Induction of exon skipping in eukaryotic cells

[0001] With the rapid development of human genome research, experts and the general public will be able to establish treatments for many severe genetic diseases in the near future, in addition to elucidating the mechanism of the disease and accurate and reliable diagnostic methods. I'm expecting that. [0002] It is expected that new insights will lead to the development of easy-to-administer small molecule gene therapy as a treatment for diseases (eg, metabolic disorders), but on the other hand, in many diseases, gene therapy other than the above, that is, , Modification, addition or substitution of abnormal gene products may ultimately be required. [0003] In recent years, research and development in this area has highlighted some technical issues that must be overcome. For example, the widespread range of genes involved in genetic disease (thus limiting the choice of the appropriate response system to which the therapeutic gene should be administered) and the reachability of the therapeutic gene to the functioning tissue (specific targeting). It is necessary to construct a technique, that is, physical targeting by restrictive insertion of genes, or biological targeting by developing a system having tissue-specific affinity), and the safety of the administration system to patients. There is a problem. It can be generally concluded that these issues are interrelated to some extent and that the smaller the therapeutic agent, the easier it is to develop a highly efficient, targetable and safe dosing system. [0004] In the present invention, these problems are addressed by inducing so-called exon skipping into cells. Exon skipping yields mature mRNAs that do not have skipped exons, so if skipped exons encode amino acids, this skipping leads to the expression of altered products. Currently, exon skipping techniques use the so-called "Anti-sense Oligonucleotide" (AON), and most of the research is on Duchenne muscular. It is performed using the mdx mouse, which is a model of dystrophy (DMD). Mdx mice with a nonsense mutation in the dystrophin gene exon 23 have been used as an animal model for Duchenne muscular dystrophy. Rarely, natural dystrophin-positive fibers are observed in mdx muscle tissue, despite having an mdx mutation that should eliminate the synthesis of functional dystrophin protein. Such dystrophin-positive fibers are thought to have been generated by a natural exon skipping mechanism due to somatic mutation or alternative splicing. AON specific for the 3'and 5'splice sites of introns 22 and 23 of the dystrophin mRNA precursor interferes with factors normally involved in the removal of intron 23, and exon 23 has also been shown to be removed from mRNA. (Wilton, 1999). In a similar study, Dunckley et al. (1998) showed that exon skipping with AON specific for 3'and 5'splice sites can have unexpected results. They observed skipping of exons 24-29 as well as exon 23, resulting in the production of mRNA with a conjugate of exon 22 and exon 30. The mechanism underlying the emergence of the unexpected 22-30 splicing variants is unknown. The appearance of such variants is believed to be due to the splice site containing a consensus sequence, resulting in non-specific hybridization with the oligonucleotides used to guide exon skipping. By hybridizing the oligonucleotide to a splice site other than the skipped exon site, it is possible, of course, to easily interfere with the accuracy of the splicing reaction. On the other hand, the reason for the inaccuracy of the splicing reaction may be that it requires the use of two oligonucleotides (for each of the 5'and 3'splice sites). Pre-mRNA with one oligonucleotide but no other oligonucleotide tends to be an unexpected splicing variant. [0005] For controlling pre-mRNA splicing in an RNA splicing reaction system, including contacting with a reagent capable of specifically inhibiting signal) (EIS) and causing the pre-mRNA to be spliced. Provide a method. The advantage of interfering with the exon inclusion signal is that such factors are present within the exon. By providing an antisense oligonucleotide against the sequence within the exon to be skipped, the exon can be effectively shielded from the splicing mechanism by interfering with the exon encapsulation signal. Exons are excluded from the final mRNA by the splicing mechanism not recognizing the exons to be skipped. The present invention does not directly interfere with the enzymatic reaction (exon binding) of the splicing mechanism. From this, it is considered that the present invention is a reliable and highly reliable method. EIS is an exon-specific structure and is thought to impart a specific molecular configuration to the splice receiving site and splice donating site. In such a concept, it is considered that the splicing mechanism recognizes exons by a specific configuration. However, the present invention is not limited to this model. We have found that exon-binding reagents inhibit EIS. Such reagents can specifically inhibit EIS, even though they can specifically contact any site of the exon. The mRNA itself obtained by the method of the present invention is useful. For example, blocking the inclusion of the exons required for mRNA can at least partially reduce the production of unwanted proteins. The method of the present invention preferably further comprises the step of translating the mRNA produced by splicing the pre-mRNA. It is also preferred that the mRNA encodes a functional protein. In a preferred embodiment of the invention, the protein comprises two or more domains and at least one of the domains. One is encoded by the mRNA produced by skipping at least some of the exons contained in the pre-mRNA. A typical example in which