Patent Publication Number: US-2021180060-A1

Title: Multiple exon skipping compositions for dmd

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61/108,416 filed Oct. 24, 2008; wherein this provisional application is incorporated herein by reference in its entirety. 
    
    
     STATEMENT REGARDING SEQUENCE LISTING 
     The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 120178_410_SEQUENCE_LISTING.txt. The text file is 157 KB, was created on Oct. 23, 2009 and is being submitted electronically via EFS-Web. 
     FIELD OF THE INVENTION 
     The present invention relates to novel antisense compounds and compositions suitable for facilitating exon skipping in the human dystrophin gene. It also provides methods for inducing exon skipping using the antisense compositions adapted for use in the methods of the invention. 
     BACKGROUND OF THE INVENTION 
     Antisense technologies are being developed using a range of chemistries to affect gene expression at a variety of different levels (transcription, splicing, stability, translation). Much of that research has focused on the use of antisense compounds to correct or compensate for abnormal or disease-associated genes in a wide range of indications. Antisense molecules are able to inhibit gene expression with specificity, and because of this, many research efforts concerning oligonucleotides as modulators of gene expression have focused on inhibiting the expression of targeted genes or the function of cis-acting elements. The antisense oligonucleotides are typically directed against RNA, either the sense strand (e.g., mRNA) or minus-strand in the case of some viral RNA targets. To achieve a desired effect of specific gene down-regulation, the oligonucleotides generally either promote the decay of the targeted mRNA, block translation of the mRNA or block the function of cis-acting RNA elements thereby effectively preventing either de novo synthesis of the target protein or replication of the viral RNA. 
     However, such techniques are not useful where the object is to up-regulate production of the native protein or compensate for mutations that induce premature termination of translation such as nonsense or frame-shifting mutations. In these cases, the defective gene transcript should not be subjected to targeted degradation or steric inhibition, so the antisense oligonucleotide chemistry should not promote target mRNA decay or block translation. 
     In a variety of genetic diseases, the effects of mutations on the eventual expression of a gene can be modulated through a process of targeted exon skipping during the splicing process. The splicing process is directed by complex multi-component machinery that brings adjacent exon-intron junctions in pre-mRNA into dose proximity and performs cleavage of phosphodiester bonds at the ends of the introns with their subsequent reformation between exons that are to be spliced together. This complex and highly precise process is mediated by sequence motifs in the pre-mRNA that are relatively short semi-conserved RNA segments to which bind the various nuclear splicing factors that are then involved in the splicing reactions. By changing the way the splicing machinery reads or recognizes the motifs involved in pre-mRNA processing, it is possible to create differentially spliced mRNA molecules. It has now been recognized that the majority of human genes are alternatively spliced during normal gene expression, although the mechanisms involved have not been identified. 
     In cases where a normally functional protein is prematurely terminated because of mutations therein, a means for restoring some functional protein production through antisense technology has been shown to be possible through intervention during the splicing processes, and that if exons associated with disease-causing mutations can be specifically deleted from some genes, a shortened protein product can sometimes be produced that has similar biological properties of the native protein or has sufficient biological activity to ameliorate the disease caused by mutations associated with the exon (Sierakowska, Sambade et al. 1996; Wilton, Lloyd et al. 1999; van Deutekom, Bremmer-Bout et al. 2001; Lu, Mann et al. 2003; Aartsma-Rus, Janson et al. 2004). Kole et al. (U.S. Pat. Nos. 5,627,274; 5,916,808; 5,976,879; and 5,665,593) disclose methods of combating aberrant splicing using modified antisense oligonucleotide analogs that do not promote decay of the targeted pre-mRNA. Bennett et al (U.S. Pat. No. 6,210,892) describe antisense modulation of wild-type cellular mRNA processing also using antisense oligonucleotide analogs that do not induce RNAse mediated cleavage of the target RNA. 
     The process of targeted exon skipping is likely to be particularly useful in long genes where there are many exons and introns, where there is redundancy in the genetic constitution of the exons or where a protein is able to function without one or more particular exons. Efforts to redirect gene processing for the treatment of genetic diseases associated with truncations caused by mutations in various genes have focused on the use of antisense oligonucleotides that either: (1) fully or partially overlap with the elements involved in the splicing process; or (2) bind to the pre-mRNA at a position sufficiently close to the element to disrupt the binding and function of the splicing factors that would normally mediate a particular splicing reaction which occurs at that element. 
     Duchenne muscular dystrophy (DMD) is caused by a defect in the expression of the protein dystrophin. The gene encoding the protein contains 79 exons spread out over more than 2 million nucleotides of DNA. Any exonic mutation that changes the reading frame of the exon, or introduces a stop codon, or is characterized by removal of an entire out of frame exon or exons or duplications of one or more exons has the potential to disrupt production of functional dystrophin, resulting in DMD. 
     A less severe form of muscular dystrophy, Becker muscular dystrophy (BMD) has been found to arise where a mutation, typically a deletion of one or more exons, results in a correct reading frame along the entire dystrophin transcript, such that translation of mRNA into protein is not prematurely terminated. If the joining of the upstream and downstream exons in the processing of a mutated dystrophin pre-mRNA maintains the correct reading frame of the gene, the result is an mRNA coding for a protein with a short internal deletion that retains some activity resulting in a Becker phenotype. 
     Deletions of an exon or exons which do not alter the reading frame of a dystrophin protein give rise to a BMD phenotype, whereas an exon deletion that causes a frame-shift will give rise to DMD (Monaco, Bertelson et al. 1988). In general, dystrophin mutations including point mutations and exon deletions that change the reading frame and thus interrupt proper protein translation result in DMD. It should also be noted that some BMD and DMD patients have exon deletions covering multiple exons. 
     Although antisense molecules may provide a tool in the treatment of Duchenne Muscular Dystrophy (DMD), attempts to induce exon skipping using antisense molecules have had mixed success. Successful skipping of dystrophin exon 19 from the dystrophin pre-mRNA was achieved using a variety of antisense molecules directed at the flanking splice sites or motifs within the exon involved in exon definition as described by Errington et al., (Errington, Mann et al, 2003). 
     The first example of specific and reproducible exon skipping in the mdx mouse model was reported by Wilton et al (Wilton, Lloyd et al. 1999). By directing an antisense molecule to the donor splice site, exon 23 skipping was induced in the dystrophin mRNA within 6 hours of treatment of the cultured cells. Wilton et al also describe targeting the acceptor region of the mouse dystrophin pre-mRNA with longer antisense oligonucleotides. While the first antisense oligonucleotide directed at the intron 23 donor splice site induced exon skipping in primary cultured myoblasts, this compound was found to be much less efficient in immortalized cell cultures expressing higher levels of dystrophin. 
     Despite these efforts, there remains a need for improved antisense oligomers targeted to multiple dystrophin exons and improved muscle delivery compositions and methods for DMD therapeutic applications. 
     BRIEF SUMMARY OF THE INVENTION 
     Embodiments of the present invention relate generally to antisense compounds capable of binding to a selected target to induce exon skipping, and methods of use thereof to induce exon skipping. In certain embodiments, it is possible to combine two or more antisense oligonucleotides of the present invention together to induce single or multiple exon skipping. 
     In certain embodiments, it is possible to improve exon skipping of a single or multiple exons by covalently linking together two or more antisense oligonucleotide molecules (see, e.g., Aartsma-Rus, Janson et al. 2004). 
     In certain embodiments, the antisense compounds of the present invention induce exon skipping in the human dystrophin gene, and thereby allow muscle cells to produce a functional dystrophin protein. 
     The antisense oligonucleotide compounds (also referred to herein as oligomers) of the present invention typically: (i) comprise morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, (ii) contain between 10-40 nucleotide bases, preferably 20-35 bases (iii) comprise a base sequence effective to hybridize to at least 12 contiguous bases of a target sequence in dystrophin pre-mRNA and induce exon skipping. 
     In certain embodiments, the antisense compounds of the present invention may comprise phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, in accordance with the following structure (i): 
     
       
         
         
             
             
         
       
     
