Antisense molecules and methods for treating medical conditions

Abstract

We provide antisense oligonucleotides that can bind to a target nucleotide sequence and modulate splicing. [Solution] The present invention provides antisense molecular compounds and compositions suitable for binding to RNA motifs involved in premRNA splicing, which are capable of specifically and efficiently inducing exon skipping, and methods for using the same. According to a first aspect, the present invention provides an antisense molecule capable of binding to a selected target and inducing exon skipping. According to a second aspect, the present invention provides an antisense molecule selected to be useful for or suitable for a prophylactic or therapeutic treatment of a genetic disorder, which comprises at least one antisense molecule in a form suitable for delivery to a patient.

Description

Technical Field

[0001] The present invention relates to novel antisense compounds and compositions suitable for promoting exon skipping. Also provided are methods for inducing exon skipping using the novel antisense compounds, and therapeutic compositions suitable for use in the methods of the present invention.

Background Art

[0002] The following discussion of background art is intended only to facilitate an understanding of the present invention. The following discussion does not constitute an admission or concession that any of the materials referred to were part of the common general knowledge at the priority date of this application.

[0003] Currently, a great deal of effort is being spent on research into methods for suppressing or compensating for disease-causing mutations in genes. Antisense technologies have been developed that use various chemical actions that affect gene expression at various different levels (transcription, splicing, stability, translation). Many of these studies focus on the use of antisense compounds to correct or compensate for abnormal genes or genes associated with diseases in countless different conditions.

[0004] Since antisense molecules can inhibit gene expression highly specifically, much research effort regarding oligonucleotides as regulators of gene expression has focused on inhibiting the expression of target genes such as oncogenes or viral genes. Antisense oligonucleotides target either RNA (sense strand) or DNA, form a triple-stranded structure with them, and inhibit transcription by RNA polymerase II.

[0005] In order to achieve a desirable effect in the downregulation of a specific gene, the oligonucleotide must either promote the degradation of the target mRNA or block the translation of the mRNA to effectively prevent the de novo synthesis of an undesirable target protein.

[0006] Such techniques are not useful when the goal is to upregulate the production of native proteins, or to compensate for mutations that induce premature translation termination, such as nonsense mutations or frameshift mutations.

[0007] Furthermore, when a normally functioning protein terminates prematurely due to mutations present in the protein, it has been shown that several means of repairing functional protein production using antisense technology are possible through interference during the splicing process (Non-Patent Documents 1-3). In these cases, since incomplete gene transcripts should not be targeted for degradation, the chemical properties of the antisense oligonucleotide should not promote the breakdown of the target mRNA.

[0008] In various genetic disorders, the impact of mutations on the final expression of a gene can be regulated through a process of skipping target exons during splicing. The splicing process is driven by a complex multiparticle mechanism that brings adjacent exon-intron junctions in premRNA closer together, cleaving phosphodiester bonds at intron ends and subsequently rearranging the exons to be spliced ​​together. This complex and highly precise process is mediated by sequence motifs in premRNA, which are relatively short, semi-conservative RNA segments that bind to various nuclear splicing factors later involved in the splicing reaction. By altering how the splicing mechanism reads or recognizes the motifs involved in premRNA, it is possible to create various spliced ​​mRNA molecules. Currently, it is known that most human genes undergo alternative splicing during normal gene expression, but the mechanisms causing this have not been identified. Antisense oligonucleotides have been shown to bypass errors and deletions in encoded mRNA or remove them from mature gene transcripts.

[0009] In nature, the extent of gene deletion during the splicing process, i.e., exon skipping, is not fully understood, but many examples have been reported indicating that it generally occurs at very low levels (Non-Patent Literature 4). However, it has been observed that when exons associated with disease-causing mutations are specifically deleted from certain genes, short protein products may be produced that have biological properties similar to the native protein or possess sufficient biological activity to alleviate diseases caused by mutations associated with the target exon (Non-Patent Literature 5 and 6).

[0010] This targeted exon skipping process may be particularly useful in long genes where there are many exons and introns, where there is duplication in the exon genetic makeup, or where the protein can function without one or more specific exons (e.g., the dystrophin gene consisting of 79 exons; perhaps several collagen genes encoding repeating sequence blocks, or the giant nebulin or titin genes consisting of more than 80 and 370 exons, respectively).

[0011] Attempts to redirect gene processing to treat gene disorders associated with tip breakage caused by mutations in various genes have focused on the use of antisense oligonucleotides that (1) completely or partially overlap with elements involved in the splicing process; or (2) bind to premRNA at a position close enough to the element to disrupt the binding and function of splicing factors that normally mediate the specific splicing reaction occurring at that element (e.g., binding to premRNA within 3, 6, or 9 nucleotides of the element to be blocked).

[0012] For example, the regulation of premRNA splicing of mutant dystrophin by antisense oligonucleotides has been reported both in vitro and in vivo. In one dystrophin mutation reported in Japan, a 52-base pair deletion mutation leads to the removal of exon 19, along with an adjacent intron, during the splicing process (Non-Patent Literature 7). Using a minigene splicing system in vitro, it has been shown that a 31-base 2'-O-methyl oligonucleotide complementary to the 5' half of the deletion sequence in exon 19 of dystrophin Kobe inhibits splicing of wild-type premRNA (Non-Patent Literature 8). Using the same nucleotide, exon skipping was induced from native dystrophin gene transcripts in human lymphoblastoid-like cultured cells.

[0013] Non-patent document 9 describes an in vitro construct for analyzing splicing around exon 23 of mutant dystrophin in the mdx mouse mutant, a model of muscular dystrophy. We considered a plan to analyze these constructs in vitro using 2'-modified oligonucleotides that target splicing within or adjacent to exon 23 of mouse dystrophin, but no target site or sequence was obtained.

[0014] Subsequently, this group reported that 2'-O-methyl oligoribonucleotide corrects dystrophin deficiency in myoblasts derived from mdx mice. Antisense oligonucleotides targeting the 3' splice site of intron 22 of mouse dystrophin were reported to induce skipping of the mutant exon and several adjacent exons, leading to the creation of a novel in-frame dystrophin transcript with a new internal deletion. This mutant dystrophin was expressed in 1%–2% of antisense-treated mdx myotubes. The use of other oligonucleotide modifications, such as 2'-O-methoxyethyl phosphodiester, has also been described (Non-Patent Literature 10).

[0015] Therefore, antisense molecules can provide a tool in the treatment of genetic disorders such as Duchenne muscular dystrophy (DMD). However, attempts to induce exon skipping using antisense molecules have not always been successful.

[0016] The study on exon 19 of dystrophin, which successfully skipped exon 19 from dystrophin premRNA, was achieved using various antisense molecules targeting adjacent splice sites or exon motifs involved in the definition of the exon, as described in Non-Patent Literature 11.

[0017] In contrast to the apparent ease of exon 19 skipping, the initial report of exon 23 skipping in MDX mice in Non-Patent Document 10 is now considered to have merely reported a spontaneously occurring reverse mutation transcript or artifact, rather than true antisense activity. Furthermore, transcripts lacking exon 23 are not consistently produced, and Non-Patent Document 10 did not show any temporal changes in induced exon skipping, nor even dose settings for antisense oligonucleotides to show a dose-dependent effect where the level of exon skipping corresponds to an increase or decrease in the amount of antisense oligonucleotide. Moreover, these results could not be reproduced by other researchers.

[0018] The first example of specific and reproducible exon skipping in the mdx mouse model is reported in Non-Patent Literature 2. By directing an antisense molecule to the donor splice site, consistent and efficient exon 23 skipping was induced in dystrophin mRNA within 6 hours after treatment of cultured cells. Non-Patent Literature 2 also describes targeting the acceptor region of mouse dystrophin premRNA with longer antisense oligonucleotides and states that it could not reproduce the results published in Non-Patent Literature 10. Using antisense oligonucleotide selection targeting the acceptor splice site of intron 22, it was not possible to reproducibly detect exon skipping that removed only exon 23 or multiple adjacent exons.

[0019] A first antisense oligonucleotide targeting the donor splice site of intron 23 consistently induced exon skipping in primary cultured myoblasts; however, this compound was found to be less efficient in immortalized cell cultures expressing high levels of dystrophin. Nevertheless, precise targeting and antisense oligonucleotide design significantly increased the efficiency of removing specific exons (see Non-Patent Literature 12).