exon skipping is effective is, but is not limited to, a protein having a wild-type structure having at least two functional domains, each domain having an individual amino acid sequence having a primary structure. It is generated from the part of. Specific examples include transcription factors. Typical transcription factors include DNA binding domains and domains that react with other proteins in the cell. Exon skipping, which encodes part of the primary structure of an amino acid that resides between two domains, leads to the production of shorter proteins that perform at least partly equivalent functions. Therefore, detrimental mutations in the intermediate region (eg frameshift mutations and stop mutations) can be at least partially repaired by inducing exon skipping and are short proteins with (partial) function. Can be synthesized. It is also possible to induce partial skipping of exons using the methods of the invention. In this embodiment, contact of the reagent with the exon activates the concealed splice site of the exon in contact with the reagent. This aspect enhances the operability of pre-mRNA that induces functional proteins. The reaction system preferably comprises cells. Cells are in vitro ( Skipping leads to the production of shorter proteins that perform at least partly equivalent functions. Therefore, detrimental mutations in the intermediate region (eg frameshift mutations and stop mutations) can be at least partially repaired by inducing exon skipping and are short proteins with (partial) function. Can be synthesized. It is also possible to induce partial skipping of exons using the methods of the invention. In this embodiment, contact of the reagent with the exon activates the concealed splice site of the exon in contact with the reagent. This aspect enhances the operability of pre-mRNA that induces functional proteins. The reaction system preferably comprises cells. Cells are in vitro ( Skipping leads to the production of shorter proteins that perform at least partly equivalent functions. Therefore, detrimental mutations in the intermediate region (eg frameshift mutations and stop mutations) can be at least partially repaired by inducing exon skipping and are short proteins with (partial) function. Can be synthesized. It is also possible to induce partial skipping of exons using the methods of the invention. In this embodiment, contact of the reagent with the exon activates the concealed splice site of the exon in contact with the reagent. This aspect enhances the operability of pre-mRNA that induces functional proteins. The reaction system preferably comprises cells. Cells are in vitro (<u style="single">in</u><u style="single">in vitro</u>) Or in vivo (<u style="single">in</u><u style="single"></u><u style="single">vivo</u>) Is preferable, and examples of typical organisms include, but are not limited to, humans and mice. [0006] In a preferred embodiment, the present invention provides a cell with a reagent capable of specifically inhibiting the exon encapsulation signal of at least one of the exons against a cell having an mRNA precursor containing an exon encoding an aberrant protein. Provided are methods for at least partially reducing the production of aberrant proteins by cells, comprising feeding and allowing translation of the mRNA produced by splicing the pre-mRNA. [0007] Any reagent capable of specifically inhibiting the exon encapsulation signal can be used in the present invention. The reagent preferably contains a nucleic acid or a molecule having a function equivalent thereto, but the nucleic acid does not necessarily have to be single-strand. Peptide nucleic acids and other molecules having similar nucleic acid binding properties can also be used, but the binding properties do not necessarily have to be the same in the amount of binding. Nucleic acids or molecules with equivalent function may be modified to exert additional functionality. For example, 2'-O-methyloligoribonucleotides can be used. Such ribonucleotides are more resistant to RNase activity than conventional oligonucleotides. [0008] In a preferred embodiment of the invention, the exon-encapsulated signal is interfered with by an antisense nucleic acid specific for the exon recognition sequence (ERS). Such sequences are relatively purine-rich and can be identified by scrutinizing the sequence information of skipped exons (Tanaka et al., 1994,<u style="single">Mol</u>. <u style="single">Cell</u><u style="single">Biol</u>. 14: p. 1347-1354). Exon recognition sequences are thought to assist in the inclusion of so-called weak exons in mRNA (Achsel et al., 1996,<u style="single">J</u>. <u style="single">Biochem</u>. 120; p. 53-60). Such weak exons include, for example, 5'and / or 3'splice sites with low recognition efficiency by the splicing mechanism. In the present invention, it has been found that exon skipping can also be induced in so-called strong exons, that is, exons that are usually efficiently recognized by the splicing mechanism of cells. From any given sequence, it is possible (in most cases) to predict whether the sequence will contain a region presumed to be an exon and to determine if the exon is strong or weak. There are several methods for determining the strength of exons. A useful technique is the NetGene splice site prediction server (Brunak et al., 1991,<u style="single">J</u>. <u style="single">Mol</u>. <u style="single">Biol</u>. 