     wherein: 
     Y 1  is —O—, —S—, —NH—, or —CH 2 —; 
     Z is O or S; 
     Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide; and 
     X is fluoro, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, amino, optionally substituted alkylamino, or optionally substituted heterocyclyl. 
     In certain embodiments, the above intersubunit linkages, which are uncharged, may be interspersed with linkages that are positively charged at physiological pH, where the total number of positively charged linkages is between 2 and no more than half of the total number of linkages. For example, the positively charged linkages may have the above structure in which X is optionally substituted 1-piperazinyl. In other embodiments, the positively charged linkages may have the above structure in which X is substituted 1-piperazynyl, wherein the 1-piperazynyl is substituted at the 4-position with an optionally substituted alkyl guanidynyl moiety. 
     Where the antisense compound administered is effective to target a splice site of preprocessed human dystrophin, it may have a base sequence complementary to a target region containing at least 12 contiguous bases in a preprocessed messenger RNA (mRNA) human dystrophin transcript. Exemplary antisense sequences include those identified by SEQ ID NOS: 1 to 569 and 612 to 633. 
     In certain embodiments, an antisense sequence of the present invention is contained within: 
     (a) any of the sequences identified by SEQ ID NOS: 1-20, preferably SEQ ID NOS: 4, 8, 11 and 12, and more preferably SEQ ID NO:12 for use in producing skipping of exon 44 in the processing of human dystrophin pre-processed mRNA; 
     (b) any of the sequences identified by SEQ ID NOS: 21-76 and 612 to 624, preferably SEQ ID NOS: 27, 29, 34 and 39, and more preferably SEQ ID NO: 34 for use in producing skipping of exon 45 in the processing of human dystrophin pre-processed mRNA; 
     (c) any of the sequences identified by SEQ ID NOS: 77-125, preferably SEQ ID NOS: 21 to 53, and more preferably SEQ ID NOS: 82, 84-87, 90 96, 98, 99 and 101, for use in producing skipping of exon 46 in the processing of human dystrophin pre-processed mRNA; 
     (d) any of the sequences identified by SEQ ID NOS: 126-169, preferably SEQ ID NOS: 126-149, and more preferably SEQ ID NOS: 126, 128-130, 132, 144 and 146-149, for use in producing skipping of exon 47 in the processing of human dystrophin pre-processed mRNA; 
     (e) any of the sequences identified by SEQ ID NOS: 170-224 and 634, preferably SEQ ID NOS: 170-201 and 634, and more preferably SEQ ID NOS: 176, 178, 181-183, 194 and 198-201, for use in producing skipping of exon 48 in the processing of human dystrophin pre-processed mRNA; 
     (f) any of the sequences identified by SEQ ID NOS: 225-266, preferably SEQ ID NOS: 225-248, and more preferably SEQ 0 NOS: 227, 229, 234, 236, 237 and 244-248, for use in producing skipping of exon 49 in the processing of human dystrophin pre-processed mRNA; 
     (g) any of the sequences identified by SEQ ID NOS: 267-308, preferably SEQ ID NOS: 277, 287 and 290, and more preferably SEQ ID NO: 287, for use in producing skipping of exon 50 in the processing of human dystrophin pre-processed mRNA; 
     (h) any of the sequences identified by SEQ ID NOS: 309-371, preferably SEQ ID NOS: 324, 326 and 327, and more preferably SEQ ID NO: 327 for use in producing skipping of exon 51 in the processing of human dystrophin pre-processed mRNA; 
     (i) any of the sequences identified by SEQ ID NOS: 372-415, preferably SEQ ID NOS: 372-397, and more preferably SEQ ID NOS: 379-382, 384, 390 and 392-395 for use in producing skipping of exon 52 in the processing of human dystrophin pre-processed mRNA; 
     (j) any of the sequences identified by SEQ ID NOS: 416-475 and 625-633, preferably SEQ ID NOS: 428, 429 and 431, and more preferably SEQ ID NO: 429, for use in producing skipping of exon 53 in the processing of human dystrophin pre-processed mRNA; 
     (k) any of the sequences identified by SEQ ID NOS: 476-519, preferably SEQ ID NOS: 476-499, and more preferably SEQ ID NOS: 479-482, 484, 489 and 491-493, for use in producing skipping of exon 54 in the processing of human dystrophin pre-processed mRNA; and 
     (l) any of the sequences identified by SEQ ID NOS: 520-569 and 635, preferably SEQ ID NOS: 520-546 and 635, and more preferably SEQ ID NOS: 524-528, 537, 539, 540, 542 and 544, for use in producing skipping of exon 55 in the processing of human dystrophin pre-processed mRNA; 
     In certain embodiments, the compound may be conjugated to an arginine-rich polypeptide effective to promote uptake of the compound into cells. Exemplary peptides include those identified by SEQ ID NOS: 570 to 578, among others described herein. 
     In one exemplary embodiment, the arginine-rich polypeptide is covalently coupled at its N-terminal or C-terminal residue to the 3′ or 5′ end of the antisense compound. Also in an exemplary embodiment, the antisense compound is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit. 
     In general, the peptide-oligomer conjugate may further comprise a homing peptide which is selective for a selected mammalian tissue, i.e., the same tissue being targeted by the cell-penetrating peptide. The conjugate may be of the form: cell penetrating peptide-homing peptide-antisense oligomer, or, more preferably, of the form: homing peptide-cell penetrating peptide-antisense oligomer. For example, a peptide conjugate compound for use in treating Duchenne muscular dystrophy, as described above, can further comprise a homing peptide which is selective for muscle tissue, such as the peptide having the sequence identified as SEQ ID NO: 579, conjugated to the cell-penetrating peptide. Exemplary conjugates of this type include those represented herein as CP06062-MSP-PMO (cell penetrating peptide-homing peptide-antisense oligomer) and as MSP-CP06062-PMO (homing peptide-cell penetrating peptide-antisense oligomer) (see SEQ ID NOs: 580-583). 
     In some embodiments, the peptide is conjugated to the oligomer via a linker moiety. In certain embodiments the linker moiety may comprise an optionally substituted piperazynyl moiety. In other embodiments, the linker moiety may further comprise a beta alanine and/or a 6-aminohexanoic acid subunit. In yet other embodiments, the peptide is conjugated directly to the oligomer without a linker moiety. 
     Conjugation of the peptide to the oligomer may be at any position suitable for forming a covalent bond between the peptide and the oligomer or between the linker moiety and the oligomer. For example, in some embodiments conjugation of the peptide may be at the 3″ end of the oligomer. In other embodiments, conjugation of the peptide to the oligomer may be at the 5′ end of the oligomer. In yet other embodiments, the peptide may be conjugated to the oligomer through any of the intersubunit linkages. 
     In some embodiments, the peptide is conjugated to the oligomer at the 5′ end of the oligomer. In embodiments comprising phosphorus-containing intersubunit linkages, the peptide may be conjugated to the oligomer via a covalent bond to the phosphorous of the terminal linkage group. Conjugation in this manner may be with or without the linker moiety described above. 
     In yet other embodiments, the peptide may be conjugated to the oligomer at the 3′ end of the oligomer. In some further embodiments, the peptide may be conjugated to the nitrogen atom of the 3′ terminal morpolino group of the oligomer. In this respect, the peptide may be conjugated to the oligomer directly or via the linker moiety described above. 
     In some embodiments, the oligomer may be conjugated to a moiety that enhances the solubility of the oligomer in aqueous medium. In some embodiments, the moiety that enhances solubility of the oligomer in aqueous medium is a polyethyleneglycol. In yet further embodiments, the moiety that enhances solubility of the oligomer in aqueous medium is triethylene glycol. For example, in some embodiments the moiety that enhances solubility in aqueous medium may be conjugated to the oligomer at the 5′ end of the oligomer. Conjugation of the moiety that enhances solubility of the oligomer in aqueous medium to the oligomer may be either directly or through the linker moiety described above. 
     Certain embodiments of the present invention provide antisense molecules selected and or adapted to aid in the prophylactic or therapeutic treatment of a genetic disorder comprising at least an antisense molecule in a form suitable for delivery to a patient. 
     Certain embodiments of the invention provide methods for treating a patient suffering from a genetic disease wherein there is a mutation in a gene encoding a particular protein and the affect of the mutation can be abrogated by exon skipping, comprising the steps of: (a) selecting an antisense molecule in accordance with the methods described herein; and (b) administering the molecule to a patient in need of such treatment. The present invention also includes the use of purified and isolated antisense oligonucleotides of the invention, for the manufacture of a medicament for treatment of a genetic disease. 
     Certain embodiments provide a method of treating muscular dystrophy, such as a condition characterized by Duchenne muscular dystrophy, which method comprises administering to a patient in need of treatment an effective amount of an appropriately designed antisense oligonucleotide, as described herein, relevant to the particular genetic lesion in that patient. Further, certain embodiments provide a method for prophylactically treating a patient to prevent or at least minimize muscular dystrophy, including Duchene muscular dystrophy, comprising the step of: administering to the patient an effective amount of an antisense oligonucleotide or a pharmaceutical composition comprising one or more of these biological molecules. 
     Certain embodiments relate to methods of treating muscular dystrophy in a subject, comprising administering to the subject an effective amount of a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS:1 to 569 and 612 to 635, and capable of forming with the complementary mRNA sequence in a dystrophin-gene exon a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C., wherein the exon is selected from the group consisting of exons 44-55. 
     In certain embodiments, the muscular dystrophy is Duchenne&#39;s muscular dystrophy (DMD). In certain embodiments, the muscular dystrophy is Becker muscular dystrophy (BMD). 
     In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 1-20, and the exon is exon 44. In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 21-76 and 612 to 624, and the exon is exon 45. 
     In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 77-125, and the exon is exon 46. In certain embodiments, the sequence selected from the group consisting SEQ ID NOS: 126-169, and the exon is exon 47. 
     In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 170-224 and 634, and the exon is exon 48. In certain embodiments, the sequence selected from the group consisting SEQ ID NOS: 225-266, and the exon is exon 49. 
     In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 267-308, and the exon is exon 50. In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 309-371, and the exon is exon 51. 
     In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 372-415, and the exon is exon 52. In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 416-475 and 625-633, and the exon is exon 53. In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 476-519, and the exon is exon 54. In certain embodiments, the sequence is selected from the group consisting SEQ ID NOS: 520-569 and 635, and the exon is exon 55. In certain embodiments, the sequence comprises or consists essentially of SEQ ID NO:287. 
     Certain embodiments provide kits for treating a genetic disease, which kits comprise at least an antisense oligonucleotide of the present invention, packaged in a suitable container and instructions for its use. 
     These and other objects and features will be more fully understood when the following detailed description of the invention is read in conjunction with the figures. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1A  shows an exemplary morpholino oligomer structure with a phosphorodiamidate linkage; 
         FIG. 1B  shows a conjugate of an arginine-rich peptide and an antisense oligomer, in accordance with an embodiment of the invention; 
         FIG. 1C  shows a conjugate as in  FIG. 1B , wherein the backbone linkages contain one or more positively charged groups; 
         FIGS. 1D-G  show the repeating subunit segment of exemplary morpholino oligonucleotides, designated D through G. 
         FIG. 2A  shows the relative location and results of an antisense oligomer exon 51 scan designed to induce skipping of human dystrophin exon 51. 
         FIG. 2B-C  shows the relative activity in cultured human rhabdomyosarcoma (RD) cells and human primary skeletal muscle cells of the three best oligomers selected from the exon 51 scan (SEQ ID NOs: 324, 326 and 327) relative to sequences (AVI-5658; SEQ ID NO: 588 and h51AON1; SEQ ID NO:594) that are effective at inducing exon 51 skipping.  FIG. 2D  shows the relative location within exon 51 of three selected oligomers compared to certain sequences. 
         FIG. 3A  shows the relative location and results of an antisense oligomer exon 50 scan designed to induce skipping of human dystrophin exon 50 compared to other sequences that induce exon 50 skipping. 
         FIG. 3B  shows the relative location and activity of antisense sequences selected from the exon 50 scan (SEQ ID NOS: 277, 287, 290 and 291) compared to other sequences (SEQ ID NOS: 584 and 585). 
         FIG. 4A  shows the relative location and results of an antisense oligomer exon 53 scan designed to induce skipping of human dystrophin exon 53. 
         FIG. 4B  shows the relative location of certain sequences used to compare the exon-skipping activity of those oligomers selected as being most active in the exon 53 scan. 
         FIGS. 4C-F  show the results of dose-ranging studies, summarized in  FIG. 4G , using the oligomers selected as being most efficacious in the exon 53 scan (SEQ ID NOS:422, 428, 429 and 431). 
         FIGS. 4H and 4I  show the relative activity of certain sequences (SEQ ID NOS: 608-611) compared to the activity of the most active exon 53-skipping oligomer (SEQ ID NO:429) in both RD cells and human primary skeletal muscle cells. 
         FIG. 5A  shows the relative location and results of an antisense oligomer exon 44 scan designed to induce skipping of human dystrophin exon 44.  FIG. 5B  shows the relative location within exon 44 of certain sequences used to compare the exon-skipping activity to those oligomers selected as being most active in the exon 44 scan. 
         FIGS. 5C-G  show the results of dose-ranging studies, summarized in  FIG. 5H , using the oligomers selected as being most efficacious in the exon 44 scan (SEQ ID NOS: 4, 8, 11, 12 and 13). 