[0020] Therefore, there is a need to provide antisense oligonucleotides that can bind to target nucleotide sequences and modulate splicing. Simply guiding an antisense oligonucleotide to a motif presumed to be important for splicing does not guarantee the efficacy of the compound in a therapeutic setting.

[0021] The foregoing discussion relating to the background of the present invention is intended solely to facilitate understanding of the invention. It should be understood that this discussion does not acknowledge or accept that any of the materials referenced are, or were, part of the general knowledge as of the priority date of this application. [Prior art documents]

Non-licensed literature

[0022] [Non-licensed document 1] Sierakowska H,et al.,(1996)Proc Natl Acad Sci USA 93,12840-12844 [Non-licensed document 2] Wilton SD, et al., (1999) Neuromusc Disorders 9, 330-338 [Non-licensed document 3] van Deutekom JC et al.,(2001)Human Mol Genet 10,1547-1554

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0023] The present invention provides an antisense molecular compound and composition suitable for binding to an RNA motif involved in pre - mRNA splicing, which can specifically and efficiently induce exon skipping, as well as methods of using the same.

[0024] Target selection plays an important role in the efficiency of exon skipping and, by extension, in the application to subsequent potential therapies. Simply designing an antisense molecule against a target region of pre - mRNA presumed to be involved in splicing does not guarantee the induction of efficient and specific exon skipping. The most obvious or easily defined targets for splicing interference are donor and acceptor splice sites, but there are also motifs such as exon splicing enhancer elements, silencing elements, and branch points that are not as clearly defined or conserved. Acceptor and donor splice sites have consensus sequences of approximately 16 bases and approximately 8 bases, respectively (see Figure 1 for an overview of the motifs and domains involved in exon recognition, intron removal, and splicing processes). [[ID=二十]][[Means for Solving the Problems]]

[0025] According to a first aspect, the present invention provides an antisense molecule that can bind to a selected target and induce exon skipping.

[0026] For example, to induce exon skipping of exons 5, 12, 17, 21, 22, 24, 43-47, 49, 50, 54-64, 66, 67, 70, and 72 in the dystrophin gene transcript, it is preferable to select an antisense molecule from the group listed in Table 1A.

[0027] In further examples, two or more antisense oligonucleotides of the present invention can be combined to more efficiently induce exon skipping in exons 3, 4, 8, 10, 26, 36, 48, 60, 66, and 68. The combination, or "cocktail," of antisense oligonucleotides is guided by the exons to efficiently induce exon skipping.

[0028] According to a second aspect, the present invention provides an antisense molecule selected to be useful for or suitable for a preventive or therapeutic treatment of a genetic disorder, comprising at least one antisense molecule in a form suitable for delivery to a patient.

[0029] According to a third aspect, the present invention provides a method for treating a patient suffering from a genetic disorder in which a mutation exists in a gene encoding a specific protein, and the effect of the mutation can be reversed by exon skipping, the method comprising: (a) selecting an antisense molecule according to a method described herein; and (b) administering the molecule to a patient in need of such treatment.

[0030] Furthermore, the present invention provides the use of purified and isolated antisense oligonucleotides for the manufacture of pharmaceuticals for treating genetic disorders.

[0031] Furthermore, the present invention provides a method for treating symptoms characteristic of Duchenne muscular dystrophy, comprising administering to a patient in need of treatment an amount of the antisense oligonucleotide of the present invention that is effective for a therapeutically designed treatment related to a specific genetic lesion in the patient. Furthermore, the present invention provides a method for preventing or at least minimizing Duchenne muscular dystrophy by prophylactically treating a patient, comprising administering to the patient a pharmaceutical composition comprising an effective amount of antisense oligonucleotide or one or more of these biological molecules.

[0032] Furthermore, the present invention provides a kit for treating a genetic disorder, comprising at least the antisense oligonucleotide of the present invention packaged in a suitable container, and instructions for its use.

[0033] Other aspects and advantages of the present invention will become apparent to those skilled in the art by examining the following description. The description will proceed with reference to the following figures. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1 is a schematic diagram of the motifs and domains involved in the exon recognition, intron removal, and splicing processes. [Figure 2] Figure 2 is a conceptual diagram of antisense oligonucleotides that induce exon skipping to bypass disease-causing mutations (scale is not accurate). Shaded boxes represent exons with mutations that prevent the rest of the mRNA from being translated into protein. Black bars represent antisense oligonucleotides that prevent the mature mRNA from containing that exon. [Figure 3] Figure 3 is a gel electrophoresis image showing a "cocktail" of antisense molecules targeting exon 3 that strongly and consistently induce exon skipping at a transfection concentration of 10 nM in normal human muscle culture cells. [Figure 4]Figure 4 is a gel electrophoresis image showing a "cocktail" of antisense molecules targeting exon 4 that strongly and consistently induce exon skipping in normal human muscle culture cells at a transfection concentration of 25 nM. [Figure 5] Figure 5 shows a gel electrophoresis image demonstrating potent and efficient human exon 5 skipping using the antisense molecule [H5A(+35+65)] targeting the internal domain of exon 5, which is presumed to be an exon splicing enhancer element. This preferred compound consistently induces exon skipping in human muscle culture cells at a transfection concentration of 25 nM. [Figure 6] Figure 6 is a gel electrophoresis image showing a "cocktail" of antisense molecules targeting exon 8 that strongly and consistently induces exon skipping in both exon 8 and exon 8 / 9 at a transfection concentration of 10 nM in normal human muscle culture cells. [Figure 7] Figure 7 shows gel electrophoresis images of various cocktails and single antisense molecules that induce skipping of exon 10 and surrounding exons. The combination of [H10A(-05+16)] and [H10A(+98+119)] or the combination of [H10A(-05+16)] and [H10A(+130+149)] induces skipping of exon 10 and exons 9-12, while [H10A(-05+16)] alone induces skipping of exons 9-14. [Figure 8] Figure 8 shows a gel electrophoresis image of exon 14 using the antisense molecule H14A(+31+61) targeting exon 14. [Figure 9] Figure 9 is a gel electrophoresis image showing exon 17 skipping using the antisense molecule H17A(+10+35) targeting exon 17. [Figure 10]Figure 10 shows gel electrophoresis images of two cocktails of antisense molecules targeting exon 26. The double cocktail of [H26A(-07+19)] and [H26A(+24+50)] effectively induces exon 26 skipping, and the skipping efficiency is not affected by the addition of further antisense molecules to the cocktail. [Figure 11] Figure 11 is a gel electrophoresis image showing a "cocktail" of antisense molecules targeting exon 36 that strongly and consistently induce exon skipping in normal human muscle culture cells at a transfection concentration of 25 nM. [Figure 12] Figure 12 shows gel electrophoresis images of normal human muscle culture cells showing strong and consistent exon 43 skipping at a transfection concentration of 25 nM using the antisense molecule H43A(+92+117). [Figure 13] Figure 13 shows a gel electrophoresis image illustrating dose-dependent exon 55 skipping using the antisense molecule H44A(+65+90). [Figure 14] Figure 14 is a gel electrophoresis image showing strong and consistent exon 45 skipping using the antisense molecule H45A(-09+25). [Figure 15] Figure 15 is a gel electrophoresis image showing strong and consistent exon 46 skipping using the antisense molecule H46A(+81+109). [Figure 16] Figure 16 is a gel electrophoresis image showing strong and consistent exon 47 skipping using the antisense molecule H47A(+01+29). [Figure 17] Figure 17 is a gel electrophoresis image showing a "cocktail" of antisense molecules targeting exon 47 that strongly and consistently induce exon skipping. [Figure 18] Figure 18 is a gel electrophoresis image showing strong and consistent exon 49 skipping using the antisense molecule H49A(+45+70). [Figure 19]Figure 19 is a gel electrophoresis image showing strong and consistent exon 50 skipping using the antisense molecule H50A(+48+74). [Figure 20] Figure 20 is a gel electrophoresis image showing strong and consistent exon 51 skipping using the antisense molecule H51A(+66+95). [Figure 21] Figure 21 is a gel electrophoresis image showing strong and consistent exon 54 skipping using the antisense molecule H54A(+67+97). [Figure 22] Figure 22 is a gel electrophoresis image showing the antisense molecule H55A(-10+20) which induces exon 55 skipping in a dose-dependent manner. [Figure 23] Figure 23 is a gel electrophoresis image showing strong and consistent exon 56 skipping using the antisense molecule H56A(+92+121). [Figure 24] Figure 24 is a gel electrophoresis image showing the antisense molecule H57A(-10+20) which induces dose-dependent skipping of exon 57. [Figure 25] Figure 25 shows gel electrophoresis images of exon 59 and exon 58 / 59 using the antisense molecule H59A(+96+120) targeting exon 59. [Figure 26] Figure 26 shows gel electrophoresis images illustrating two different cocktails that induce exon skipping of exon 60. [Figure 27] Figure 27 is a gel electrophoresis image showing exon 63 skipping using the antisense molecule H63A(+20+49). [Figure 28] Figure 28 is a gel electrophoresis image showing exon 64 skipping using the antisense molecule H64A(+34+62). [Figure 29] Figure 29 is a gel electrophoresis image showing a "cocktail" of antisense molecules targeting exon 66 that induces dose-dependent exon skipping. [Figure 30]Figure 30 is a gel electrophoresis image showing exon 67 skipping using the antisense molecule H67A(+17+47). [Figure 31] Figure 31 is a gel electrophoresis image showing a "cocktail" of antisense molecules targeting exon 68 that induces dose-dependent exon skipping. [Figure 32] Figure 32 is a gel electrophoresis image showing a "cocktail" of antisense molecules that strongly and consistently induce exon skipping of exons 69 / 70 at a transfection concentration of 25 nM. [Figure 33] Figure 33 is a gel electrophoresis image showing various "cocktails" of antisense molecules that induce skipping of exon 50 at different levels. [Figure 34] Figure 34 is a gel electrophoresis image showing a cocktail of three antisense molecules that induce efficient skipping of exons 50 / 51. [Figure 35] Figure 35 is a graph of concentration measurement results showing exon skipping of various efficiencies. The antisense molecules tested were exon 3 [H3A (+30+60) and H3A (+61+85)]; exon 4 [H4D (+14-11) and H4A (+11+40)]; exon 14 [H14A (+32+61)]; exon 17 [H17A (+10+35)]; exon 26 [H26A (-07+19), H26A (+24+50) and H26A (+68+92)]; and exon 36 [H36A (-16+09) and H36A (+22+51)]. [Figure 36] Figure 36 is a graph of concentration measurement results showing exon skipping of various efficiencies. The antisense molecules tested were exon 46[H46A(+81+109)]; exon 47[H47A(+01+29)]; exon 48[H48A(+01+28) and H48A(+40+67)]; and exon 49[H49A(+45+70)]. [Figure 37] Figure 37 is a gel electrophoresis image showing exon 11 skipping using the antisense molecule H11A(+50+79). [Figure 38]Figure 38 is a gel electrophoresis image showing exon 12 skipping using the antisense molecule H12A(+30+57). [Figure 39] Figure 39 is a gel electrophoresis image showing exon 44 skipping using the antisense molecule H44A(+59+85). [Figure 40] Figure 40 is a gel electrophoresis image showing exon 45 skipping using the antisense molecule H45A(-03+25). [Figure 41] Figure 41 is a gel electrophoresis image showing exon 51 skipping using the antisense molecule H51A(+71+100). [Figure 42] Figure 42 is a gel electrophoresis image showing exon 52 skipping using the antisense molecule H52A(+09+38). [Figure 43] Figure 43 is a gel electrophoresis image showing exon 53 skipping using the antisense molecule H53A(+33+65). [Figure 44] Figure 44 is a gel electrophoresis image showing exon 46 skipping using the antisense molecule H46A(+93+122). [Figure 45] Figure 45 is a gel electrophoresis image showing exon 73 skipping using the antisense molecule H73A(+02+26). [Figure 46A] Figure 46A shows the arrangement of antisense molecules. [Figure 46B] Figure 46B shows the arrangement of antisense molecules. [Modes for carrying out the invention]