220: p. You can search by 49-65). Exon skipping by the methods of the invention can be induced in (almost) all exons, depending on whether the exon is a weak or strong exon and whether the exon embraces ERS. There is no. In a preferred embodiment, the exon targeted for skipping is a strong exon. In one other preferred embodiment, the exons subject to skipping do not include ERS. [0009] The method of the present invention can be used for various purposes. In one embodiment, the methods of the invention are used to at least partially reduce the production of aberrant proteins. Examples of such proteins include tumor proteins or viral proteins. In many tumors, not only the presence of the tumor protein, but also its relative expression is related to the phenotype of the tumor cells. Similarly, not only the presence of viral protein, but also the amount of viral protein in the cell determines the virulence of a particular virus. In addition, whether the virus propagates efficiently or inefficiently, depending on the timing of expression in the life cycle of the virus and the quantitative balance of certain viral proteins in the cell, for the effective increase and spread of the virus. Is determined. By reducing the amount of aberrant protein in cells using the methods of the invention, for example, tumorigenicity (metastatic) of tumor cells and / or viral growth of virus-infected cells can be reduced. [0010] In a preferred embodiment, the method of the invention is used to convert an aberrant protein into a functional protein. In one embodiment, functional proteins such as those described above exert the function of proteins that are normally present in cells but not in the cells being treated. In most cases, even a partial repair of function will significantly improve the performance of the treated cells. Due to the improved performance, treated cells can show a selective advantage over untreated cells, increasing the effectiveness of the treatment. [0011] The aspects of the invention described above are particularly suitable for restoring the expression of a deleted gene. It specifically skips the target exon and removes harmful mutations (typically stop mutations or frameshift point mutations, or deletions or insertions of one or more exons that result in translational arrest). Achieved by avoiding or modifying. [0012] Compared with the gene transfer method, the novel splice-modulation gene of the present invention therapy) requires the administration of a smaller therapeutic agent, but not limited to, a therapeutic agent consisting of 14-40 nucleotides as a typical example. In a preferred embodiment, a molecule consisting of 14-25 nucleotides is used because it is easy to produce and more effectively taken up by cells. The method of the present invention makes it possible to design an effective and safe administration system associated with a therapeutic agent more flexibly. An important additional advantage of such an embodiment of the invention is that it restores (at least some) of the endogenous gene activity that still retains most or all of the gene regulatory circuits of tissue-specific isoforms. Ensuring appropriate expression levels and synthesis. [0013] The aspects of the invention described above can, in principle, be applied to any genetic disease or genetic predisposition, provided that the translation of the slightly shorter protein within the sequence is fully or partially functional. , Skipping that targets a specific exon can restore the translation reading frame that was originally destroyed by the mutation. Aspects in which therapeutic value is found in the application of this method include disease predisposition caused by secondary mutations in tumor suppressor genes, for example, in breast cancer, colon cancer, tuberous sclerosis, neurofibromatosis, etc. A (partial) restoration of activity eliminates the expression of variability due to secondary mutations and prevents tumorigenesis. Other preferred embodiments include (partial) repair of genes in which deletion of a gene product has a direct disease-inducing effect. Examples include hemophilia A (due to a deficiency of blood coagulation factor VIII), some forms of congenital thyroid dysfunction (due to tyroglobulin synthesis deficiency), and Duchenne muscular dystrophy (DMD). DMD is a disease in which deletions, duplications and arrest mutations of the X-linked dystrophin gene caused by frameshifts result in severe progressive weakness. DMD usually leads to death in late adolescence or early adulthood, while Becker muscular dystrophy, which develops with deletions or duchenne muscular mutations other than frameshifting at the same locus, is mild. Yes, life expectancy is the same as normal people from 35 to 40 years. In aspects of the invention applied to DMDs, existing deletion regions can be extended by exon skipping (or modifying the mRNA product of existing duplicate mutants) by the methods of the invention. This is done by restoring the reading frame and skipping the number of close exons required to produce a slightly shorter but functional protein inside the sequence. Based on the mild clinical symptoms of BMD patients with mutations equivalent to the induction of such deletions, the symptoms of DMD patients who received AON therapy may be alleviated. [0014] Various mutations in the dystrophin gene lead to dysfunctional proteins. (See http://www.dmd.nl, an internationally recognized database of DMDs and related diseases, for a union catalog). The exons that should actually be skipped to produce the functional dystrophin protein are altered by mutations. Table 1 is a list of exons that can be skipped, but the present application is not limited to these. In this table, the frequently occurring dystrophin gene mutations observed in humans for the above-mentioned exons that can be treated by the method of the present invention are also shown. The exon skipping shown in the table results in the production of altered dystrophin protein, which at least functions as a Becker-type muscle dystrophin protein. Thus, in one embodiment of the invention, a method