         FIGS. 5I and 5J  show the relative activity of certain sequences (SEQ ID NOS: 600-603) compared to the activity of the most active exon 53-skipping oligomer (SEQ ID NO:12) in both RD cells and human primary skeletal muscle cells. 
         FIG. 6A  shows the relative location and results of an antisense oligomer exon 45 scan designed to induce skipping of human dystrophin exon 45.  FIG. 6B  shows the relative location within exon 45 of certain sequences used to compare the exon-skipping activity to those oligomers selected as being most active in the exon 45 scan. 
         FIGS. 6C-F  show the results of dose-ranging studies, summarized in  FIG. 6H , using the oligomers selected as being most efficacious in the exon 45 scan (SEQ ID NOS: 27, 29, 34 and 39).  FIG. 6G  uses a relatively inactive oligomer (SEQ ID NO: 49) as a negative control. 
         FIGS. 6I and 6J  show the relative activity of certain sequences (SEQ ID NOS: 604-607) compared to the activity of the most active exon 53-skipping oligomer (SEQ ID NO: 34) in both RD cells and human primary skeletal muscle cells. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Embodiments of the present invention relate generally to improved antisense compounds, and methods of use thereof, which are specifically designed to induce exon skipping in the dystrophin gene. Dystrophin plays a vital role in muscle function, and various muscle-related diseases are characterized by mutated forms of this gene. Hence, in certain embodiments, the improved antisense compounds described herein induce exon skipping in mutated forms of the human dystrophin gene, such as the mutated dystrophin genes found in Duchenne&#39;s muscular dystrophy (DMD) and Becker&#39;s muscular dystrophy (BMD). 
     Due to aberrant mRNA splicing events caused by mutations, these mutated human dystrophin genes either express defective dystrophin protein or express no measurable dystrophin at all, a condition that leads to various forms of muscular dystrophy. To remedy this condition, the antisense compounds of the present invention typically hybridize to selected regions of a pre-processed RNA of a mutated human dystrophin gene, induce exon skipping and differential splicing in that otherwise aberrantly spliced dystrophin mRNA, and thereby allow muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein. In certain embodiments, the resulting dystrophin protein is not necessarily the “wild-type” form of dystrophin, but is rather a truncated, yet functional or semi-functional, form of dystrophin. 
     By increasing the levels of functional dystrophin protein in muscle cells, these and related embodiments may be useful in the prophylaxis and treatment of muscular dystrophy, especially those forms of muscular dystrophy, such as DMD and BMD, that are characterized by the expression of defective dystrophin proteins due to aberrant mRNA splicing. The specific oligomers described herein further provide improved, dystrophin-exon-specific targeting over other oligomers in use, and thereby offer significant and practical advantages over alternate methods of treating relevant forms of muscular dystrophy. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. 
     Definitions 
     The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. 
     By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. 
     By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene. 
     Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. 
     By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements. 
     The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic adds&#39; bases are matched according to the base pairing rules. Cr, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often desired, some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the target RNA. Variations at any location within the oligomer are included. In certain embodiments, variations in sequence near the termini of an oligomer are generally preferable to variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 nucleotides of the 5′ and/or 3′ terminus. 
     The terms “cell penetrating peptide” or “CPP” are used interchangeably and refer to cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains. The peptides, as shown herein, have the capability of inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration. 
     The terms “antisense oligomer” or “antisense compound” are used interchangeably and refer to a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence. The cyclic subunits are based on ribose or another pentose sugar or, in a preferred embodiment, a morpholino group (see description of morpholino oligomers below). 
     Such an antisense oligomer can be designed to block or inhibit translation of mRNA or to inhibit natural pre-mRNA splice processing, and may be said to be “directed to” or “targeted against” a target sequence with which it hybridizes. In certain embodiments, the target sequence includes a region including an AUG start codon of an mRNA, a 3′ or 5′ splice site of a pre-processed mRNA, or a branch point. The target sequence may be within an exon or within an intron. The target sequence for a splice site may include an mRNA sequence having its 5′ end 1 to about 25 base pairs downstream of a normal splice acceptor junction in a preprocessed mRNA. A preferred target sequence for a splice is any region of a preprocessed mRNA that includes a splice site or is contained entirely within an exon coding sequence or spans a splice acceptor or donor site. An oligomer is more generally said to be “targeted against” a biologically relevant target, such as a protein, virus, or bacteria, when it is targeted against the nucleic acid of the target in the manner described above. Included are antisense oligomers that comprise, consist essentially of, or consist of one or more of SEQ ID NOS:1 to 569 and 612 to 635. Also included are variants of these antisense oligomers, including variant oligomers having 80%, 85%, 90%, 95%, 97%, 98%, or 99% (including all integers in between) sequence identity or sequence homology to any one of SEQ ID NOS:1 to 569 and 612 to 635, and/or variants that differ from these sequences by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, preferably those variants that induce exon skipping of one or more selected human dystrophin exons. Also included are oligomers of any on or more of SEQ ID NOS:584-611 and 634-635, which comprise a suitable number of charged linkages, as described herein, e.g. up to about 1 per every 2-5 uncharged linkages, such as about 4-5 per every 10 uncharged linkages, and/or which comprise an Arg-rich peptide attached thereto, as also described herein. 
     The terms “morpholino oligomer” or “PMO” (phosphoramidate- or phosphorodiamidate morpholino oligomer) refer to an oligonucleotide analog composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing linkages, one to three atoms long, preferably two atoms long, and preferably uncharged or cationic, joining the morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring bears a purine or pyrimidine base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base in a polynucleotide. See, e.g., the structure in  FIG. 1A , which shows a preferred phosphorodiamidate linkage type. Variations can be made to this linkage as long as they do not interfere with binding or activity. For example, the oxygen attached to phosphorus may be substituted with sulfur (thiophosphorodiamidate). The 5′ oxygen may be substituted with amino or lower alkyl substituted amino. The pendant nitrogen attached to phosphorus may be unsubstituted, monosubstituted, or disubstituted with (optionally substituted) lower alkyl. See also the discussion of cationic linkages below. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, and PCT Appn. No. PCT/US07/11435 (cationic linkages), all of which are incorporated herein by reference. 
     The purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine. Also included are bases such as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trime115thoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, β-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonyhnethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman &amp; Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as illustrated above; such bases can be used at any position in the antisense molecule. Persons skilled in the art will appreciate that depending on the uses of the oligomers. Ts and Us are interchangeable. For instance, with other antisense chemistries such as 2′-O-methyl antisense oligonucleotides that are more RNA-like, the T bases may be shown as U (see, e.g., Sequence ID Listing). 
     An “amino acid subunit” or “amino acid residue” can refer to an α-amino acid residue (e.g., —CO—CHR—NH—) or a β- or other amino acid residue (e.g., —CO—(CH 2 ) n CHR—NH—), where R is a side chain (which may include hydrogen) and n is 1 to 6, preferably 1 to 4. 
     The term “naturally occurring amino acid” refers to an amino acid present in proteins found in nature, such as the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine. The term “non-natural amino acids” refers to those amino acids not present in proteins found in nature, examples include beta-alanine (β-Ala; or B), 6-aminohexanoic acid (Ahx) and 6-aminopentanoic acid. Additional examples of “non-natural amino acids” include, without limitation, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. 
     An “effective amount” or “therapeutically effective amount” refers to an amount of therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, which is effective to produce a desired physiological response or therapeutic effect in the subject. One example of a desired physiological response includes increased expression of a relatively functional or biologically active form of the dystrophin protein, mainly in muscle tissues or cells that contain a defective dystrophin protein or no dystrophin, as compared no antisense oligomer or a control oligomer. Examples of desired therapeutic effects include, without limitation, improvements in the symptoms or pathology of muscular dystrophy, reducing the progression of symptoms or pathology of muscular dystrophy, and slowing the onset of symptoms or pathology of muscular dystrophy, among others. Examples of such symptoms include fatigue, mental retardation, muscle weakness, difficulty with motor skills (e.g., running, hopping, jumping), frequent falls, and difficulty walking. The pathology of muscular dystrophy can be characterized, for example, by muscle fibre damage and membrane leakage. For an antisense oligomer, this effect is typically brought about by altering the splice-processing of a selected target sequence (e.g., dystrophin), such as to induce exon skipping. 
     An “exon” refers to a defined section of nucleic acid that encodes for a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after either portions of a pre-processed (or precursor) RNA have been removed by splicing. The mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA. The human dystrophin gene has about 75 exons. 
     An “intron” refers to a nucleic acid region (within a gene) that is not translated into a protein. An intron is a non-coding section that is transcribed into a precursor mRNA (pre-mRNA), and subsequently removed by splicing during formation of the mature RNA. 
     “Exon skipping” refers generally to the process by which an entire exon, or a portion thereof, is removed from a given pre-processed RNA, and is thereby excluded from being present in the mature RNA, such as the mature mRNA that is translated into a protein. Hence, the portion of the protein that is otherwise encoded by the skipped exon is not present in the expressed form of the protein, typically creating an altered, though still functional, form of the protein. In certain embodiments, the exon being skipped is an aberrant exon from the human dystrophin gene, which may contain a mutation or other alteration in its sequence that otherwise causes aberrant splicing. In certain embodiments, the exon being skipped is any one or more of exons 1-75 of the dystrophin gene, though any one or more of exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and/or 55 of the human dystrophin gene are preferred. 
     “Dystrophin” is a rod-shaped cytoplasmic protein, and a vital part of the protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. Dystrophin contains multiple functional domains. For instance, dystrophin contains an actin binding domain at about amino acids 14-240 and a central rod domain at about amino acids 253-3040. This large central domain is formed by 24 spectrin-like triple-helical elements of about 109 amino acids, which have homology to alpha-actinin and spectrin. The repeats are typically interrupted by four proline-rich non-repeat segments, also referred to as hinge regions. Repeats 15 and 16 are separated by an 18 amino acid stretch that appears to provide a major site for proteolytic cleavage of dystrophin. The sequence identity between most repeats ranges from 10-25%. One repeat contains three alpha-helices: 1, 2 and 3. Alpha-helices 1 and 3 are each formed by 7 helix turns, probably interacting as a coiled-coil through a hydrophobic interface. Alpha-helix 2 has a more complex structure and is formed by segments of four and three helix turns, separated by a Glycine or Proline residue. Each repeat is encoded by two exons, typically interrupted by an intron between amino acids 47 and 48 in the first part of alpha-helix 2. The other intron is found at different positions in the repeat, usually scattered over helix-3. Dystrophin also contains a cysteine-rich domain at about amino acids 3080-3360), including a cysteine-rich segment (i.e., 15 Cysteines in 280 amino acids) showing homology to the C-terminal domain of the slime mold ( Dictyostelium discoideum ) alpha-actinin. The carboxy-terminal domain is at about amino acids 3361-3685. 
     The amino-terminus of dystrophin binds to F-actin and the carboxy-terminus binds to the dystrophin-associated protein complex (DAPC) at the sarcolemma. The DAPC includes the dystroglycans, sarcoglycans, integrins and caveolin, and mutations in any of these components cause autosomally inherited muscular dystrophies. The DAPC is destabilized when dystrophin is absent, which results in diminished levels of the member proteins, and in turn leads to progressive fibre damage and membrane leakage. In various forms of muscular dystrophy, such as Duchenne&#39;s muscular dystrophy (DMD) and Becker&#39;s muscular dystrophy (BMD), muscle cells produce an altered and functionally defective form of dystrophin, or no dystrophin at all, mainly due to mutations in the gene sequence that lead to incorrect splicing. The predominant expression of the defective dystrophin protein, or the complete lack of dystrophin or a dystrophin-like protein, leads to rapid progression of muscle degeneration, as noted above. In this regard, a “defective” dystrophin protein may be characterized by the forms of dystrophin that are produced in certain subjects with DMD or BMD, as known in the art, or by the absence of detectable dystrophin. 
     Table A provides an illustration of the various dystrophin domains, the amino acid residues that encompass these domains, and the exons that encode them, 
     