[0035] A brief explanation of sequence listings [Table 1A] [Table 1B]

[0036] General Overview Those skilled in the art will understand that variations and modifications other than those specifically described are possible in the inventions described herein. It should be understood that the present invention includes all such variations and modifications. Furthermore, the present invention includes all of the steps, features, compositions, and compounds mentioned or indicated individually or collectively in the specification, as well as any and all combinations or any two or more of the aforementioned steps or features.

[0037] The present invention is not limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention as described herein.

[0038] The sequence identification numbers (SEQ ID NOs), including the nucleotide and amino acid sequence information contained herein, are listed together at the end of this document. These were created using the PatentIn program version 3.0. Each nucleotide or amino acid sequence is given a numerical heading. <210> and subsequent sequence specifiers (for example, <210> 1. <210> Identified in the sequence listing by (2) the length, type, and origin of each nucleotide or amino acid sequence, respectively, are given a numerical heading. <211> , <212> , and <213> The information provided is indicated. The sequences of nucleotides and amino acids mentioned in the specification are indicated by numerical headings. <400> and subsequent sequence specifiers (for example, <400> 1. <400> Defined by the information provided by (2nd class).

[0039] To identify various antisense molecules, a naming system for antisense molecules has been proposed and published (Mann et al., (2002)). J Gen Med (See 4,644–654). This nomenclature is particularly relevant when testing several slightly different antisense molecules that all target the same target region, as shown below: H#A / D(x:y) The first letter indicates the species (for example, H: human, M: mouse, C: dog). The "#" represents the target dystrophin exon number. "A / D" indicates the acceptor splice site or donor splice site at the beginning and end of the exon, respectively. (xy) represents the annealing coordinate where "-" or "+" indicates an intron sequence or an exon sequence, respectively. For example, A(-6+18) represents the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. The nearest splice site is the acceptor, so "A" is written before these coordinates. The annealing coordinate at a donor splice site can be written as D(+2-18), representing the last 2 exon bases and the first 18 intron bases corresponding to the annealing site of the antisense molecule. The annealing coordinate represented by A(+65+85), which is an exon as a whole, is the site between the 65th and 85th nucleotides from the beginning of that exon.

[0040] All disclosures of publications cited herein (including patents, patent applications, articles, laboratory manuals, books, or other documents) are incorporated herein by reference. No one is authorized to constitute prior art or to be part of the general knowledge of research in the field to which this invention relates.

[0041] As used herein, the terms “origin” and “derived from” indicate that a particular object (integer) can be obtained from a particular origin, even if it is not necessarily directly obtained from such origin.

[0042] Throughout this specification, unless context requires otherwise, the term “comprise” or its variations (such as “comprises” or “comprising”) shall be understood to mean including the specified subject or group of subjects, but not excluding any other subject or group of subjects.

[0043] Other definitions of the selected terms used herein can be found in the detailed description of the invention and apply throughout the specification. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains.

[0044] Description of Preferred Embodiments When an antisense molecule targets a nucleotide sequence involved in splicing at an exon within a premRNA sequence, normal exon splicing can be inhibited, causing the splicing mechanism to bypass the mature mRNA and the entire mature exon. Figure 2 illustrates the concept of an antisense oligonucleotide that induces exon skipping.

[0045] In many genes, deletion of an entire exon results in the loss of a key functional domain or the collapse of the leading frame, producing a non-functional protein. However, in some proteins, it is possible to shorten the protein by deleting one or more exons from within the protein without the collapse of the leading frame and without significantly altering the protein's biological activity. Generally, such proteins have a structural role, a functional domain at the terminal, or both a structural role and a functional domain at the terminal. This invention describes an antisense molecule that can bind to a specific dystrophin premRNA and redirect the processing of that gene.

[0046] A preferred objective of antisense molecule-based therapies is to achieve maximum exon skipping with the lowest possible concentration of antisense molecules. Generally, antisense molecules may cause strong and robust exon skipping; weak and scattered exon skipping; or no exon skipping at all. It is preferable to develop antisense molecules (single or in combination) that can produce strong, robust, and consistent exon skipping at low therapeutic doses.

[0047] Antisense molecule According to a first aspect of the present invention, an antisense molecule capable of binding to a selected target and inducing exon skipping is provided. To induce exon skipping in the exons of the dystrophin gene transcript, it is preferable to select an antisense molecule from the group of compounds shown in Table 1A.

[0048] Furthermore, a combination of two or more antisense oligonucleotides capable of binding to a selected target and inducing exon skipping, i.e., a "cocktail," is provided. In order to induce exon skipping in the exons of the dystrophin gene transcript, it is preferable to select an antisense molecule from the group of compounds shown in Table 1B that is included in the "cocktail."