characterized in that an exon inclusion signal is present at exon 2,8,43,44,45,46,50,51,52 or 53 of the human dystrophin gene. Provided. Occurrences of certain deletion / insertion mutations occur more frequently than others. Inducing skipping of exon 46 by the method of the present invention was successfully treated in about 7% of patients carrying a DMD deletion, revealing that dystrophin-positive muscle fibers were detected in dystrophin patients. We also succeeded in treating about 15% of patients carrying a DMD deletion by inducing skipping of exon 51 by the method of the present invention. This method of treatment resulted in the patient having at least a small amount of dystrophin-positive muscle fibers. Skipping exons 46 or 51 using the methods of the invention can treat approximately 22% of patients carrying a dystrophin gene deletion. Thus, in a preferred embodiment of the invention, the exon encapsulation signal is present at exon 46 or exon 51. In a particularly preferred embodiment, the reagents used include the following nucleic acid sequences: At least one nucleic acid sequence selected from the group consisting of hAON # 4, hAON # 6, hAON # 8, hAON # 9, hAON # 11 and / or hAON # 21-30; / Or an analog. The functional region, derivative and / or analog has the same exon skipping activity as hAON # described above, but in the method of the present invention, the amount of the activity does not necessarily have to be the same. [0015] Inducing exon skipping of one or more exons contained in pre-mRNA is effective. For example, given the diversity of mutations and certain properties such as exon length and amino acid sequences adjacent to the mutation, it is possible that one or more exons must be skipped to restore function. Preferred examples of such situations found in DMD deletion databases include, but are not limited to, deletions of exons 46-50. Patients lacking exons 46-50 do not produce functional dystrophin protein. However, by inducing skipping of exon 45 and exon 51, dystrophin proteins with at least partial function can be produced. Other preferred examples include, but are not limited to, patients with exon 2 duplication. By giving a reagent capable of inhibiting the EIS of exon 2, one or both of exon 2 is partially skipped, and next to the region consisting of a partial sequence of exon 2 or the region lacking two exon 2. It is possible to obtain wild-type protein from. Other preferred examples include, but are not limited to, skipping exons 45-50. This skipping creates a Becker-like variant within the reading frame. Becker-like variants can be induced to treat any mutation present in exons 45,46,47,48,49 and / or 50 or combinations thereof. In another aspect of the present invention, there is provided a method further comprising the step of giving the cell another reagent that inhibits the exon encapsulation signal of another exon contained in the pre-mRNA. The use of two or more reagents to induce exon skipping on two or more different genes of pre-mRNA is, of course, within the scope of the invention. [0016] In another aspect, the present invention provides a method for selecting a reagent suitable for splicing controlled gene therapy and a method for confirming the effectiveness of the selected reagent as a specific exon skipping reagent in a preliminary experiment. To do. A method for confirming that a reagent complementary to a part of the exon of interest can specifically inhibit the exon encapsulation signal of the exon, and is an mRNA precursor containing the exon of interest. A method is provided comprising feeding a cell having a body a test reagent, culturing the cell to generate an mRNA from the pre-mRNA, and confirming that the exon is absent in the produced mRNA. .. In a preferred embodiment, the reagent contains a nucleic acid or a molecule having equivalent function, and the nucleic acid is complementary to a portion of the exon of interest. Reagents capable of inducing specific exon skipping can be identified by the methods of the invention. The method also includes performing preliminary screening to see if the reagent binds to an exon-containing nucleic acid, preferably RNA, with a relatively high affinity. As a result, a method for confirming that the reagent can specifically inhibit the exon encapsulation signal possessed by the exon is provided, and the method performs the nucleic acid or its equivalent function under in vitro conditions. It further includes the step of measuring the relative binding affinity between the molecule having the molecule and the mRNA molecule containing the target exon. [0017] In another embodiment, the reagent obtained by the method of the present invention is provided. In a preferred embodiment, the reagent comprises a nucleic acid or a molecule having equivalent function. When the reagent is used to induce exon skipping in a cell, it is preferable to reduce the amount of abnormal protein in the cell at least partially, and the protein capable of exerting a function in the cell by the exon skipping. It is further preferred to yield RNA encoding. In a particularly preferred embodiment, the pre-mRNA is derived from the dystrophin gene. Functional proteins preferably include mutant dystrophin proteins or normal dystrophin proteins. Further, it is preferable that the mutant dystrophin protein has at least the function of the dystrophin protein in patients with Becker-type muscular dystrophy. In a particularly preferred embodiment, the reagents used include the following nucleic acid sequences: selected from the group consisting of hAON # 4, hAON # 6, hAON # 8, hAON # 9, hAON # 11 and / or hAON # 21-30. At least one nucleic acid sequence; or the functional region, derivative and / or analog of hAON # described