       
         
           
               
               
               
               
             
               
                 TABLE A 
               
               
                   
               
               
                   
                   
                 Residue 
                   
               
               
                 Domain 
                 Sub Domain 
                 Nos 
                 Exons 
               
               
                   
               
             
            
               
                 actin binding 
                   
                  14-240 
                 2-8 
               
               
                 domain 
                   
                   
                   
               
               
                 central rod 
                   
                  253-3040 
                  8-61 
               
               
                 domain 
                   
                   
                   
               
               
                   
                 hinge 1 
                 253-327 
                 (8)-9  
               
               
                   
                 repeat 1 
                 337-447 
                 10-11 
               
               
                   
                 repeat 2 
                 448-556 
                 12-14 
               
               
                   
                 repeat 3 
                 557-667 
                 14-16 
               
               
                   
                 hinge 2 
                 668-717 
                 17 
               
               
                   
                 repeat 4 
                 718-828 
                 (17)-20  
               
               
                   
                 repeat 5 
                 829-934 
                 20-21 
               
               
                   
                 repeat 6 
                  935-1045 
                 22-23 
               
               
                   
                 repeat 7 
                 1046-1154 
                 (23)-(26) 
               
               
                   
                 repeat 8 
                 1155-1263 
                 26-27 
               
               
                   
                 repeat 9 
                 1264-1367 
                  28-(30) 
               
               
                   
                 repeat 10 
                 1368-1463 
                 30-32 
               
               
                   
                 repeat 11 
                 1464-1568 
                  32-(34) 
               
               
                   
                 repeat 12 
                 1569-1676 
                 34-35 
               
               
                   
                 repeat 13 
                 1677-1778 
                 36-37 
               
               
                   
                 repeat 14 
                 1779-1874 
                  38-(40) 
               
               
                   
                 repeat 15 
                 1875-1973 
                 40-41 
               
               
                   
                 interruption 
                 1974-1991 
                 42 
               
               
                   
                 repeat 16 
                 1992-2101 
                 42-43 
               
               
                   
                 repeat 17 
                 2102-2208 
                 44-45 
               
               
                   
                 repeat 18 
                 2209-2318 
                 46-48 
               
               
                   
                 repeat 19 
                 2319-2423 
                 48-50 
               
               
                   
                 hinge 3 
                 2424-2470 
                 50-51 
               
               
                   
                 repeat 20 
                 2471-2577 
                 51-53 
               
               
                   
                 repeat 21 
                 2578-2686 
                  53-(55) 
               
               
                   
                 repeat 22 
                 2687-2802 
                  55-(57) 
               
               
                   
                 repeat 23 
                 2803-2931 
                 57-59 
               
               
                   
                 repeat 24 
                 2932-3040 
                  59-(61) 
               
               
                   
                 hinge 4 
                 3041-3112 
                 61-64 
               
               
                 Cysteine-rich 
                   
                 3080-3360 
                 63-69 
               
               
                 domain 
                   
                   
                   
               
               
                   
                 dystrogiycan binding site 
                 3080-3408 
                 63-70 
               
               
                   
                 WW domain 
                 3056-3092 
                 62-63 
               
               
                   
                 EF-hand 1 
                 3130-3157 
                 65 
               
               
                   
                 EF-hand 2 
                 3178-3206 
                 65-66 
               
               
                   
                 ZZ domain 
                 3307-3354 
                 68-69 
               
               
                 Carboxy-terminal 
                   
                 3361-3685 
                 70-79 
               
               
                 domain 
                   
                   
                   
               
               
                   
                 alpha1-syntrophin binding 
                 3444-3494 
                 73-74 
               
               
                   
                 site 
                   
                   
               
               
                   
                 ß1-syntrophin binding site 
                 3495-3535 
                 74-75 
               
               
                   
                 (Leu)6-heptad repeat 
                 3558-3593 
                 75 
               
               
                   
               
            
           
         
       
     