[0049] Even if an antisense molecule is designed to completely cover the consensus splice site, target exon skipping does not necessarily occur. Furthermore, the inventors have found that the size, i.e., length, of the antisense oligonucleotide itself is not always the primary factor in designing an antisense molecule. For some targets, such as exon 19, a short 12-nucleotide antisense oligonucleotide was able to induce exon skipping, while longer (20-31 nucleotides) oligonucleotides were less efficient. For several other targets, such as mouse dystrophin exon 23, an antisense oligonucleotide with only 17 residues in length was able to induce skipping more efficiently than another overlapping compound of 25 nucleotides. However, the inventors have found that, generally, longer antisense molecules often induce exon skipping more efficiently than shorter molecules. Therefore, the antisense molecule of the inventors is preferably 24-30 nucleic acids, preferably about 28 nucleotides in length. For example, a 20-base antisense oligonucleotide (H16A(-07+13)) is ineffective in inducing exon 16 exon skipping, but a 31-base oligonucleotide (H16A(-06+25)) that completely contains the aforementioned 20-base oligonucleotide has already been found to be effective in inducing skipping (Harding et al (2007) Mol Ther 15:157-166).

[0050] Furthermore, the inventors found that there are no arbitrary standard motifs that can be blocked or masked by antisense molecules to redirect splicing. For several exons, such as exon 23 of mouse dystrophin, the donor splice site was the most likely target for redirecting exon skipping. It should be noted that the design and testing of a series of exon 23-specific antisense molecules annealing to the overlapping region of the donor splice site showed considerable variability in the efficiency of exon skipping induction. As reported by Mann et al. (2002), the bypass efficiency of nonsense mutations varied greatly depending on the annealing of the antisense molecule ("Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy"). J Gen Med (4:644-654). No consistent exon 23 skipping induction targeting exon 23 or acceptor sites in any internal domain has been found.

[0051] In other exons targeted for removal, masking of the donor splice site did not induce exon skipping at all. However, directing an antisense molecule to the acceptor splice site (human exon 8, discussed below) induced potent and persistent exon skipping. It should be noted that the removal of human exon 8 is closely related to the simultaneous removal of exon 9. Since the sequence homology between the antisense oligonucleotide of exon 8 and the corresponding region of exon 9 is low, this is not considered a cross-reactivity issue. Rather, the splicing of these two exons is generally related. This is not the only example; similar effects have been observed in canine cells, where targeting to remove exon 8 also results in the skipping of exon 9. Furthermore, targeting to remove exon 23 in mouse dystrophin premRNA often also removes exon 22. This effect is dose-dependent and indicates that the two adjacent exons are closely cooperative in their processing.

[0052] In other targeted exons, antisense molecules targeting donor or acceptor splice sites did not induce exon skipping, or only slightly did. However, annealing of antisense molecules to the exon region (i.e., the exon splicing enhancer element in human dystrophin exon 4) was the most efficient in inducing exon skipping. Several exons, such as mouse and human exon 19, are readily skipped by antisense molecules targeting various motifs. Specifically, targeted exon skipping is induced after masking the donor and acceptor splice sites or the exon splicing enhancer element using antisense oligonucleotides.

[0053] Furthermore, it is impossible to predict which antisense molecule cocktails will induce exon skipping. For example, combining two antisense molecules that are very effective individually in inducing exon skipping may not necessarily cause exon skipping when combined as a cocktail. For instance, H50A(+02+30) and H50A(+66+95) individually induce good skipping of exons 50 and 51, respectively. However, when combined as a cocktail, they only induce a slight skipping of the same two exons. Similarly, the combination of H50A(+02+30) and H51A(+66+90), or the combination of H50A(+02+30) and H51A(+61+90), did not efficiently induce exon skipping of exons 50 and 51, despite the effectiveness of the individual antisense molecules. However, by introducing a third antisense molecule ([H51D(+16-07)]) which does not cause skipping on its own, and creating a cocktail of the three elements ([H50A(+02+30)], H51A(+66+90), and [H51D(+16-07)]), we were able to skip exons 50 and 51 down to 1 nM.

[0054] Alternatively, excellent skipping can occur when two or three antisense molecules that are ineffective or have very little effect individually are combined. For example, H26A(-07+19)[SEQ ID NO: 39], H26A(+24+50)[SEQ ID NO: 40], and H26A(+68+92)[SEQ ID NO: 41] individually produce inefficient skipping of exon 26 and induce multiple exon skipping (26-29 or 27-30). However, when the three exons are combined as a cocktail, exon 26 skipping occurs very efficiently.

[0055] From the above examples and discussion, it is clear that there is no way to accurately predict whether a given combination will work.

[0056] Antisense molecules can induce exon skipping in a dose-dependent or dose-independent manner. Dose-dependent means that a larger amount of antisense molecule skips the exon more effectively, while dose-independent antisense molecules can induce skipping even at very low doses. For example, Figure 15 shows that H46A(+81+109)[SEQ ID NO: 12] skips exon 46 equally well regardless of the amount of antisense molecule present (600 nM to 25 nM). In contrast, H57A(-10+20)[SEQ ID NO: 20](Figure 24) strongly induces exon 57 skipping at 100 nM, but the degree of skipping decreases at 50 nM and decreases even more significantly at 25 nM.

[0057] It is preferable to select antisense molecules that induce skipping in a dose-independent manner, because therapeutic effects can be obtained even when these molecules are administered at very low concentrations. However, it is also acceptable to select antisense molecules that induce skipping in a dose-dependent manner, particularly molecules that induce good or excellent skipping at low concentrations, as preferred molecules. The antisense molecules of the present invention are preferably able to induce good or excellent exon skipping at low concentrations of less than 500 nM, preferably less than 200 nM, more preferably 100 nM, 50 nM, or even as low as 25 nM. The oligonucleotide molecules of the present invention are most preferably able to induce skipping at a level of more than 30% at a concentration of 100 nM.

[0058] To identify and select suitable antisense oligonucleotides for use in regulating exon skipping, the nucleic acid sequence whose function should be regulated must first be identified. This may be, for example, a gene (or mRNA transcribed from such gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule derived from an infectious agent. In the context of the present invention, preferred target sites are those involved in mRNA splicing (i.e., splice donor sites, splice acceptor sites, or exon splicing enhancer elements). Splice branch points and exon recognition sequences or splice enhancer elements can also be target sites for regulating mRNA splicing.

[0059] Preferably, the present invention aims to provide an antisense molecule capable of binding to a selected target in dystrophin premRNA to induce efficient and consistent exon skipping. Duchenne muscular dystrophy results from mutations that disrupt the synthesis of the functional gene product of dystrophin. These defects in the Duchenne muscular dystrophy gene are generally nonsense mutations or genomic rearrangements, such as deletions, duplications, microdeletions, or insertions that disrupt the reading frame. Because the human dystrophin gene is a large and complex gene (79 exons are spliced ​​together to produce a mature mRNA containing an open reading frame of approximately 11,000 bases), there are numerous locations where these mutations can occur. Consequently, comprehensive antisense oligonucleotide-based therapies that address many of the mutations in the dystrophin gene that cause various diseases require that many exons be targets for removal during the splicing process.

[0060] In the context of the present invention, preferred target sites are those involved in mRNA splicing (i.e., splice donor sites, splice acceptor sites, or exon splicing enhancer elements). Splice branch points and exon recognition sequences or splice enhancer elements can also be target sites for regulating mRNA splicing.

[0061] Oligonucleotides and DNA or RNA are complementary if a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen-bond with each other. Therefore, the terms “specifically hybridizable” and “complementary” are used to indicate that the oligonucleotide and the DNA or RNA target are sufficiently complementary or precisely paired to enable stable and specific binding. It is understood in the art that the sequence of an antisense molecule does not need to be 100% complementary to the specifically hybridizable target sequence. An antisense molecule is specifically hybridizable if there is sufficient complementarity to prevent nonspecific binding of the antisense compound to non-target sequences under conditions where specific binding is desirable—that is, under physiological conditions in the case of an in vivo assay or therapeutic treatment, or under the conditions under which the assay is performed in the case of an in vitro assay.