above. The functional region, derivative and / or analog has the same exon skipping activity as hAON # described above, but in the method of the present invention, the amount of the activity does not necessarily have to be the same. [0018] Various methods for introducing reagents into cells are known in the art. In particular, nucleic acid introduction methods have been extensively developed. One of ordinary skill in the art can fully ascertain whether a method of introduction is suitable for carrying out the present invention. An example of such a method includes, but is not limited to, encapsulating the reagent of the present invention in a liposome and feeding the liposome to a cell having a target mRNA precursor, but the present invention is not limited to this example. Liposomes are particularly suitable as carriers for introducing nucleic acids into cells. An antisense molecule capable of inducing exon skipping can introduce a nucleic acid containing a transcription unit for producing antisense RNA and cause the cell to produce it. As a suitable transfer unit<u style="single">Small nuclear RNA</u>Alternatively, tRNA transcription units can be mentioned, but are not limited thereto. Therefore, the present invention further provides a nucleic acid carrier containing a nucleic acid of the present invention capable of inhibiting an exon encapsulation signal or a molecule having a function equivalent thereto. In one embodiment, the carrier is capable of expressing the nucleic acids of the invention. When a single-strand virus is used as a carrier, it goes without saying that even if such a virus includes only the antisense sequence of the reagent of the present invention, it is included in the scope of the present invention. Also, in another aspect of such a single-strand virus, the AON of the invention is encoded by a small nuclear RNA or tRNA transcription unit present in the viral nucleus encapsulated in the virus as a carrier. Has been done. The preferred single-strand virus is an adeno-associated virus. [0019] In a further aspect, the invention provides a nucleic acid or nucleic acid carrier for use in the preparation of a medicament. In a preferred embodiment, the drug is used in the treatment of a genetic disease. More preferably, the drug is used in the treatment of Duchenne muscular dystrophy. [0020] Example [0021] [0021] Example 1 Since exon 45 is the most frequently deleted exon in DMD, we first attempted to induce specific skipping of exon 46 (Fig. 1). This induction produces the shorter but more functional dystrophin protein found in BMD patients with deletions of exons 45 and 46. An experimental system was first constructed to control the splicing of the dystrophin pre-mRNA of the mouse dystrophin gene. Then, we aimed at the human dystrophin gene with the aim of restoring the translation reading frame and dystrophin synthesis in muscle cells derived from DMD patients carrying the exon 45 deletion. [0022]<u style="single">mAONs and hAONs design</u>A mouse-specific AON sequence and a human-specific AON sequence (mAON and hAON) for the internal region of exon 46 were designed (Fig. 2). The internal region of the exon 46 used has a purine base-rich sequence and is considered to have a putative function related to the splicing control of the exon 46. Unmodified DNA oligonucleotides (combined by EuroGentec, Belgium) were used for initial screening of AON by gel shift assay (see below). In the actual muscle cell transfection experiment, 2'-O-methylphosphorothioate antisense oligoribonucleotide (synthesized by EuroGentec, Belgium) was used. Such modified RNA oligonucleotides are known to have resistance to endonucleases and RNase H and to bind RNA with high affinity. Ultimately recognized as valid,<u style="single">in</u><u style="single">in vitro</u> The sequence of AON given to muscle cells in is shown below. The corresponding mouse-specific AON and human-specific AON are highly homologous but not exactly the same. [0023] The deoxy form of AON used is shown below, but in the final used 2'-O-methylribonucleotide, T is replaced with U. [0024]<img file="JP4846965B2_D0001.tif" /> 【0025】<img file="JP4846965B2_D0002.tif" /> 【0026】<u style="single">Gel shift assay</u>The effectiveness of AON was determined based on its binding affinity for the target sequence. Despite the recent developments in computer simulation programs for predicting RNA folding, it is difficult to infer which of the designed AONs binds to the target sequence with high affinity. Therefore, a gel shift assay was performed (according to the protocol described in Bruice et al., 1997). Exon 46 target RNA fragment from PCR fragment (amplified from mouse or human muscle mRNA using sense primer with T7 promoter sequence)³²In the presence of P-CTP<u style="single">in</u><u style="single">in vitro</u> It was produced by the T7 transcription method. The binding affinity of individual AONs (0.5 pmol) to the target transcription fragment was measured by hybridization at 37 ° C for 30 minutes followed by polyacrylamide (8%) gel electrophoresis. These analyzes were performed to screen for mouse-specific and human-specific AON (Fig. 3). Changes in mobility in at least 5 different mouse-specific AONs (mAON # 4, 6, 8, 9 and 11) and their corresponding 4 human-specific AONs (hAON # 4, 6, 8 and 9) Was observed, indicating that it has a binding affinity for the target RNA. [0027]<u style="single">Transfection of cultured muscle cells</u>By AON<u style="single">in</u><u style="single">in vitro</u> To analyze the skipping induction efficiency in muscle cells, exon 46-specific AON, which showed the highest binding affinity for the target exon, was selected using a gel shift assay. Non-specific AON was used as a negative control for specific skipping of exon 46 in all transfection experiments. As mentioned above, we first constructed an experimental system of mouse muscle cells. Both cultured proliferative myoblasts (high levels of dystrophin expression) derived from the mouse muscle cell line C2C12 cells and cultured post-mitotic myoblasts were used. Subsequent experiments with cultured human-derived muscle cells included muscle biopsy material from one unaffected muscle biopsy material and muscle biopsy material from two unrelated DMD patients carrying a deletion of Exon 45. Isolated cultured primary muscle cells were used. These heterologous cultures contained approximately 20-40% myogenic cells. 