     As used herein, the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function. 
     A “functional” dystrophin protein refers generally to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that is otherwise characteristic of muscular dystrophy, typically as compared to the altered or “defective” form of dystrophin protein that is present in certain subjects with DMD or BMD. In certain embodiments, a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers in between) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to routine techniques in the art. As one example, dystrophin-related activity in muscle cultures in vitro can be measured according to myotube size, myofibril organization (or disorganization), contractile activity, and spontaneous clustering of acetylcholine receptors (see, e.g., Brown et al.,  Journal of Cell Science.  112:209-216, 1999). Animal models are also valuable resources for studying the pathogenesis of disease, and provide a means to test dystrophin-related activity. Two of the most widely used animal models for DMD research are the mdx mouse and the golden retriever muscular dystrophy (GRMD) dog, both of which are dystrophin negative (see, e.g., Collins &amp; Morgan,  Int J Exp Pathol  84: 165-172, 2003). These and other animal models can be used to measure the functional activity of various dystrophin proteins. Included are truncated forms of dystrophin, such as those forms that are produced by certain of the exon-skipping antisense compounds of the present invention. 
     By “gene” is meant a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5′ and 3′ untranslated sequences). 
     By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated polynucleotide,” as used herein, may refer to a polynucleotide that has been purified or removed from the sequences that flank it in a naturally-occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment. 
     By “enhance” or “enhancing,” or “increase” or “increasing,” or “stimulate” or “stimulating,” refers generally to the ability of one or antisense compounds or compositions to produce or cause a greater physiological response (i.e., downstream effects) in a cell or a subject, as compared to the response caused by either no antisense compound or a control compound. A measurable physiological response may include increased expression of a functional form of a dystrophin protein, or increased dystrophin-related biological activity in muscle tissue, among other responses apparent from the understanding in the art and the description herein. Increased muscle function can also be measured, including increases or improvements in muscle function by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The percentage of muscle fibres that express a functional dystrophin can also be measured, including increased dystrophin expression in about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of muscle fibres. For instance, it has been shown that around 40% of muscle function improvement can occur if 25-30% of fibers express dystrophin (see, e.g., DelloRusso et al,  Proc Natl Acad Sci USA  99: 12979-12984, 2002). An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1), e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no antisense compound (the absence of an agent) or a control compound. 
     The term “reduce” or “inhibit” may relate generally to the ability of one or more antisense compounds of the invention to “decrease” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic art. Relevant physiological or cellular responses (in viva or in vitro) will be apparent to persons skilled in the art, and may include reductions in the symptoms or pathology of muscular dystrophy, or reductions in the expression of defective forms of dystrophin, such as the altered forms of dystrophin that are expressed in individuals with DMD or BMD. A “decrease” in a response may be statistically significant as compared to the response produced by no antisense compound or a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between. 
     “Homology” refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984,  Nucleic Acids Research  12, 387-395). In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example; by the comparison algorithm used by GAP. 
     The recitations “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g.; Ala, Pro; Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. 
     Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity”. A “reference sequence” is at least 8 or 10 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul of al., 1997 , Nucl. Acids Res.  25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., “Current Protocols in Molecular Biology,” John Wiley &amp; Sons Inc, 1994-1998, Chapter 15. 
     “Treatment” or “treating” of an individual (e.g., a mammal, such as a human) or a cell may include any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent. Treatment includes any desirable effect on the symptoms or pathology of a disease or condition associated with the dystrophin protein, as in certain forms of muscular dystrophy, and may include, for example, minimal changes or improvements in one or more measurable markers of the disease or condition being treated. Also included are “prophylactic” treatments, which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof. 
     Hence, included are methods of treating muscular dystrophy, such as DMD and BMD, by administering one or more antisense oligomers of the present invention (e.g., SEQ ID NOS: 1 to 569 and 612 to 635, and variants thereof), optionally as part of a pharmaceutical formulation or dosage form, to a subject in need thereof. Also included are methods of inducing exon-skipping in a subject by administering one or more antisense oligomers, in which the exon is one of exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and/or 55 from the dystrophin gene, preferably the human dystrophin gene. A “subject,” as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated with an antisense compound of the invention, such as a subject that has or is at risk for having DMD or BMD, or any of the symptoms associated with these conditions (e.g., muscle fibre loss). Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included. 
     Also included are vector delivery systems that are capable of expressing the oligomeric, dystrophin-targeting sequences of the present invention, such as vectors that express a polynucleotide sequence comprising any one or more of SEQ ID NOS: 1 to 569 and 612 to 635, or variants thereof, as described herein. By “vector” or “nucleic acid construct” is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. 
     A vector or nucleic acid construct system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector or nucleic acid construct is preferably one which is operably functional in a mammalian cell, such as a muscle cell. The vector can also include a selection marker such as an antibiotic or drug resistance gene, or a reporter gene (i.e., green fluorescent protein, luciferase), that can be used for selection or identification of suitable transformants or transfectants. Exemplary delivery systems may include viral vector systems (i.e., viral-mediated transduction) including, but not limited to, retroviral (e.g., lentiviral) vectors, adenoviral vectors, adeno-associated viral vectors, and herpes viral vectors, among others known in the art. 
     The term “operably linked” as used herein means placing an oligomer-encoding sequence under the regulatory control of a promoter, which then controls the transcription of the oligomer. 
     A wild-type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene. 
     “Alkyl” or “alkylene” both refer to a saturated straight or branched chain hydrocarbon radical containing from 1 to 18 carbons. Examples include without limitation methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl and n-hexyl. The term “lower alkyl” refers to an alkyl group, as defined herein, containing between 1 and 8 carbons. 
     “Alkenyl” refers to an unsaturated straight or branched chain hydrocarbon radical containing from 2 to 18 carbons and comprising at least one carbon to carbon double bond. Examples include without limitation ethenyl, propenyl, iso-propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyl and n-hexenyl. The term “lower alkenyl” refers to an alkenyl group, as defined herein, containing between 2 and 8 carbons. 
     “Alkynyl” refers to an unsaturated straight or branched chain hydrocarbon radical containing from 2 to 18 carbons comprising at least one carbon to carbon triple bond. Examples include without limitation ethynyl, propynyl, iso-propynyl, butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl. The term “lower alkynyl” refers to an alkynyl group, as defined herein, containing between 2 and 8 carbons. 
     “Cycloalkyl” refers to a mono- or poly-cyclic alkyl radical. Examples include without limitation cyclobutyl, cycopentyl, cyclohexyl, cycloheptyl and cyclooctyl. 
     “Aryl” refers to a cyclic aromatic hydrocarbon moiety containing from 5 to 18 carbons having one or more dosed ring(s). Examples include without limitation phenyl, benzyl, naphthyl, anthracenyl, phenanthracenyl and biphenyl. 
     “Aralkyl” refers to a radical of the formula RaRb where Ra is an alkylene chain as defined above and Rb is one or more aryl radicals as defined above, for example, benzyl, diphenylmethyl and the like. 
     “Thioalkoxy” refers to a radical of the formula —SRc where Rc is an alkyl radical as defined herein. The term “lower thioalkoxy” refers to an alkoxy group, as defined herein, containing between 1 and 8 carbons. 
     “Alkoxy” refers to a radical of the formula —ORda where Rd is an alkyl radical as defined herein. The term “lower alkoxy” refers to an alkoxy group, as defined herein, containing between 1 and 8 carbons. Examples of alkoxy groups include, without limitation, methoxy and ethoxy. 
     “Alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group. 
     “Carbonyl” refers to the —C(═O)— radical. 
     “Guanidynyl” refers to the H 2 N(C═NH 2 )—NH— radical. 
     “Amidinyl” refers to the H 2 N(C═NH 2 )CH— radical. 
     “Amino” refers to the —NH 2  radical. 
     “Alkylamino” refers to a radical of the formula —NHRd or —NRdRd where each Rd is, independently, an alkyl radical as defined herein. The term “lower alkylamino” refers to an alkylamino group, as defined herein, containing between 1 and 8 carbons. 
     “Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Thus, in addition to the heteroaryls listed below, heterocycles also include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiopyranyl, and the like. 
     “Heteroaryl” means an aromatic heterocycle ring of 5- to 10 members and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and bicyclic ring systems. Representative heteroaryls are pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. 
     The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkoxy”, “optionally substituted thioalkoxy”, “optionally substituted alkyl amino”, “optionally substituted lower alkyl”, “optionally substituted lower alkenyl”, “optionally substituted lower alkoxy”, “optionally substituted lower thioalkoxy”, “optionally substituted lower alkyl amino” and “optionally substituted heterocyclyl” mean that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include: deuterium, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, oxo, halogen, —CN, —ORx, NRxRy, NRxC(═O)Ry, NRxSO2Ry, —NRxC(═O)NRxRy, C(═O)ORx, C(═O)NRxRy, —SOmRx and —SOmNRxRy, wherein m is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle or optionally substituted cycloalkyl and each of said optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle and optionally substituted cycloalkyl substituents may be further substituted with one or more of oxo, halogen, —CN, —ORx, NRxRy, NRxC(═O)Ry, NRxSO2Ry, —NRxC(═O)NRxRy, C(═O)Rx, C(═O)ORx, C(═O)NRxRy, —SOmRx and —SOmNRxRy. 
     Constructing Antisense Oligonucleotides 
     Examples of morpholino oligonucleotides having phosphorus-containing backbone linkages are illustrated in  FIGS. 1A-1C . Especially preferred is a phosphorodiamidate-linked morpholino oligonucleotide such as shown in  FIG. 1C , which is modified, in accordance with one aspect of the present invention, to contain positively charged groups at preferably 10%-50% of its backbone linkages. Morpholino oligonucleotides with uncharged backbone linkages and their preparation, including antisense oligonucleotides, are detailed, for example, in (Summerton and Weller 1997) and in co-owned U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521,063, and 5,506,337, all of which are expressly incorporated by reference herein. 
     Important properties of the morpholino-based subunits include: 1) the ability to be linked in a oligomeric form by stable, uncharged or positively charged backbone linkages; 2) the ability to support a nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil and inosine) such that the polymer formed can hybridize with a complementary-base target nucleic acid, including target RNA, Tm values above about 45° C. in relatively short oligonucleotides (e.g., 10-15 bases); 3) the ability of the oligonucleotide to be actively or passively transported into mammalian cells; and 4) the ability of the antisense oligonucleotide:RNA heteroduplex to resist RNAse and RNaseH degradation, respectively. 
     Exemplary backbone structures for antisense oligonucleotides of the claimed subject matter include the morpholino subunit types shown in  FIGS. 1D-G , each linked by an uncharged or positively charged, phosphorus-containing subunit linkage.  FIG. 1D  shows a phosphorus-containing linkage which forms the five atom repeating-unit backbone, wherein the morpholino rings are linked by a 1-atom phosphoamide linkage.  FIG. 1E  shows a linkage which produces a 6-atom repeating-unit backbone. In this structure, the atom Y linking the 5′ morpholino carbon to the phosphorus group may be sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety pendant from the phosphorus may be fluorine, an alkyl or substituted alkyl, an alkoxy or substituted alkoxy, a thioalkoxy or substituted thioalkoxy, or unsubstituted, monosubstituted, or disubstituted nitrogen, including cyclic structures, such as morpholines or piperidines. Alkyl, alkoxy and thioalkoxy preferably include 1-6 carbon atoms. The Z moieties are sulfur or oxygen, and are preferably oxygen. 
     The linkages shown in  FIGS. 1F and 1G  are designed for 7-atom unit-length backbones. In structure 1F, the X moiety is as in Structure 1E, and the Y moiety may be methylene, sulfur, or, preferably, oxygen. In Structure 1G, the X and Y moieties are as in Structure 1E. Particularly preferred morpholino oligonucleotides include those composed of morpholino subunit structures of the form shown in  FIG. 1E , where X=NH 2 , N(CH 3 ) 2 , optionally substituted 1-piperazinyl, or other charged group, Y=O, and Z=O. 
     As noted above, the uncharged or substantially uncharged oligonucleotide may be modified, in accordance with an aspect of the invention, to include charged linkages, e.g. up to about 1 per every 2-5 uncharged linkages, such as about 4-5 per every 10 uncharged linkages. Optimal improvement in antisense activity may be seen when about 25% of the backbone linkages are cationic, including about 20% to about 30%. Also included are oligomers in which about 35%, 40%, 45%, 50%, 55%, 60% (including all integers in between), or more of the backbone linkages are cationic. Enhancement is also seen with a small number, e.g., 5% or 10-20%, of cationic linkages. 
     A substantially uncharged, phosphorus containing backbone in an oligonucleotide analog is typically one in which a majority of the subunit linkages, e.g., between 50%-100%, typically at least 60% to 100% or 75% or 80% of its linkages, are uncharged at physiological pH and contain a single phosphorous atom. 
     Additional experiments conducted in support of the present invention indicate that the enhancement seen with added cationic backbone charges may, in some cases, be further enhanced by distributing the bulk of the charges dose to the “center-region” backbone linkages of the antisense oligonucleotide, e.g., in a 20mer oligonucleotide with 8 cationic backbone linkages, having at least 70% of these charged linkages localized in the 10 centermost linkages. 
     The antisense compounds can be prepared by stepwise solid-phase synthesis, employing methods detailed in the references cited above, and below with respect to the synthesis of oligonucleotides having a mixture of uncharged and cationic backbone linkages. In some cases, it may be desirable to add additional chemical moieties to the antisense compound, e.g. to enhance pharmacokinetics or to facilitate capture or detection of the compound. Such a moiety may be covalently attached, typically to a terminus of the oligomer, according to standard synthetic methods. For example, addition of a polyethyleneglycol moiety or other hydrophilic polymer, e.g., one having 10-100 monomeric subunits, may be useful in enhancing solubility. One or more charged groups, e.g., anionic charged groups such as an organic acid, may enhance cell uptake. A reporter moiety, such as fluorescein or a radiolabeled group, may be attached for purposes of detection. Alternatively, the reporter label attached to the oligomer may be a ligand, such as an antigen or biotin, capable of binding a labeled antibody or streptavidin. In selecting a moiety for attachment or modification of an antisense compound, it is generally of course desirable to select chemical compounds of groups that are biocompatible and likely to be tolerated by a subject without undesirable side effects. 
     As noted above, the antisense compound can be constructed to contain a selected number of cationic linkages interspersed with uncharged linkages of the type described above. The intersubunit linkages, both uncharged and cationic, preferably are phosphorus-containing linkages, having the structure (II): 
     
