanoDrop ND-1000 (manufactured by LMS Co., Ltd.). ) was used for measurement. For 400 ng of extracted total RNA, the QIAGEN OneStep RT-PCR Kit (manufactured by Qiagen) was used. One-Step RT-PCR was performed. The reaction mixture was prepared according to the protocol included with the kit. The thermal cycler used was the PTC-100 (manufactured by MJ Research). The RT-PCR procedure used was... The ram is as follows: 50°C, 30 minutes: Reverse transcription reaction 95°C, 15 minutes: Thermal denaturation [94°C, 30 seconds; 60°C, 30 seconds; 72°C, 1 minute] x 35 cycles: PCR amplification 72°C, 10 minutes: Thermal inactivation of polymerase

[0155] The nucleotide sequences of the forward and reverse primers used in RT-PCR are as follows: That is the case. Forward primer: 5'-CATCAAGCAGAAGGCAACAA-3' (SEQ ID NO: 42) Reverse primer: 5'-GAAGTTTCAGGGCCAAGTCA-3' (SEQ ID NO: 43)

[0156] One μl of the reaction product from the above PCR was analyzed using a Bioanalyzer (Agilent). The amount of polynucleotides "A" in the band skipped by exon 53, and the amount of polynucleotides "A" in the band skipped by exon 53. The polynucleotide amount "B" of the bands that did not show up was measured. Based on constant values, the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / (A + B) x 100

[0157] Experimental results The results are shown in Figures 18 and 19. This experiment shows that the oligomer of the present invention with PMO No. 8 is PMO No. Compared to antisense oligomers 15 and 16, it skips exon 53 with significantly higher efficiency. It was found that this causes (Figure 18). Furthermore, the oligomers of the present invention of PMO No. 3 and 8 are PMO No. . Skipping exon 53 with significantly higher efficiency than the oligomers of the present invention 13 and 14. It was found that (Figure 19). From this result, even with the same sequence, the one with an -OH group at the 5' end is the one that However, it is shown to have high skipping efficiency. [Industrial applicability]

[0158] From the experimental results shown in the test examples, the oligomers (PMO No. 1-10) of the present invention are related to the prior art. Compared to the oligomers (PMO No. 11, 12, 15, and 16), it exhibits remarkable differences in all cellular environments. It was shown that exon 53 could be skipped with high efficiency. Also, the 5 used in Test Example 2 Cell O17 is a cell collected from a DMD patient, and also the fibroblast used in Experimental Examples 3 and 5. These are also exon 53 skipping target cells derived from DMD patients. In particular, in Test Examples 3 and 5 The oligomer of the present invention is used in cells derived from DMD patients that are subject to exon 53 skipping. Therefore, because it exhibits an exon 53 skipping efficiency of 90% or more, the oligomer of the present invention is actually It can be said that this drug also efficiently skips exon 53 when administered to DMD patients. Therefore, the oligomer of the present invention is extremely useful in the treatment of DMD. [Sequence Listing Free Text]