3 equivalent ratios of cationic polymer PEI (MBI) to different AON (concentration: 1 μM) Cells were transfected with AON using Fermentas). The AONs transfected in these experiments have a 5'fluorescein group, so transfection efficiency can be measured by counting the fluorescent nuclei. In general, over 60% of cells showed nuclear-specific uptake of AON. RNA was isolated using RNAzol B (CamPro Scientific, The Netherlands) 24 hours after transfection to facilitate RT-PCR analysis. [0028]<u style="single">RT-PCR and sequencing</u>Reverse transcription of RNA<u style="single">C</u>. <u style="single">therm</u>.. This was done using polymerase (Roche) and exon 48-specific reverse transcription primers. Two cycles of PCR were performed to amplify the cDNA to detect exon 46 skipping of the dystrophin gene. PCR is nested by using primers contained in exons 44 and 47 (human experimental system) or primers contained in exons 45 and 47 (mouse experimental system). Amplification) is included. In cultured mouse myoblasts and cultured mouse myotube cells, partial products of a size corresponding to the product in which exon 45 was directly spliced to exon 47 were detected (Fig. 4). Subsequent sequencing confirmed the specific skipping of exon 46 in the mouse dystrophin transcript. The efficiency of exon skipping was different for each AON, with mAON # 4 and # 11 showing the highest efficiency. Based on these favorable results, we conducted an experiment focusing on controlling the splicing of dystrophin in cultured human muscle cells as well. As a result, a partial product corresponding to the product exon 45 spliced to exon 47 was detected in the control muscle cells. Interestingly, short fragments of exon 44 spliced into exon 47 were detected in patient-derived muscle cells. Specific skipping of exon 46 was confirmed by sequence data of human dystrophin transcripts. Such regulation of splicing found in both mouse and human dystrophin transcripts was not found in untransfected or non-specific AON-transfected cultured cells. [0029]<u style="single">Immunohistochemical analysis</u>To restore translation and synthesis of the dystrophin protein, we attempted to induce exon 46 skipping in muscle cells from patients carrying a deletion of exon 45. Two dystrophin monoclonal antibodies (Mandys-1 and Dys) created against each of the adjacent and distal domains of the target region of the dystrophin protein to detect dystrophin products when transfected with hAON # 8. Using -2), two patient-derived cultured muscle cells were subjected to immunocytochemical analysis. Fluorescence analysis revealed that dystrophin synthesis was restored in all patient-derived cultured cells (Fig. 5). In the treated sample, about 80% or more of the muscle fibers were positively stained for dystrophin. [0030] The experimental results of the present inventors show for the first time the restoration of dystrophin synthesis by the endogenous DMD gene of muscle cells derived from DMD patients. This demonstrates the principle for performing purpose-appropriate splicing control of dystrophin pre-mRNA for therapeutic purposes. [0031]<u style="single">Exon 51 Target Skipping</u><u style="single">Simultaneous skipping of dystrophin exons</u>Exon 51 target skipping. The inventors of the present invention<u style="single">in</u><u style="single">in vitro</u> The possibility of AON regulation of dystrophin exon 46 in mouse and human muscle cells was clarified. These findings provided the basis for further studies evaluating AON as a therapeutic reagent for DMD. The majority of DMD-inducing deletions occur intensively at two sites in the gene that are prone to mutations, and target skipping of one particular exon screens a series of patients with various mutations. It can be restored (see Table 1). Exons 51 are interesting target exons. This exon skipping can be used to treat patients with deletions in regions ranging from exon 50, exon 45-50, exon 48-50, exon 49-50, exon 52, or exon 52-63. , The total number of such patients reaches 15% of the total number of patients registered in our Leiden database. [0032] Ten human-specific AON species (hAON # 21-30, see below) were designed for various purine-rich regions present in exon 51 of dystrophin. Such purine base-rich regions suggested the existence of factors presumed to be responsible for controlling the exon splicing reaction, namely regions that were attempted to be shielded to induce exon removal during the splicing reaction. All experiments were performed according to the protocol used for skipping exon 46 (see above). A gel shift assay was performed to confirm hAON, which has a high binding affinity for the target RNA. Five types of hAON showing the highest affinity were selected. Exon 51 skipping potential<u style="single">in</u><u style="single">in vitro</u> These hAONs were transfected into control human muscle cells for analysis in. RNA was isolated 24 hours after transfection and cDNA was generated using exon 53 or 65 specific reverse transcription primers. PCR amplification of the target region was performed by combining