[0062] Using the above method, an antisense molecule can be selected from which any exon can be deleted from a protein that can be shortened without affecting its biological function, but the deletion of the exon must not result in a reading frameshift in the shortened transcription mRNA. Therefore, in a linear sequence of three exons, if the first exon is at the end of which two of the three nucleotides in the codon are encoded, and the next exon is deleted, the third exon in the linear sequence must begin with a single nucleotide that can complete the nucleotide triplet of the codon. If the third exon does not begin with the single nucleotide, a reading frameshift occurs that leads to the production of a truncated or non-functional protein.

[0063] It is understood that codon rearrangement at the end of an exon in structural proteins does not always occur at the end of the codon. As a result, it may be necessary to delete more than one exon from the premRNA to ensure in-frame reading of mRNA. In such situations, multiple antisense oligonucleotides would need to be selected by the present invention, each targeting different regions involved in inducing splicing at the exon to be deleted.

[0064] The length of the antisense molecule may vary as long as it can selectively bind to the intended site within the premRNA molecule. The length of such a sequence can be determined by the selection procedure described herein. Generally, antisense molecules are about 10 to 50 nucleotides in length. However, it is understood that nucleotides of any length within this range may be used in the method described above. The length of the antisense molecule is preferably 17 to 30 nucleotides. Surprisingly, it has been found that longer antisense molecules are often more efficient in inducing exon skipping. Therefore, the length of the antisense molecule is most preferably 24 to 30 nucleotides.

[0065] To determine which exons within the dystrophin gene are connectable, an exon boundary map should be consulted. The connection between one exon and another is based on the number of exons present at the 3' boundary being equal to the number present at the 5' boundary of the exon being connected. Therefore, if exon 7 is deleted, exon 6 must connect with exon 12 or 18 to maintain the reading frame. Thus, an antisense oligonucleotide must be selected to redirect splicing for exons 7-11 in the former case, and for exons 7-17 in the latter case. Another somewhat simpler approach to restoring the reading frame before and after exon 7 deletion is to remove two adjacent exons. Inducing skipping of exons 6 and 8 requires splicing exons 5 through 9 to produce an in-frame transcript. However, in practice, targeting exon 8 for removal from premRNA also removes exon 9, resulting in a transcript with exons 5 and 10 joined. The reading frame remains unchanged whether or not exon 9 is present.

[0066] Once the antisense molecule to be tested is identified, it is prepared according to standard techniques in the art. The most common method for generating an antisense molecule is methylation of the 2' position of hydroxyribose and introduction of a phosphorothioate skeleton. This produces a molecule that is superficially similar to RNA but far more resistant to nuclease degradation.

[0067] To prevent premRNA degradation while forming a double helix with the antisense molecule, the antisense molecule used in the method may be adapted to minimize or avoid cleavage by endogenous RNaseH. This property is highly desirable because the presence of unmethylated RNA oligonucleotides in the intracellular environment or in contact with crude extracts containing RNaseH leads to degradation of the premRNA:antisense oligonucleotide double helix. Any modified antisense molecule that can bypass or does not induce such degradation can be used in this method. This nuclease resistance can be achieved by modifying the antisense molecule of the present invention to include a partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups, including carboxylic acid groups, ester groups, and alcohol groups.

[0068] An example of an antisense molecule that is not cleaved by intracellular RNaseH when forming a double helix with RNA is a 2'-O-methyl derivative. 2'-O-methyl-oligoribonucleotides are highly stable in the intracellular environment and animal tissues, and their double helix with RNA has a higher Tm value than their ribo or deoxyribo counterparts. Alternatively, the nuclease-resistant antisense molecule of the present invention may have at least one of its last 3' terminal nucleotides that is fluorinated. Alternatively, the nuclease-resistant antisense molecule of the present invention may have a phosphorothioate bond linking at least two of its last 3' terminal nucleotide bases, preferably a phosphorothioate bond linking the last four 3' terminal nucleotide bases.

[0069] Antisense molecules that do not activate RNaseH can be prepared according to known techniques (see, for example, U.S. Patent No. 5,149,797). Such antisense molecules may be deoxyribonucleotide sequences or ribonucleotide sequences, and simply include as one member of their sequence any structural modification that sterically hindrances or prevents the binding of RNaseH to a double-strand molecule containing oligonucleotides, wherein the structural modification does not substantially hinder or disrupt the formation of the double helix. Since the oligonucleotide portion involved in the formation of the double helix is ​​substantially different from the portion involved in the binding of RNaseH to the oligonucleotide, many antisense molecules that do not activate RNaseH are available. For example, such an antisense molecule may be an oligonucleotide in which at least one or all of the phosphate residues cross-linking the nucleotides are modified phosphates such as methylphosphonate, methylphosphorothioate, phosphoromolholide, phosphoropiperadide, and forholoamidate. For example, the phosphate residues cross-linking the nucleotides may be modified as described above, every other residue. In another non-limiting example, such an antisense molecule contains at least one or all of its nucleotides as a 2' lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, e.g., methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, the nucleotides may be modified alternately as described above.

[0070] Antisense oligonucleotides are a preferred form of antisense molecule, but the present invention includes other oligomeric antisense molecules, including but not limited to oligonucleotide pseudosubstances such as those described below.

[0071] Specific examples of preferred antisense compounds useful in the present invention include oligonucleotides containing a modified skeleton or internucleoside links that do not exist in nature. As defined herein, oligonucleotides having a modified skeleton include those that retain a phosphorus atom in the skeleton and those that do not. For the purposes of this specification, and as sometimes referred to in the art, modified nucleotides that do not have a phosphorus atom in the internucleoside skeleton can also be considered oligonucleosides.

[0072] In other preferred oligonucleotide pseudomaterials, the sugar and nucleoside linkages, i.e., the backbone, of the nucleotide unit are substituted with novel groups. The basic unit maintains hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an oligonucleotide pseudomaterial that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is substituted with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobase is retained and bonded directly or indirectly to the aza nitrogen atom of the amide portion of the backbone.

[0073] Furthermore, the modified oligonucleotide may contain one or more substituted sugar moieties. The oligonucleotide may also contain nucleobase (often simply referred to as "base" in the art) modification or substitution. Certain nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the double-strand stability of nucleic acids by 0.6°C to 1.2°C and is a preferred base substitution in the present invention, and is particularly preferred when combined with 2'-O-methoxyethyl sugar modification.

[0074] Another modification of the oligonucleotide of the present invention involves chemically linking one or more moieties or conjugates to the oligonucleotide to enhance its activity, intracellular distribution, or intracellular uptake. Such moieties include, but are not limited to, cholesterol moieties, cholic acid, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chains, or lipid moieties such as adamantane acetate, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0075] It is not necessarily required that all positions in a given compound be uniformly modified; in fact, more than one of the aforementioned modifications may be incorporated into a single compound, or even into a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds that are chimeric compounds. A "chimeric" antisense compound, i.e., a "chimera," in the context of the present invention, is an antisense molecule, particularly an oligonucleotide, that contains two or more chemically distinct regions, each of which is composed of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides generally contain at least one region in which the oligonucleotide is modified to increase resistance to nuclease degradation and increase uptake into cells, and further regions to increase binding affinity to a target nucleic acid.

[0076] Method for manufacturing antisense molecules Antisense molecules used in accordance with the present invention can be easily and routinely produced through well-known solid-phase synthesis techniques. Equipment for such synthesis is available from several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.

[0077] In addition to or instead of the above, any other means known in the art for such synthesis may be used. Similar techniques are well known to be used to prepare oligonucleotides such as phosphorothioates and alkylated derivatives. In one such automated embodiment, diethyl phosphoramidite is used as a starting material and can be synthesized as described in Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

[0078] The antisense molecules of the present invention are synthesized in vitro and do not involve antisense compositions of biological origin or gene vector constructs designed to lead to the in vivo synthesis of antisense molecules. Furthermore, to aid in uptake, distribution, and / or absorption, the molecules of the present invention may be mixed, encapsulated, conjugated, or bound together with other molecules, molecular structures, or mixtures of compounds, for example, in liposomes, receptor-targeted molecules, or in oral, rectal, topical, or other formulations.

[0079] Therapeutic drugs Furthermore, the present invention may be used as a preventive or therapeutic agent for the purpose of treating genetic disorders.

[0080] Accordingly, in one embodiment, the present invention provides an antisense molecule that binds to a selected target in dystrophin premRNA in a therapeutically effective amount, mixed with a pharmaceutically acceptable carrier, diluent, or excipient, to induce the efficient and consistent exon skipping described herein.