[0159] Sequence ID 2: Synthetic nucleic acid Sequence ID 3: Synthetic nucleic acid Sequence ID 4: Synthetic nucleic acid Sequence ID 5: Synthetic nucleic acid Sequence ID 6: Synthetic nucleic acid Sequence ID 7: Synthetic nucleic acid Sequence ID 8: Synthetic nucleic acid Sequence ID 9: Synthetic nucleic acid Sequence ID 10: Synthetic nucleic acid Sequence ID 11: Synthetic Nucleic Acid Sequence ID 12: Synthetic nucleic acid Sequence ID 13: Synthetic Nucleic Acid Sequence ID 14: Synthetic nucleic acid Sequence ID 15: Synthetic nucleic acid Sequence ID 16: Synthetic Nucleic Acid Sequence ID 17: Synthetic nucleic acid Sequence ID 18: Synthetic Nucleic Acid Sequence ID 19: Synthetic nucleic acid Sequence ID 20: Synthetic nucleic acid Sequence ID 21: Synthetic Nucleic Acid Sequence ID 22: Synthetic nucleic acid Sequence ID 23: Synthetic nucleic acid Sequence ID 24: Synthetic nucleic acid Sequence ID 25: Synthetic nucleic acid Sequence ID 26: Synthetic nucleic acid Sequence ID 27: Synthetic nucleic acid Sequence ID 28: Synthetic nucleic acid Sequence ID 29: Synthetic nucleic acid Sequence ID 30: Synthetic nucleic acid Sequence ID 31: Synthetic nucleic acid Sequence ID 32: Synthetic nucleic acid Sequence ID 33: Synthetic nucleic acid Sequence ID 34: Synthetic nucleic acid Sequence ID 35: Synthetic nucleic acid Sequence ID 36: Synthetic nucleic acid Sequence ID 37: Synthetic nucleic acid Sequence ID 38: Synthetic nucleic acid Sequence ID 39: Synthetic nucleic acid Sequence ID 40: Synthetic nucleic acid Sequence ID 41: Synthetic nucleic acid Sequence ID 42: Synthetic nucleic acid Sequence ID 43: Synthetic nucleic acid Sequence ID 45: Synthetic nucleic acid Sequence ID 46: Synthetic nucleic acid Sequence ID 47: Synthetic nucleic acid Sequence ID 48: Synthetic nucleic acid Sequence ID 49: Synthetic nucleic acid Sequence ID 50: Synthetic nucleic acid Sequence ID 51: Synthetic nucleic acid Sequence ID 52: Synthetic nucleic acid Sequence ID 53: Synthetic nucleic acid Sequence ID 54: Synthetic nucleic acid Sequence ID 55: Synthetic nucleic acid Sequence ID 56: Synthetic nucleic acid Sequence ID 57: Synthetic nucleic acid Sequence ID 58: Synthetic nucleic acid Sequence ID 59: Synthetic nucleic acid Sequence ID 60: Synthetic nucleic acid Sequence ID 61: Synthetic nucleic acid Sequence ID 62: Synthetic nucleic acid Sequence ID 63: Synthetic nucleic acid Sequence ID 64: Synthetic nucleic acid Sequence ID 65: Synthetic nucleic acid Sequence ID 66: Synthetic nucleic acid Sequence ID 67: Synthetic nucleic acid Sequence ID 68: Synthetic nucleic acid Sequence ID 69: Synthetic nucleic acid Sequence ID 70: Synthetic nucleic acid Sequence ID 71: Synthetic nucleic acid Sequence ID 72: Synthetic nucleic acid Sequence ID 73: Synthetic nucleic acid Sequence ID 74: Synthetic nucleic acid Sequence ID 75: Synthetic nucleic acid Sequence ID 76: Synthetic nucleic acid Sequence ID 77: Synthetic nucleic acid Sequence ID 78: Synthetic nucleic acid Sequence ID 79: Synthetic nucleic acid Sequence ID 80: Synthetic nucleic acid Sequence ID 81: Synthetic nucleic acid Sequence ID 82: Synthetic nucleic acid Sequence ID 83: Synthetic nucleic acid Sequence ID 84: Synthetic nucleic acid Sequence ID 85: Synthetic nucleic acid Sequence ID 86: Synthetic nucleic acid Sequence ID 87: Synthetic nucleic acid Sequence ID 88: Synthetic nucleic acid Sequence ID 89: Synthetic nucleic acid Sequence ID 90: Synthetic nucleic acid Sequence ID 91: Synthetic nucleic acid Sequence ID 92: Synthetic nucleic acid Sequence ID 93: Synthetic nucleic acid Sequence ID 94: Synthetic nucleic acid Sequence ID 95: Synthetic nucleic acid Sequence ID 96: Synthetic nucleic acid Sequence ID 97: Synthetic nucleic acid Sequence ID 98: Synthetic nucleic acid Sequence ID 99: Synthetic nucleic acid Sequence ID 100: Synthetic nucleic acid Sequence ID 101: Synthetic nucleic acid Sequence ID 102: Synthetic nucleic acid Sequence ID 103: Synthetic Nucleic Acid Sequence ID 104: Synthetic nucleic acid Sequence ID 105: Synthetic nucleic acid Sequence ID 106: Synthetic nucleic acid Sequence ID 107: Synthetic nucleic acid Sequence ID 108: Synthetic nucleic acid Sequence ID 109: Synthetic nucleic acid Sequence ID 110: Synthetic nucleic acid Sequence ID 111: Synthetic nucleic acid Sequence ID 112: Synthetic nucleic acid Sequence ID 113: Synthetic nucleic acid Sequence ID 114: Synthetic nucleic acid Sequence ID 115: Synthetic nucleic acid Sequence ID 116: Synthetic nucleic acid Sequence ID 117: Synthetic nucleic acid Sequence ID 118: Synthetic nucleic acid Sequence ID 119: Synthetic nucleic acid Sequence ID 120: Synthetic nucleic acid Sequence ID 121: Synthetic Nucleic Acid Sequence ID 122: Synthetic nucleic acid Sequence ID 123: Synthetic Nucleic Acid