various primers adjacent to exon 51. Human dystrophin transcripts revealed that RT-PCR and sequencing induced specific skipping of exon 51. Subsequently, two hAONs (# 23 and # 29) that have been shown to induce exon skipping were transfected into six cultured muscle cells from DMD patients with one of the above mutations. did. Exon 51 skipping in these cultured cells was confirmed by RT-PCR and sequencing (Fig. 7). More importantly, immunohistochemical analysis using multiple antibodies made against different parts of the dystrophin protein showed that all analysis results restored dystrophin protein synthesis by exon 51 skipping. It was. [0033]<img file="JP4846965B2_D0003.tif" />[0034] Simultaneous skipping of multiple dystrophin exons. Skipping one exon, such as exon 46 or exon 51, in addition to the deletion mutation can restore the reading frame for a wide variety of DMD mutations. The range of mutations to which this method is applicable can be expanded by skipping one or more exons at the same time. For example, in DMD patients with a deletion of exon 46 to exon 50, it was possible to reconstruct the translation reading frame simply by skipping both exons 45 and 51 adjacent to the deletion region. [0035] ERS independent exon skipping. Exon 29 mutations in two Becker muscular dystrophy patients resulted in exon 29 skipping (Ginjaar et al., 2000, EJHG, vol. 8: p. 793-796). We investigated the possibility of inducing skipping of exon 29 by mutation site targeting using AON. The mutation is located in a purine base-rich region that may be associated with ERS activity. We designed AON sequences for the inside (h29AON # 1 ~ h29AON # 6) and outside (h29AON # 7 ~ h29AON # 11) of the region considered to be ERS (see below). A gel shift assay was performed (as described above) to identify AONs with high affinity for the target RNA (Figure 8). Subsequently, h29AON # 1, # 2, # 4, # 6, # 9, # 10 and # 11 were transfected into control cultured human myotubes using a PEI transfection reagent. RNA was isolated 24 hours after transfection and reverse transcription of the cDNA was performed using exon 31-specific reverse transcription primers. PCR amplification of the target region was performed by combining various primers adjacent to exon 29. This RT-PCR followed by sequencing (FIGS. 8B and C) revealed that exon 29 skipping was induced in the human dystrophin transcript. However, these AONs that promote exon 29 skipping were designed based on both the inner and outer sequences of the ERS to which the AONs are thought to bind (h29AON # 1, # 2, # 4, # 6, # 9 and # 11). These results suggest that skipping exon 29 does not depend on whether exon 29 contains or does not contain ERS, so binding of AON to exon 29 inactivates the exon encapsulation signal more than ERS. The ERS-independent exon skipping demonstrated herein has the potential to extend the entire scope of application of the therapeutic methods of the invention to exons that do not contain ERS. [0036]<img file="JP4846965B2_D0004.tif" /> 【0037】<img file="JP4846965B2_D0005.tif" />[0038] In mouse muscle tissue<u style="single">in</u><u style="single"></u><u style="single">vivo</u> AON Inducible Exon 46 Skipping. Experiments with cultured muscle cells gave promising results, so next<u style="single">in</u><u style="single">vivo</u> We tested various mouse dystrophin exon 46 specific AONs. The test was performed by intramuscular injection of mouse dystrophin exon 46-specific AON bound to polyethyleneimine (PEI) into the gastrocnemius muscle of a control mouse.<u style="single">in</u><u style="single">in vitro</u> MAON # 4, # 6 and # 11, whose effectiveness in mouse muscle cells has already been clarified, can be determined by RT-PCR and sequencing.<u style="single">in</u><u style="single">vivo</u> It was found to induce skipping of exon 46 in the muscle tissue of (Fig. 9).<u style="single">in</u><u style="single">vivo</u> Exon 46 skipping was dose-dependent and showed the highest efficiency (up to 10%) with a 2-day injection of 20 μg / muscle / day. [0039] References Achsel et al., 1996, <u style="single">J</u>. <u style="single">Biochem</u>. 120: p. 53-60. Bruice TW and Lima, WF (1997) <u style="single">Biochemistry</u> 36 (16): p. 5004-5019. Brunak et al., 1991, <u style="single">J</u>. <u style="single">Mol</u>. <u style="single">Biol</u>. 220: p. 49-65. Dunckley, MG et al, (1998) <u style="single">Human</u><u style="single">molecular</u><u style="single">genetics</u> 7: p. 1083-1090. Ginjaar et al., 2000, <u style="single">EJHG</u>, vol. 8, p. 793-796. Mann et al., 2001, <u style="single">PNAS</u> vol. 98, p. 42-47. Tanaka et al., 1994, <u style="single">Mol</u>. <u style="single">Cell</u>. <u style="single">Biol</u>. 14: p. 1347-1354. Wilton SD et al., (1999) <u style="single">Neuromuscular</u><u style="single">disorders</u> 9: p. 330-338. [0040] Details and background on Duchenne muscular dystrophy and related diseases can be found on the website http://www.dmd.nl. [0041] [table 1]<img file="JP4846965B2_D0006.tif" />[Simple explanation of drawings] FIG. 1. Exon 45 deletion is one of the most frequent DMD mutations. This deletion splics exon 44 to exon 46, cleaves the translation reading frame and creates a stop codon inside exon 46, resulting in a dystrophin deficiency. An object of the present inventors is a milder disease carrying a deletion of exons 45 and 46 by artificially inducing skipping of further exons, that is, exons 46, to reconstruct the reading frame. It restores the synthesis of the slightly shorter but highly functional dystrophin protein found in patients with Becker muscular dystrophy. [Figure 2] Exon 46 has a region rich in purine bases that is thought to potentially be responsible for pre-mRNA splicing control. A primer sequence consisting of overlapping 2'-O-methylphosphorothioate antisense oligoribonucleotides (AON) was designed for the purine base-rich region existing inside the mouse dystrophin exon 46. These AONs differ in both length and sequence. Chemical modification of AON confers resistance to endonucleases and RNase H in muscle cells.