[0081] The term "pharmaceutically acceptable" refers to molecular entities and compositions that, when administered to a patient, are physiologically tolerable and generally do not cause allergic reactions or similar adverse reactions such as acute gastric motility. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with the compound. Such pharmaceutical carriers may be oils containing petroleum, animal, plant, or synthetic oils such as peanut oil, soybean oil, mineral oil, and sesame oil, and inert liquids such as water. Water or physiological saline, as well as aqueous solutions of dextrose and glycerol, are preferred as carriers, particularly as injectable solutions. Preferred pharmaceutical carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA, (1990).

[0082] In a more specific embodiment of the present invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of an antisense molecule together with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such a composition includes additives such as various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength diluents, detergents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and bulk substances (e.g., lactose, mannitol). These substances may be incorporated into particulate preparations of polymer compounds such as polyacetic acid and polyglycolic acid, or into liposomes. Hyaluronic acid may also be used. Such a composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the protein and its derivatives. For example, see Martin, Remington's Pharmaceutical Sciences, 18th, incorporated herein by reference. See Ed. (1990, Mack Publishing Co., Easton, PA 18042), pages 1435-1712. The composition may be prepared in liquid form or in dry powder form such as freeze-dried form.

[0083] The pharmaceutical compositions provided in accordance with the present invention are recognized as being administerable by any application known in the art. The pharmaceutical compositions for administration are preferably administered by infusion, oral, pulmonary, or nasal route. The antisense molecules are more preferably delivered by intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous administration route.

[0084] Antisense molecule-based therapy Furthermore, the use of the antisense molecule of the present invention for manufacturing pharmaceuticals that regulate genetic disorders is also addressed by the present invention.

[0085] The delivery of therapeutically useful amounts of antisense molecules can be carried out by methods already disclosed. For example, intracellular delivery of antisense molecules can be carried out via a composition comprising a mixture of the antisense molecule and an effective amount of a block copolymer. An example of this method is described in U.S. Patent Application Publication No. 2004 / 0248833.

[0086] Other methods for delivering antisense molecules to the nucleus include Mann CJ et al., (2001) ["Antisense-induced exon skipping and the synthesis of dystrophin in the mdx mouse”.Proc., Natl.Acad.Science,98(1)42-47] and Gebski et al.,(2003).Human Molecular This is described in Genetics, 12(15):1801-1811.

[0087] A method for introducing nucleic acid molecules into cells using an expression vector, either as naked DNA or DNA complexed with a liquid carrier, is described in U.S. Patent No. 6,806,084.

[0088] In some cases, it is desirable to deliver antisense molecules in a colloidal dispersion system. Examples of colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and liquid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposomal formulations.

[0089] Liposomes are artificial membrane vesicles useful as delivery vesicles in vitro and in vivo. These formulations may have net cationic, anionic, or neutral charge properties, which are useful for delivery methods in vitro, in vivo, and ex vivo. Large monolamellar vesicles (LUVs) with a size of 0.2 PHI.m to 4.0 PHI.m can encapsulate a significant proportion of aqueous buffer containing macromolecules. RNA and DNA may be encapsulated within the aqueous buffer and delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981).

[0090] For liposomes to be effective gene transport vehicles, they must have the following characteristics: (1) highly efficient encapsulation of target antisense molecules without reducing the biological activity of the antisense molecules; (2) preferential and substantial binding to target cells compared to non-target cells; (3) highly efficient delivery of the aqueous contents of the vesicle to the cytoplasm of target cells; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).

[0091] Liposome compositions are typically combinations of phospholipids, particularly combinations of phospholipids with high phase transition temperatures, and are usually used in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0092] Alternatively, the antisense construct may be combined with other pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic salines, such as phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, or transdermal administration.

[0093] The listed routes of administration are intended for guidance only, as those skilled in the art can easily determine the optimal route of administration and any dosage for any specific animal and condition.

[0094] The antisense molecules of the present invention include any pharmaceutically acceptable salts, esters, salts of such esters, or any other compounds that can provide (directly or indirectly) biologically active metabolites or residues thereof when administered to animals, including humans. Accordingly, for example, prodrugs and pharmaceutically acceptable salts of the compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents are also included in the disclosure.

[0095] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of the compound of the present invention; that is, a salt that retains the desirable biological activity of the compound and does not impart any undesirable toxic effects to the compound.

[0096] In the case of oligonucleotides, preferred examples of pharmaceutically acceptable salts include, but are not limited to, (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, and polyamines (e.g., spermine and spermidine); (b) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid; (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. The pharmaceutical compositions of the present invention can be administered in a number of ways depending on whether topical or systemic treatment is preferred and the area to be treated. Administration may be local (including administration to mucous membranes, such as ocular and rectal delivery), pulmonary administration by inhalation or blowing of powder or aerosol (e.g., intratracheal, intranasal, epidermal, and transdermal via nebulizer), oral, or parenteral administration. Parenteral administration may include injection or infusion into veins, arteries, subcutaneous tissue, abdominal cavity, or muscle; or intracranial administration, such as intrathecal or intracardiac administration. Oligonucleotides having at least one 2'-O-methoxyethyl modification are considered particularly useful for oral administration.

[0097] The pharmaceutical formulations of the present invention may be presented in convenient unit dosage forms and can be prepared according to commonly used techniques well known in the pharmaceutical industry. Such techniques include a step of associating the active ingredient with a pharmaceutical carrier or excipient. Generally, the formulations are prepared by homogeneously and tightly associating the active ingredient with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, shaping the product.

[0098] The present invention kit Furthermore, the present invention provides a kit for treating patients with genetic disorders, comprising at least one antisense molecule packaged in a suitable container and instructions for its use.

[0099] In a preferred embodiment, the kit comprises at least one antisense molecule shown in Table 1A, or a cocktail of antisense molecules shown in Table 1B. The kit may also contain non-essential reagents such as buffers and stabilizers.

[0100] The contents of the kit may be lyophilized, and the kit may further contain a suitable solvent for reconstituting the lyophilized components. The individual components of the kit may be packaged in separate containers, and such containers may be accompanied by a notice in the form prescribed by the government agency that regulates the manufacture, use, or sale of pharmaceutical or biological products, which reflects the authorization of the agency that regulates the manufacture, use, or sale of such products for human administration.

[0101] When the components of the kit are provided in one or more liquid solutions, the liquid solutions may be aqueous solutions, such as sterile aqueous solutions. When used in vivo, the expression construct may be formulated into a pharmaceutically acceptable injectable composition. In this case, the container means may be an inhaler, syringe, pipette, eye dropper, or other similar device, from which the formulation can be applied to the affected area of ​​an animal, such as the lungs, injected into the animal, or applied to and mixed with other components of the kit.

[0102] Furthermore, the components of the kit may be provided in a dry or lyophilized form. When reagents or components are provided in a dry form, they are generally reconstituted by the addition of a suitable solvent. It is also conceivable that the solvent may be provided in a separate container. Regardless of the number or type of containers, the kit of the present invention may include, or be packaged with, equipment for assisting in the injection / administration or placement of the final complex composition into the body of an animal. Such equipment may be an inhaler, syringe, pipette, forceps, measuring spoon, eyedropper, or any medically approved delivery vehicle.

[0103] Those skilled in the art should recognize that the application of the above method has a wide range of applications for identifying antisense molecules suitable for use in the treatment of many other diseases. [Examples]

[0104] The following examples provide further detail on the procedures for using the present invention and describe the best possible embodiments for carrying out various aspects of the invention. These examples are presented for illustrative purposes only and should not be seen as limiting the true scope of the invention in any way. References cited herein are expressly incorporated herein by reference.

[0105] Methods for cloning molecules, immunological methods, and protein chemistry methods not explicitly described in the following examples are reported in the literature and are known to those skilled in the art. General textbooks describing conventional molecular biology, microbiology, and recombinant DNA techniques in the art include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II, MRL Press, Ltd., Oxford, UK (1985); and Ausubel, F., Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, Struhl, K. Current Protocols in Molecular Biology. Greene Publishing Associates / Wiley Intersciences, New York (2002).