       
         
         
             
             
         
       
     
     wherein: 
     W is —S— or —O—, and is preferably —O—, 
     X=—NR 1 R 2  or —OR 6 , 
     Y═—O— or —NR′, and 
     each said linkage in the oligomer is selected from: 
     (a) an uncharged linkage (a), wherein each of R 1 , R 2 , R 6  and R 7  is independently selected from hydrogen and lower alkyl; 
     (b1) a cationic linkage (b1), wherein X=—NR 1 R 2  and Y=—O—, and —NR 1 R 2  represents an optionally substituted piperazinyl moiety, such that R 1 R 2 =—CHRCHRN(R 3 )(R 4 )CHRCHR—, wherein: 
     each R is independently H or —CH 3 , 
     R 4  is H, —CH 3 , or an electron pair, and 
     R 3  is selected from H, optionally substituted lower alkyl, —C(═NH)NH 2 , —Z-L-NHC(═NH)NH 2 , and [—C(═O)CHR′NH] m H, where: 7 is —C(═O)— or a direct bond, L is an optional linker up to 18 atoms in length, preferably up to 12 atoms, and more preferably up to 8 atoms in length, having bonds selected from optionally substituted alkyl, optionally substituted alkoxy, and optionally substituted alkylamino, R′ is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6, preferably 1 to 4; 
     (b2) a cationic linkage (b2), wherein X=—NR 1 R 2  and Y=—O—, R 1 =H or —CH 3 , and R 2 =LNR 3 R 4 R 5 , wherein L, R 3 , and R 4  are as defined above, and R 5  is H, optionally substituted lower alkyl, or optionally substituted lower (alkoxy)alkyl; and 
     (b3) a cationic linkage (b3), wherein Y=—NR 7  and X=—OR 6 , and R 7 =-LNR 3 R 4 R 5 , wherein L, R 3 , R 4  and R 5  are as defined above, and R 6  is H or optionally substituted lower alkyl; and 
     at least one said linkage is selected from cationic linkages (b1), (b2), and (b3). 
     Preferably, the oligomer includes at least two consecutive linkages of type (a) (i.e. uncharged linkages). In further embodiments, at least 5% of the linkages in the oligomer are cationic linkages (i.e. type (b1), (b2), or (b3)); for example, 10% to 60%, and preferably 20-50% linkages may be cationic linkages. 
     In one embodiment, at least one linkage is of type (b1), where, preferably, each R is H, R 4  is H, —CH 3 , or an electron pair, and R 3  is selected from H, optionally substituted lower alkyl, —C(═NH)NH 2 , and —C(═O)-L-NHC(═NH)NH 2  The latter two embodiments of R 3  provide a guanidino moiety, either attached directly to the piperazine ring, or pendant to a linker group L, respectively. For ease of synthesis, the variable Z in R 3  is preferably —C(═O)—, as shown. 
     The linker group L, as noted above, contains bonds in its backbone selected from optionally substituted alkyl, optionally substituted alkoxy, and optionally substituted alkylamino, wherein the terminal atoms in L (e.g., those adjacent to carbonyl or nitrogen) are carbon atoms. Although branched linkages are possible, the linker is preferably unbranched. In one embodiment, the linker is a linear alkyl linker. Such a linker may have the structure —(CH 2 ) n —, where n is 1-12, preferably 2-8, and more preferably 2-6. 
     The morpholino subunits have the following structure (III): 
     
       
         
         
             
             
         
       
     
     wherein Pi is a base-pairing moiety, and the linkages depicted above connect the nitrogen atom of (III) to the 5 carbon of an adjacent subunit. The base-pairing moieties Pi may be the same or different, and are generally designed to provide a sequence which binds to a target nucleic acid. 
     The use of embodiments of linkage types (b1), (b2) and (b3) above to link morpholino subunits (III) may be illustrated graphically as follows: 
     
       
         
         
             
             
         
       
     
     Preferably, all cationic linkages in the oligomer are of the same type; i.e. all of type (b1), all of type (b2), or all of type (b3). 
     In further embodiments, the cationic linkages are selected from linkages (b1″) and (b1″) as shown below, where (b1′) is referred to herein as a “Pip” linkage and (b1″) is referred to herein as a “GuX” linkage: 
     
       
         
         
             
             
         
       
     