<u style="single">in</u><u style="single">in vitro</u> To investigate transfection efficiency in this study, AON contained a 5'fluorescein group that allowed the identification of AON-positive cells. FIG. 3 A gel shift assay was performed to examine the binding affinities of various AONs for the target exon 46 RNA. The figure shows 5 mAONs (mAON # 4,6,8,9 and 11) that have the highest affinity for the target RNA. When AON binds to RNA, a complex with reduced gel mobility is formed and can be detected by band changes. The binding of AON to the target is sequence-specific. Random mAONs, ie mAONs non-specific to exon 46, did not cause band changes. [Fig. 4] Mouse-specific AON and human-specific AON, which showed the highest binding affinity in the gel shift assay, were transfected into mouse cultured myotubes and human cultured myotubes, respectively. Figure 4A: RT-PCR analysis of cultured mouse cells transfected with mAON # 4,6,9 and 11 showed a partial product of a size corresponding to the product in which exon 45 was directly spliced to exon 47. Exon 46 skipping was not detected when random AON was transfected. Figure 4B: RT- of cultured human muscle cells from each of one unaffected (C in the figure) and two unrelated DMD patients (P1 and P2 in the figure). PCR analysis revealed that partial products were produced when transfecting hAON # 4 and hAON # 8. In control, this product corresponds to the exon 45 spliced to exon 47, while the fragment size seen in the patient corresponds to the exon 44 spliced to exon 47. Exon 46 skipping was not detected in untransfected and random hAON-transfected cells. The highest exon 46 skipping efficiency was obtained with hAON # 8. FIG. 5 is sequence data of RT-PCR products obtained from patient DL279.1 (corresponding to P1 in Figure 4), which lacks patient exon 45 (upper panel). ), It was confirmed that the transfection of hAON # 8 further caused skipping of Exon 46 (lower panel). The skipping of exon 46 is specific, exon 44 is accurately spliced to exon 47 and reconstructs the translation reading frame. FIG. 6: Immunohistochemical analysis of cultured muscle cells derived from patient DL279.1 transfected with hAON # 8. The cells were contacted with the two dystrophin antibodies prepared for different regions of the protein. Antibodies used were antibodies against regions close to the target exon 46 (ManDys-1, ex. -31-32) and antibodies away from exon 46 (Dys-2, ex. -77-79). is there. The lower panel shows the absence of dystrophin protein in the myotube, whereas the hAON # 8 inducible skipping of exon 46 clearly results in the synthesis of dystrophin protein, as dystrophin protein is detected in both antibodies. Restored (upper panel). FIG. 7A: RT-PCR analysis of RNA isolated from control cultured human muscle cells treated with hAON # 23, # 24, # 27, # 28 or # 29. Subproducts of a size corresponding to the exon 50 spliced to exon 52 were detected in cells treated with hAON # 23 and # 28. When the sequences of these products were determined, it was confirmed that exon 51 was skipped accurately (Fig. 7B). Further abnormal splicing products were obtained from cells treated with hAON # 28 and # 29. Sequence determination revealed the presence of concealed splice sites within the frame of exon 51, which is used infrequently with AON treatment. The products produced by the AON treatment included a partial product of exon 51 consisting of a restored reading frame, confirming further therapeutic value. FIG. 8A: Gel shift assay was performed to confirm the binding affinity of various hAON # 29s for exon 29 target RNA. Compared to non-hybridized RNA (none in the figure), h29AON # 1, # 2, # 4, # 6, # 9, # 10 and # 11 form a complex with low gel mobility. , Bound to RNA. Random AON from dystrophin exon 19 did not form a complex. Figure 8B: RT-PCR analysis of RNA isolated from cultured human muscle cells treated with h29AON # 1, # 2, # 4, # 6, # 9, # 10 or # 11 shows that exon 28 is exon. We revealed the existence of a partial product of a size corresponding to the product spliced to 30. These results are AON (h29AON # 1, # 2, # 4 or # 6) for the inner sequence of the region considered to be the ERS in exon 29 or AON (h29AON # 9, # 10 or # 6) for the outer sequence. We show that it is possible to specifically skip exon 29 using # 11). Further anomalous splicing products resulting from both exon 28 and 29 skipping were detected (confirmed by sequence data but not shown here). This aberrant splicing product was also present in untreated cells, presumably due to the natural selective skipping promoted by AON treatment. AON19 obtained from dystrophin exon 19 did not induce skipping of exon 29. Figure 8C: Specific skipping of exon 29 was confirmed by sequence data of RT-PCR fragments, which are partial sequences. The nucleotide sequence of the exon 29 skipping product in cells treated with h29AON # 1 is shown here. FIG. 9A: RT-PCR analysis of mouse gastrocnemius-derived RNA, RNA isolated on day 2 after administration of 5, 10 or 20 μg of mAON # 4, # 6 or # 11, respectively. .. A partial product of a size corresponding to the product exon 45 spliced to exon 47 was detected in all treated muscles. "-RT", "-RNA", "AD-1" and "AD-2" were analyzed as negative controls for the RT-PCR reaction. Figure 9B: Sequences of the partial products produced by mAON # 4 and # 6 (# 11 was also tested, but no data are shown) confirmed the exact skipping of exon 46.