[0106] Determination of exon skipping induced in human muscle cells Since no consistent trend applicable to all exons is considered to exist, our attempts to develop a rational approach to the design of antisense molecules were not entirely successful. Therefore, the identification of the most effective and thus most therapeutically useful antisense molecular compounds was the result of empirical research.

[0107] These empirical studies involved using computer programs to identify motifs potentially involved in the splicing process. Other computer programs were used to identify pre-mRNA regions that may lack extensive secondary structures and therefore could serve as annealing sites for antisense molecules. None of these approaches proved entirely reliable for designing antisense oligonucleotides for reliable and efficient exon skipping induction.

[0108] First, annealing sites in human dystrophin premRNA were selected for testing based on known or predicted motifs or regions involved in splicing. 2OMe antisense oligonucleotides were designed to be complementary to the target sequences under investigation and synthesized using an Expedite 8909 nucleic acid synthesizer. After synthesis, the oligonucleotides were cleaved from the support column, deprotected with ammonium hydroxide, and then desalted. The quality of oligonucleotide synthesis was monitored by the intensity of the trityl signal at each deprotection step during synthesis, when detected in a synthesis log. The concentration of the antisense oligonucleotide was estimated by measuring the absorbance of diluted aliquots at 260 nm.

[0109] Next, specific amounts of antisense molecules were tested for their ability to induce exon skipping in in vitro assays as described below.

[0110] In short, primary cultures of normal myoblasts were prepared from human muscle biopsies obtained after informed consent. The cells were grown and differentiated into myotubes using standard culture techniques. The cells were then transfected with antisense oligonucleotides by delivering the oligonucleotides as a cationic lipoplex, a mixture of antisense molecules, or a cationic liposome preparation.

[0111] Next, after the cells were grown for another 24 hours, total RNA was extracted and molecular analysis was initiated. Reverse transcriptase amplification (RT-PCR) was performed to test for the induction of rearrangement of the target region or exons of dystrophin premRNA.

[0112] For example, in the testing of antisense molecules to induce exon 19 skipping, several exons were scanned by RT-PCR to detect the presence of any adjacent exons. For instance, when inducing exon 19 skipping, RT-PCR was performed using primers that amplified across exons 17 to 21. Furthermore, larger products in this region (i.e., exons 13-26) were amplified to ensure minimal amplification bias for shorter induced and skipped transcripts. Shorter products or exon-skipped products tend to amplify more efficiently, potentially biasing estimates of normal and induced transcripts.

[0113] The size of the amplified reaction products was estimated on an agarose gel and compared to an appropriate size standard. Final confirmation of the identity of these products was performed by directly sequencing the DNA to confirm that accurate or predicted exon junctions were maintained.

[0114] Once exon skipping is efficiently induced with one antisense molecule, the next duplicate antisense molecule can be synthesized and then evaluated in the assay as described above. An efficient antisense molecule is defined as one that induces potent and sustained exon skipping at transfection concentrations of approximately 300 nM or less. The oligonucleotide molecules of the present invention are most preferably capable of inducing skipping at a level of more than 30% at a concentration of 100 nM.

[0115] Concentration measurement method To determine which antisense molecule achieved the desired efficiency, concentration-measuring analysis was performed on the results of the exon skipping procedure. The amplified products were fractionated on a 2% agarose gel, stained with ethidium bromide, and imaged using a Chemi-Smart 3000 gel documentation system (Vilber Lourmat, Marne La Vallee). The bands were then analyzed using a gel documentation system (Bio-Profil, Bio-1D version 11.9, Vilber Lourmat, Marne La Vallee) according to the manufacturer's instructions.

[0116] The following antisense molecules were subjected to concentration measurements.

number

[0117] Antisense oligonucleotides targeting exon 17 Antisense oligonucleotides targeting exon 17 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above.

[0118] Table 2 below shows that the effects of antisense molecules targeting the same site (the acceptor splice site of exon 17) can be very different, even though the binding sites of the two antisense molecules overlap. H17A(-07+23)[SEQ ID NO: 3], which anneals to the last 7 bases of intron 16 and the first 23 bases of exon 17, induces exon 17 skipping when delivered to cells at a concentration of 25 nM. In contrast, the antisense molecule H17A(-12+18), which anneals to the last 12 bases of the intron and the first 18 bases of exon 17, and therefore overlaps with the binding site of H17A(-07+23), was unable to induce exon skipping at all. Furthermore, H17A(-07+16), which anneals to the last 7 bases of intron 16 and the first 16 bases of exon 17, skipped both exon 17 and exon 18 at 200 nM. The antisense molecule H17A(+61+86)[SEQ ID NO: 4], which binds to the intra-exon splicing enhancer element motif of exon 17, was also able to successfully induce exon skipping. This indicates that the ability of an antisense molecule to induce exon skipping cannot be simply predicted from the binding site and must be determined through rigorous testing. [Table 2]

[0119] This data indicates that while some specific antisense molecules efficiently induce exon skipping, other antisense molecules targeting nearby or overlapping regions may exhibit significantly lower induction efficiency. Titeral studies show that some molecules can induce targeted exon skipping at concentrations of 20 nM–25 nM, while less efficient antisense molecules could only induce exon skipping at concentrations above 300 nM. Therefore, targeting antisense molecules to motifs involved in the splicing process is shown to play a crucial role in the overall efficacy of the compound.

[0120] Effectiveness refers to the ability to induce consistent skipping of the target exon. However, sometimes the skipping of the target exon is consistently related to adjacent exons; that is, the splicing of several exons has been found to be closely linked. For example, when targeting exon 23 in a mouse model of muscular dystrophy using an antisense molecule targeting the exon donor site, dystrophins lacking exons 22 and 23 are often detected. As another example, when using an antisense molecule targeting exon 8 of the human dystrophin gene, many induced transcripts lack both exons 8 and 9.

[0121] Antisense oligonucleotide targeting exon 2 Antisense oligonucleotides targeting exon 2 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 3]

[0122] Antisense oligonucleotide targeting exon 3 Antisense oligonucleotides targeting exon 3 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above.

[0123] The antisense molecules H3A(+30+60)[SEQ ID NO: 31] and H3A(+61+85)[SEQ ID NO: 32] induce exon 3 skipping when used individually, but when combined, the two molecules are even more effective in inducing skipping (Figure 3), and can also induce exon 4 and 5 skipping at 300 nM and 600 nM. This result cannot be determined, or inferred, from the results obtained when each antisense molecule is used individually. In the above further products, amplification by the outer primers carried over from RT-PCR produces a transcript lacking exon 3, forming a heteroduplex. [Table 4]

[0124] Antisense oligonucleotides targeting exon 4 Antisense oligonucleotides targeting exon 4 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. Figure 4 shows exon 4 skipping using a cocktail of H4A(+11+40)[SEQ ID NO: 33] and H4D(+14-11)[SEQ ID NO: 34]. [Table 5]

[0125] Antisense oligonucleotide targeting exon 5 Antisense oligonucleotides targeting exon 5 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. H5D(+26-05) was not able to induce even low levels of exon 5 skipping and is therefore considered an undesirable antisense molecule. However, H5A(+35+65)[SEQ ID NO: 1], which may target the exon skipping enhancer element, was found to be very efficient in inducing the skipping of the target exon shown in Figure 5 and is therefore considered a preferred compound for inducing exon 5 skipping. [Table 6]

[0126] Antisense oligonucleotide targeting exon 6 Antisense oligonucleotides targeting exon 6 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 7]

[0127] Antisense oligonucleotide targeting exon 7 Antisense oligonucleotides targeting exon 7 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 8]

[0128] Antisense oligonucleotide targeting exon 8 Antisense oligonucleotides targeting exon 8 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 6. [Table 9]

[0129] Antisense oligonucleotides targeting exon 9 Antisense oligonucleotides targeting exon 9 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 10]

[0130] Antisense oligonucleotides targeting exon 10 Antisense oligonucleotides targeting exon 10 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 7 for examples of single antisense oligonucleotide molecules and cocktails that induce skipping of exon 10 and surrounding exons. The single antisense oligonucleotide molecule H10A(-05+16) [SEQ ID NO: 37] was able to induce skipping of exons 9-14, while the combination with H10A(+98+119) [SEQ ID NO: 38] was able to induce skipping of exon 10 alone and exons 9-12 (partially skipping of exons 10-12). The combination of H10A(-05+16) and H10A(+130+149) was able to induce skipping of exon 10 and exons 9-12. [Table 11]