     In the structures above, W is S or O, and is preferably O; each of R 1  and R 2  is independently selected from hydrogen and optionally substituted lower alkyl, and is preferably methyl; and A represents hydrogen or a non-interfering substituent (i.e. a substituent that does not adversely affect the ability of an oligomer to bind to its intended target) on one or more carbon atoms in (b1′) and (b1″). Preferably, the ring carbons in the piperazine ring are unsubstituted; however, the ring carbons of the piperazine ring may include non-interfering substituents, such as methyl or fluorine. Preferably, at most one or two carbon atoms is so substituted. 
     In further embodiments, at least 10% of the linkages are of type (b1′) or (b1″); for example, 10%-60% and preferably 20% to 50%, of the linkages may be of type (b1′) or (b1″). 
     In other embodiments, the oligomer contains no linkages of the type (b1′) above. Alternatively, the oligomer contains no linkages of type (b1) where each R is H, R 3  is H or —CH 3 , and R 4  is H, —CH 3 , or an electron pair. 
     The morpholino subunits may also be linked by non-phosphorus-based intersubunit linkages, as described further below, where at least one linkage is modified with a pendant cationic group as described above. 
     Other oligonucleotide analog linkages which are uncharged in their unmodified state but which could also bear a pendant amine substituent could be used. For example, a 5′nitrogen atom on a morpholino ring could be employed in a sulfamide linkage or a urea linkage (where phosphorus is replaced with carbon or sulfur, respectively) and modified in a manner analogous to the 5′-nitrogen atom in structure (b3) above. 
     Oligomers having any number of cationic linkages are provided, including fully cationic-linked oligomers. Preferably, however, the oligomers are partially charged, having, for example, 10%-80%. In preferred embodiments, about 10% to 60%, and preferably 20% to 50% of the linkages are cationic. 
     In one embodiment, the cationic linkages are interspersed along the backbone. The partially charged oligomers preferably contain at least two consecutive uncharged linkages; that is, the oligomer preferably does not have a strictly alternating pattern along its entire length. 
     Also considered are oligomers having blocks of cationic linkages and blocks of uncharged linkages; for example, a central block of uncharged linkages may be flanked by blocks of cationic linkages, or vice versa. In one embodiment, the oligomer has approximately equal-length 5′, 3′ and center regions, and the percentage of cationic linkages in the center region is greater than about 50%, preferably greater than about 70%. 
     Oligomers for use in antisense applications generally range in length from about 10 to about 40 subunits, more preferably about 10 to 30 subunits, and typically 15-25 bases, including those having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases. In certain embodiments, an oligomer of the invention having 19-20 subunits, a useful length for an antisense compound, may ideally have two to ten, e.g., four to eight, cationic linkages, and the remainder uncharged linkages. An oligomer having 14-15 subunits may ideally have two to seven, e.g., 3 to 5, cationic linkages, and the remainder uncharged linkages. 
     Each morpholino ring structure supports a base pairing moiety, to form a sequence of base pairing moieties which is typically designed to hybridize to a selected antisense target in a cell or in a subject being treated. The base pairing moiety may be a purine or pyrimidine found in native DNA or RNA (e.g., A, G, C, T or U) or an analog, such as hypoxanthine (the base component of the nucleoside inosine) or 5-methyl cytosine. 
     Peptide Transporters 
     The antisense compounds of the invention may include an oligonucleotide moiety conjugated to an arginine-rich peptide transport moiety effective to enhance transport of the compound into cells. The transport moiety is preferably attached to a terminus of the oligomer, as shown, for example, in  FIGS. 1B and 1C . The peptide transport moiety preferably comprises 6 to 16 subunits selected from X′ subunits, Y′ subunits, and Z′ subunits, wherein: 
     (a) each X′ subunit independently represents lysine, arginine or an arginine analog, said analog being a cationic α-amino acid comprising a side chain of the structure R 1 N═C(NH 2 )R 2 , where R 1  is H or R; R 2  is R, —NH 2 , —NHR, or —NR 2 , where R is optionally substituted lower alkyl or optionally substituted lower alkenyl; R 1  and R 2  may join together to form a ring; and the side chain is linked to said amino acid via R 1  or R 2 ; 
     (b) each Y′ subunit independently represents a neutral amino acid —C(═O)—(CHR) n —NH—, where n is 2 to 7 and each R is independently H or methyl; and 
     (c) each subunit independently represents an α-amino acid having a neutral aralkyl side chain; 
     wherein the peptide comprises a sequence represented by at least one of (X′Y′X′) p , (X′Y′) m , and/or (X′Z′Z′) p , where p is 2 to 5 and m is 2 to 8. Certain embodiments include various combinations selected independently from (X′Y′X′) p , (X′Y′) m , and/or (X′Z′Z′) p , including, for example, peptides having the sequence (X′Y′X′)(X′Z′Z′)(X′Y′X′)(X′Z′Z′) (SEQ ID NO:637). 
     In selected embodiments, for each X′, the side chain moiety is guanidyl, as in the amino acid subunit arginine (Arg). In certain embodiments, each Y is independently —C(═O)—(CH 2 ) n —CHR—NH—, where n is 2 to 7 and R is H. For example, when n is 5 and R is H, Y′ is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx; when n is 2 and R is H, is a β-alanine subunit, abbreviated herein as B. Certain embodiments relate to carrier peptides having a combination of different neutral amino acids, including, for example, peptides comprising the sequence —RahxRRBRRAhxRRBRAhxB— (SEQ ID NO:578), which contains both β-alanine and 6-aminohexanoic acid. 
     Preferred peptides of this type include those comprising arginine dimers alternating with single Y′ subunits, where Y′ is preferably Ahx or B or both. Examples include peptides having the formula (RY′R) p  and/or the formula (RRY′) p , where p is 1 to 2 to 5 and where Y′ is preferably Ahx. In one embodiment, Y′ is a 6-aminohexanoic acid subunit, R is arginine and p is 4. Certain embodiments include various linear combinations of at least two of (RY′R) p  and (RRY′) p , including, for example, illustrative peptides having the sequence (RY′R)(RRY′)(RY′R)(RRY′) (SEQ ID NO:638), or (RRY′)(RY′R)(RRY′) (SEQ ID NO:639). Other combinations are contemplated. In a further illustrative embodiment, each Z′ is phenylalanine, and m is 3 or 4. 
     The conjugated peptide is preferably linked to a terminus of the oligomer via a linker Ahx-B, where Ahx is a 6-aminohexanoic acid subunit and B is a β-alanine subunit, as shown, for example, in  FIGS. 1B and 1C . 
     In selected embodiments, for each X′, the side chain moiety is independently selected from the group consisting of guanidyl (HN═C(NH 2 )NH—), amidinyl (HN═C(NH 2 )CH—), 2-aminodihydropyrimidyl, 2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl, and it is preferably selected from guanidyl and amidinyl. In one embodiment, the side chain moiety is guanidyl, as in the amino acid subunit arginine (Arg). 
     In certain embodiments, the Y′ subunits may be contiguous, in that no X′ subunits intervene between Y′ subunits, or interspersed singly between X′ subunits. In certain embodiments, the linking subunit may be between Y′ subunits. In one embodiment, the Y′ subunits are at a terminus of the transporter; in other embodiments, they are flanked by X′ subunits. In further preferred embodiments, each Y′ is —C(═O)—(CH 2 ) n —CHR—NH—, where n is 2 to 7 and R is H. For example, when n is 5 and R is H, Y′ is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx. In selected embodiments of this group, each X′ comprises a guanidyl side chain moiety, as in an arginine subunit. Preferred peptides of this type include those comprising arginine dimers alternating with single Y′ subunits, where Y′ is preferably Ahx. Examples include peptides having the formula (RY′R) 4  or the formula (RRY′) 4  where Y′ is preferably Ahx. In the latter case, the nucleic acid analog is preferably linked to a terminal Y′ subunit, preferably at the C-terminus, as shown, for example, in  FIGS. 1B and 1C . The preferred linker is of the structure AhxB, where Ahx is a 6-aminohexanoic acid subunit and B is a β-alanine subunit. 
     The transport moieties as described above have been shown to greatly enhance cell entry of attached oligomers, relative to uptake of the oligomer in the absence of the attached transport moiety, and relative to uptake by an attached transport moiety lacking the hydrophobic subunits Y′. Such enhanced uptake is preferably evidenced by at least a two-fold increase, and preferably a four-fold increase, in the uptake of the compound into mammalian cells relative to uptake of the agent by an attached transport moiety lacking the hydrophobic subunits Y′. Uptake is preferably enhanced at least twenty fold, and more preferably forty fold, relative to the unconjugated compound. 
     A further benefit of the transport moiety is its expected ability to stabilize a duplex between an antisense compound and its target nucleic acid sequence, presumably by virtue of electrostatic interaction between the positively charged transport moiety and the negatively charged nucleic acid. The number of charged subunits in the transporter is less than 14, as noted above, and preferably between 8 and 11, since too high a number of charged subunits may lead to a reduction in sequence specificity. 
     The use of arginine-rich peptide transporters (i.e., cell-penetrating peptides) is particularly useful in practicing the present invention. Certain peptide transporters have been shown to be highly effective at delivery of antisense compounds into primary cells including muscle cells (Marshall, Oda et al. 2007; Jearawiriyapaisarn, Moulton et al. 2008; Wu, Moulton et al. 2008). Furthermore, compared to other peptide transporters such as Penetratin and the Tat peptide, the peptide transporters described herein, when conjugated to an antisense PMO, demonstrate an enhanced ability to alter splicing of several gene transcripts (Marshall, Oda et al. 2007). Especially preferred are the P007, CP06062 and CP04057 transport peptides listed below in Table 3 (SEQ ID NOS: 573, 578 and 577, respectively). 
     Exemplary peptide transporters, including linkers (B or AhxB) are given below in Table B below. Preferred sequences are those designated CP06062 (SEQ ID NO: 578), P007 (SEQ ID NO: 573) and CP04057 (SEQ ID NO: 577). 
     
       
         
           
               
             
               
                 TABLE B 
               
             
            
               
                   
               
               
                 Exemplary Peptide Transporters for Intracellular 
               
               
                 Delivery of PMO 
               
            
           
           
               
               
               
            
               
                   
                 Sequence 
                 SEQ ID 
               
               
                 Peptide 
                 (N-terminal to C-terminal) 
                 NO: 
               
               
                   
               
               
                 rTAT 
                 RRRQRRKKRC 
                 570 
               
               
                   
               
               
                 R 9 F 2   
                 RRRRRRRRRFFC 
                 571 
               
               
                   
               
               
                 (RRAhx) 4 B 
                 RRAhxRRAhxRRAhxRRAhxB 
                 572 
               
               
                   
               
               
                 (RAhxR) 4 AhxB; 
                 RAhxRRAhxRRAhxRRAhxRAhxB 
                 573 
               
               
                 (P007) 
                   
                   
               
               
                   
               
               
                 (AhxRR) 4 AhxB 
                 AhxRRAhxRRAhxRRAhxRRAhxB 
                 574 
               
               
                   
               
               
                 (RAhx) 6 B 
                 RAhxRAhxRAhxRAhxRAhxRAhxB 
                 575 
               
               
                   
               
               
                 (RAhx) 8 B 
                 RAhxRAhxRAhxRAhxRAhxRAhxR 
                 576 
               
               
                   
                 AhxB 
                   
               
               
                   
               
               
                 (RAhxR) 5 AhxB 
                 RAhxRRAhxRRAhxRRAhxRRAhxR 
                 577 
               
               
                 (CP05057) 
                 AhxB 
                   
               
               
                   
               
               
                 (RAhxRRBR) 2 AhxB; 
                 RAhxRRBRRAhxRRBRAhxB 
                 578 
               
               
                 (CP06062) 
                   
                   
               
               
                   
               
               
                 MSP 
                 ASSLNIA 
                 579 
               
               
                   
               
            
           
         
       
     