Claims

1. Antiseptics enable skipping of exon 53 of the human dystrophin gene. It is an oligomer, and is located at the 5' end of the 53rd exon of the human dystrophin gene. The 31st to 53rd, 31st to 54th, 31st to 55th, 31st to 56th, 31st to 57th, 31st to 58th 1st, 32nd-53rd, 32nd-54th, 32nd-55th, 32nd-56th, 32nd-57th, 32nd ~58th, 33rd to 53rd, 33rd to 54th, 33rd to 55th, 33rd to 56th, 33rd to 57th, Numbers 33-58, 34-53, 34-54, 34-55, 34-56, and 34-57 The 34th to 58th, the 35th to 53rd, the 35th to 54th, the 35th to 55th, the 35th to 56th, the 35th 57th, 35th-58th, 36th-53rd, 36th-54th, 36th-55th, 36th-56th, A base complementary to either the sequence consisting of nucleotides 36-57 or 36-58. An antisense oligomer consisting of a sequence.

2. The antisense oligomer according to claim 1, which is an oligonucleotide.

3. The sugar portion and / or of at least one nucleotide constituting the oligonucleotide The antisense oligomer according to claim 2, wherein the nic acid bond portion is modified.

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

5. The phosphate bond portion of at least one nucleotide constituting the oligonucleotide , phosphorothioate bond, phosphorodithioate bond, alkylphosphonate bond, ho Either selected from the group consisting of sphoramidate bonds and boranophosphate bonds. The antisense oligomer according to claim 3 or 4, which is one of the following.

6. The antisense oligomer according to claim 1, which is a morpholino oligomer.

7. The antisense of phosphorodiamidate morpholino oligomer according to claim 6. Ligomar.

8. The 5' end is a group of any of the following chemical formulas (1) to (3), as described in claim 6 or 7. Intestinal oligomer. 【Chemistry 24】

9. From the 5' end of the 53rd exon of the human dystrophin gene, positions 32-56 or 36 A sequence consisting of a base sequence complementary to the sequence of nucleotides ~56, any one of claims 1 to 8 The antisense oligomer described in item 1.

10. Claim comprising a base sequence selected from any one of the group consisting of Sequence IDs 2 to 37. An antisense oligomer as described in any one of items 1 to 8.

11. From any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 17, 23, 29, and 35 The antisense oligomer according to any one of claims 1 to 8.

12. The nucleotide sequence comprising either SEQ ID NO: 11 or 35, as described in any one of claims 1 to 8. Antisense oligomer.

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

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