[0131] Antisense oligonucleotides targeting exon 11 Antisense oligonucleotides targeting exon 11 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 37. [Table 12]

[0132] Antisense oligonucleotides targeting exon 12 Antisense oligonucleotides targeting exon 12 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 38. [Table 13]

[0133] Antisense oligonucleotides targeting exon 13 Antisense oligonucleotides targeting exon 13 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 14]

[0134] Antisense oligonucleotides targeting exon 14 Antisense oligonucleotides targeting exon 14 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 8. [Table 15]

[0135] Antisense oligonucleotides targeting exon 16 Antisense oligonucleotides targeting exon 16 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 16]

[0136] Antisense oligonucleotides targeting exon 17 Antisense oligonucleotides targeting exon 17 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 64]

[0137] Antisense oligonucleotides targeting exon 18 Antisense oligonucleotides targeting exon 18 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 9. [Table 17]

[0138] Antisense oligonucleotides targeting exon 19 Antisense oligonucleotides targeting exon 19 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 18]

[0139] Antisense oligonucleotides targeting exon 20 Antisense oligonucleotides targeting exon 20 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 19]

[0140] Antisense oligonucleotide targeting exon 23 Antisense oligonucleotides targeting exon 23 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. The H23(+69+98)-SNP contains a already documented single nucleotide polymorphism (SNP). [Table 65]

[0141] Antisense oligonucleotides targeting exon 24 Antisense oligonucleotides targeting exon 24 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 20]

[0142] Antisense oligonucleotide targeting exon 25 Antisense oligonucleotides targeting exon 25 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. The oligonucleotide H25A(+95+119)-DupA is a patient-specific antisense molecule. [Table 21]

[0143] Antisense oligonucleotide targeting exon 26 Antisense oligonucleotides targeting exon 26 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 10. [Table 22]

[0144] Antisense oligonucleotides targeting exon 31 Antisense oligonucleotides targeting exon 31 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 23]

[0145] Antisense oligonucleotide targeting exon 32 Antisense oligonucleotides targeting exon 32 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 24]

[0146] Antisense oligonucleotide targeting exon 34 Antisense oligonucleotides targeting exon 34 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 25]

[0147] Antisense oligonucleotide targeting exon 35 Antisense oligonucleotides targeting exon 35 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 26]

[0148] Antisense oligonucleotide targeting exon 36 Antisense oligonucleotides targeting exon 36 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 11. [Table 27]

[0149] Antisense oligonucleotide targeting exon 38 Antisense oligonucleotides targeting exon 38 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 28]

[0150] Antisense oligonucleotide targeting exon 39 Antisense oligonucleotides targeting exon 39 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 29]

[0151] Antisense oligonucleotides targeting exon 41 Antisense oligonucleotides targeting exon 41 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 30]

[0152] Antisense oligonucleotide targeting exon 42 Antisense oligonucleotides targeting exon 42 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 31]

[0153] Antisense oligonucleotide targeting exon 43 Antisense oligonucleotides targeting exon 43 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 12. [Table 32]

[0154] Antisense oligonucleotides targeting exon 44 Antisense oligonucleotides targeting exon 44 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figures 13 and 39. [Table 33]

[0155] Antisense oligonucleotide targeting exon 45 Antisense oligonucleotides targeting exon 45 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figures 14 and 40. [Table 34]

[0156] Antisense oligonucleotide targeting exon 46 Antisense oligonucleotides targeting exon 46 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figures 15 and 44. [Table 35]

[0157] Antisense oligonucleotides targeting exons 44-46 Antisense oligonucleotides targeting exons 44-46 were prepared, and their ability to induce exon skipping in human muscle cells was tested using the same method as described above. [Table 36]

[0158] Antisense oligonucleotide targeting exon 47 Antisense oligonucleotides targeting exon 47 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 16. [Table 37]

[0159] Antisense oligonucleotide targeting exon 48 Antisense oligonucleotides targeting exon 48 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 17. [Table 38]

[0160] Antisense oligonucleotide targeting exon 49 Antisense oligonucleotides targeting exon 49 were prepared and tested for their ability to induce exon skipping in human muscle cells using a method similar to the above method. Please refer to Figure 18.

Table 39

[0161] Antisense oligonucleotides targeting exon 50 Antisense oligonucleotides targeting exon 50 were prepared and tested for their ability to induce exon skipping in human muscle cells using a method similar to the above method. Please refer to Figures 19 and 33.

Table 40

[0162] Antisense oligonucleotides targeting exon 51 Antisense oligonucleotides targeting exon 51 were prepared and tested for their ability to induce exon skipping in human muscle cells using a method similar to the above method. Please refer to Figures 20 and 41.

Table 41

[0163] Antisense oligonucleotides targeting exon 52 Antisense oligonucleotides targeting exon 52 were prepared and tested for their ability to induce exon skipping in human muscle cells using a method similar to the above method. Please refer to Figure 42.

Table 42

[0164] Antisense oligonucleotides targeting exon 53 Antisense oligonucleotides targeting exon 53 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 43. [Table 43]

[0165] Antisense oligonucleotide targeting exon 54 Antisense oligonucleotides targeting exon 54 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 21. [Table 44]

[0166] Antisense oligonucleotide targeting exon 55 Antisense oligonucleotides targeting exon 55 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 22. [Table 45]

[0167] Antisense oligonucleotide targeting exon 56 Antisense oligonucleotides targeting exon 56 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 23. [Table 46]

[0168] Antisense oligonucleotide targeting exon 57 Antisense oligonucleotides targeting exon 57 were prepared and tested for their ability to induce exon skipping in human muscle cells using a method similar to the above method. See Figure 24.

Table 47

[0169] Antisense oligonucleotides targeting exon 59 Antisense oligonucleotides targeting exon 59 were prepared and tested for their ability to induce exon skipping in human muscle cells using a method similar to the above method. See Figure 25.

Table 48

[0170] Antisense oligonucleotides targeting exon 60 Antisense oligonucleotides targeting exon 60 were prepared and tested for their ability to induce exon skipping in human muscle cells using a method similar to the above method. See Figure 26.

Table 49

[0171] Antisense oligonucleotides targeting exon 61 Antisense oligonucleotides targeting exon 61 were prepared and tested for their ability to induce exon skipping in human muscle cells using a method similar to the above method.

Table 50

[0173] Antisense oligonucleotide targeting exon 63 Antisense oligonucleotides targeting exon 63 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 27. [Table 52]

[0174] Antisense oligonucleotides targeting exon 64 Antisense oligonucleotides targeting exon 64 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 28. [Table 53]

[0175] Antisense oligonucleotide targeting exon 65 Antisense oligonucleotides targeting exon 65 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 54]

[0176] Antisense oligonucleotide targeting exon 66 Antisense oligonucleotides targeting exon 66 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 29. [Table 55]

[0177] Antisense oligonucleotide targeting exon 67 Antisense oligonucleotides targeting exon 67 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 30. [Table 56]

[0178] Antisense oligonucleotide targeting exon 68 Antisense oligonucleotides targeting exon 68 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 31. [Table 57]

[0179] Antisense oligonucleotide targeting exon 69 Antisense oligonucleotides targeting exon 69 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. See Figure 32, which shows cocktails of H69A(+32+60) and H70A(-06+18) for removing both exons 69 and 70. [Table 58]

[0180] Antisense oligonucleotide targeting exon 70 Antisense oligonucleotides targeting exon 70 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 59]

[0181] Antisense oligonucleotides targeting exon 71 Antisense oligonucleotides targeting exon 71 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 60]

[0182] Antisense oligonucleotide targeting exon 72 Antisense oligonucleotides targeting exon 72 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 61]

[0183] Antisense oligonucleotide targeting exon 73 Antisense oligonucleotides targeting exon 73 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 62]

[0184] Antisense oligonucleotide targeting exon 74 Antisense oligonucleotides targeting exon 74 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 66]

[0185] Antisense oligonucleotide targeting exon 76 Antisense oligonucleotides targeting exon 76 were prepared and tested for their ability to induce exon skipping in human muscle cells using the same method as described above. [Table 63]

[0186] Modifications of the above embodiments for carrying out various embodiments of the present invention will become apparent to those skilled in the art based on the above teachings relating to the disclosed invention. The above embodiments of the present invention are merely illustrative and should not be construed as limiting in any way.

Claims

[Claim 1] The kit or method described in the specification.

Images