     Formulations 
     In certain embodiments, the present invention provides formulations or compositions suitable for the therapeutic delivery of antisense oligomers, as described herein. Hence, in certain embodiments, the present invention provides pharmaceutically acceptable compositions that comprise a therapeutically-effective amount of one or more of the oligomers described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. While it is possible for an oligomer of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). 
     Methods for the delivery of nucleic add molecules are described, for example, in Akhtar et al., 1992 , Trends Cell Bio.,  2:139; and  Delivery Strategies for Antisense Oligonucleotide Therapeutics , ed. Akhtar; Sullivan el al., PCT WO 94/02595. These and other protocols can be utilized for the delivery of virtually any nucleic acid molecule, including the isolated oligomers of the present invention. 
     As detailed below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually: (6) ocularly; (7) transdermally; or (8) nasally. 
     The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. 
     The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. 
     Some examples of materials that can serve as pharmaceutically-acceptable carriers include, without limitation: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose; ethyl cellulose and cellulose acetate; (4) powdered tragacanth: (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils; such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer&#39;s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. 
     Additional non-limiting examples of agents suitable for formulation with the antisense oligomers of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues; biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, D F et al., 1999 , Cell Transplant,  8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms ( Prog Neuropsychopharmacol Biol Psychiatry,  23, 941-949, 1999). 
     The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, branched and unbranched or combinations thereof, or long-circulating liposomes or stealth liposomes). Oligomers of the invention can also comprise covalently attached PEG molecules of various molecular weights. These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al.  Chem. Rev.  1995, 95, 2601-2627; Ishiwata et al.,  Chem, Pharm. Bull.  1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995 , Biochim. Biophys. Acta,  1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. 
     In a further embodiment, the present invention incudes oligomer compositions prepared for delivery as described in U.S. Pat. Nos. 6,692,911, 7,163,695 and 7,070,807. In this regard, in one embodiment, the present invention provides an oligomer of the present invention in a composition comprising copolymers of lysine and histidine (HK) as described in U.S. Pat. Nos. 7,163,695, 7,070,807, and 6,692,911 either alone or in combination with PEG (e.g., branched or unbranched PEG or a mixture of both), in combination with PEG and a targeting moiety or any of the foregoing in combination with a crosslinking agent. In certain embodiments, the present invention provides antisense oligomers in compositions comprising gluconic-acid-modified polyhistidine or gluconylated-polyhistidine/transferrin-polylysine. One skilled in the art will also recognize that amino acids with properties similar to His and Lys may be substituted within the composition. 
     Certain embodiments of the oligomers described herein may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term “pharmaceutically-acceptable salts” in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). 
     The pharmaceutically acceptable salts of the subject oligomers include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. 
     In certain embodiments, the oligomers of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically-acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, e.g., Berge et al., supra). 
     Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. 
     Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic add, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric add, and the like. 
     Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. 
     In certain embodiments, a formulation of the present invention comprises an excipient selected from cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and an oligomer of the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable an oligomer of the present invention. 
     Methods of preparing these formulations or compositions include the step of bringing into association an oligomer of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. 
     Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. An oligomer of the present invention may also be administered as a bolus, electuary or paste. 
     In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules, trouches and the like), the active ingredient may be mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic add; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamer and sodium lauryl sulfate; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic add, and mixtures thereof; (10) coloring agents; and (11) controlled release agents such as crospovidone or ethyl cellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. 
     A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (e.g., gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. 
     The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. 
     Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. 
     Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. 
     Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. 
     Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. 
     Formulations or dosage forms for the topical or transdermal administration of an oligomer as provided herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active oligomers may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. 
     Powders and sprays can contain, in addition to an oligomer of the present invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. 
     Transdermal patches have the added advantage of providing controlled delivery of an oligomer of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the oligomer in the proper medium. Absorption enhancers can also be used to increase the flux of the agent across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the agent in a polymer matrix or gel, among other methods known in the art. 
     Pharmaceutical compositions suitable for parenteral administration may comprise one or more oligomers of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. 
     These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject oligomers may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. 
     In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility, among other methods known in the art. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. 
     Injectable depot forms may be made by forming microencapsule matrices of the subject oligomers in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of oligomer to polymer, and the nature of the particular polymer employed, the rate of oligomer release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. 
     When the oligomers of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier. 
     As noted above, the formulations or preparations of the present invention may be given orally, parenterally, topically, or rectally. They are typically given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. 
     The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. 
     The phrases “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient&#39;s system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. 
     Regardless of the route of administration selected, the oligomers of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, may be formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being unacceptably toxic to the patient. 
     The selected dosage level will depend upon a variety of factors including the activity of the particular oligomer of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular oligomer being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular oligomer employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. 
     A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day. 
     If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain situations, dosing is one administration per day. In certain embodiments, dosing is one or more administration per every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to maintain the desired expression of a functional dystrophin protein. 
     Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, as described herein and known in the art. In certain embodiments, microemulsification technology may be utilized to improve bioavailability of lipophilic (water insoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo, S. K., et al.,  Drug Development and Industrial Pharmacy,  17(12), 1685-1713, 1991 and REV 5901 (Sheen, P. C., et al.,  J Pharm Sci  80(7), 712-714, 1991). Among other benefits, microemulsification provides enhanced bioavailability by preferentially directing absorption to the lymphatic system instead of the circulatory system, which thereby bypasses the liver, and prevents destruction of the compounds in the hepatobiliary circulation. 
     In one aspect of invention, the formulations contain micelles formed from an oligomer as provided herein and at least one amphiphilic carrier, in which the micelles have an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter less than about 30 nm, or even less than about 20 nm. 
     While all suitable amphiphilic carriers are contemplated, the presently preferred carriers are generally those that have Generally-Recognized-as-Safe (GRAS) status, and that can both solubilize the compound of the present invention and microemulsify it at a later stage when the solution comes into a contact with a complex water phase (such as one found in human gastro-intestinal tract). Usually, amphiphilic ingredients that satisfy these requirements have HLB (hydrophilic to lipophilic balance) values of 2-20, and their structures contain straight chain aliphatic radicals in the range of C-6 to C-20. Examples are polyethylene-glycolized fatty glycerides and polyethylene glycols. 
     Examples of amphiphilic carriers include saturated and monounsaturated polyethyleneglycolyzed fatty acid glycerides, such as those obtained from fully or partially hydrogenated various vegetable oils. Such oils may advantageously consist of tri-, di-, and mono-fatty acid glycerides and di- and mono-polyethyleneglycol esters of the corresponding fatty acids, with a particularly preferred fatty acid composition including capric acid 4-10, capric acid 3-9, lauric acid 40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%. Another useful class of amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, with saturated or mono-unsaturated fatty acids (SPAN-series) or corresponding ethoxylated analogs (TWEEN-series). 
     Commercially available amphiphilic carriers may be particularly useful, including Gelucire-series, Labrafil, Labrasol, or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurate and di-laurate. Lecithin, Polysorbate 80, etc (produced and distributed by a number of companies in USA and worldwide). 
     In certain embodiments, the delivery may occur by use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present invention into suitable host cells. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticle or the like. The formulation and use of such delivery vehicles can be carried out using known and conventional techniques. 
     Hydrophilic polymers suitable for use in the present invention are those which are readily water-soluble, can be covalently attached to a vesicle-forming lipid, and which are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolic acid copolymer, and polyvinyl alcohol. In certain embodiments, polymers have a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, or from about 300 daltons to about 5,000 daltons. In other embodiments, the polymer is polyethyleneglycol having a molecular weight of from about 100 to about 5,000 daltons, or having a molecular weight of from about 300 to about 5,000 daltons. In certain embodiments, the polymer is polyethyleneglycol of 750 daltons (PEG(750)). Polymers may also be defined by the number of monomers therein; a preferred embodiment of the present invention utilizes polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons). 
     Other hydrophilic polymers which may be suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose. 
     In certain embodiments, a formulation of the present invention comprises a biocompatible polymer selected from the group consisting of polyamides polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, celluloses, polypropylene, polyethylenes, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or copolymers thereof. 
     Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8 glucose units, designated by the Greek letter α, β, or γ, respectively. The glucose units are linked by α-1,4-glucosidic bonds. As a consequence of the chair conformation of the sugar units, all secondary hydroxyl groups (at C-2, C-3) are located on one side of the ring, while all the primary hydroxyl groups at C-6 are situated on the other side. As a result, the external faces are hydrophilic, making the cyclodextrins water-soluble. In contrast, the cavities of the cyclodextrins are hydrophobic, since they are lined by the hydrogen of atoms C-3 and C-5, and by ether-like oxygens. These matrices allow complexation with a variety of relatively hydrophobic compounds, including, for instance, steroid compounds such as 17α-estradiol (see, e.g., van Uden et al. Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). The complexation takes place by Van der Waals interactions and by hydrogen bond formation. For a general review of the chemistry of cyclodextrins, see, Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994). 
     The physico-chemical properties of the cyclodextrin derivatives depend strongly on the kind and the degree of substitution. For example, their solubility in water ranges from insoluble (e.g., triacetyl-beta-cyclodextrin) to 147% soluble (w/v) (G-2-beta-cyclodextrin). In addition, they are soluble in many organic solvents. The properties of the cyclodextrins enable the control over solubility of various formulation components by increasing or decreasing their solubility. 
     Numerous cyclodextrins and methods for their preparation have been described. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259) and Gramera, et al, (U.S. Pat. No. 3,459,731) described electroneutral cyclodextrins. Other derivatives include cyclodextrins with cationic properties [Parmeter (II), U.S. Pat. No. 3,453,257], insoluble crosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), and cyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No. 3,426,011]. Among the cyclodextrin derivatives with anionic properties, carboxylic acids, phosphorous acids, phosphinous adds, phosphonic acids, phosphoric acids, thiophosphonic adds, thiosulphinic acids, and sulfonic acids have been appended to the parent cyclodextrin [see, Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrin derivatives have been described by Stella, et al. (U.S. Pat. No. 5,134,127). 
     Liposomes consist of at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes may be characterized by membrane type and by size. Small unilamellar vesicles (SUVs) have a single membrane and typically range between 0.02 and 0.05 μm in diameter; large unilamellar vesicles (LUVS) are typically larger than 0.05 μM. Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes with several nonconcentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are termed multivesicular vesicles. 
     One aspect of the present invention relates to formulations comprising liposomes containing an oligomer of the present invention, where the liposome membrane is formulated to provide a liposome with increased carrying capacity. Alternatively or in addition, the compound of the present invention may be contained within, or adsorbed onto, the liposome bilayer of the liposome. An oligomer of the present invention may be aggregated with a lipid surfactant and carried within the liposome&#39;s internal space; in these cases, the liposome membrane is formulated to resist the disruptive effects of the active agent-surfactant aggregate. 
     According to one embodiment of the present invention, the lipid bilayer of a liposome contains lipids derivatized with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space encapsulated by the liposome, and extend from the exterior of the lipid bilayer into the surrounding environment. 
     Active agents contained within liposomes of the present invention are in solubilized form. Aggregates of surfactant and active agent (such as emulsions or micelles containing the active agent of interest) may be entrapped within the interior space of liposomes according to the present invention. A surfactant acts to disperse and solubilize the active agent, and may be selected from any suitable aliphatic, cycloaliphatic or aromatic surfactant, including but not limited to biocompatible lysophosphatidylcholines (LPCs) of varying chain lengths (for example, from about C14 to about C20). Polymer-derivatized lipids such as PEG-lipids may also be utilized for micelle formation as they will act to inhibit micelle/membrane fusion, and as the addition of a polymer to surfactant molecules decreases the CMC of the surfactant and aids in micelle formation. Preferred are surfactants with CMCs in the micromolar range; higher CMC surfactants may be utilized to prepare micelles entrapped within liposomes of the present invention. 
     Liposomes according to the present invention may be prepared by any of a variety of techniques that are known in the art. See, e.g., U.S. Pat. No. 4,235,871; Published PCT applications WO 96/14057; New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104; Lasic D D, Liposomes from physics to applications, Elsevier Science Publishers BV, Amsterdam, 1993. For example, liposomes of the present invention may be prepared by diffusing a lipid derivatized with a hydrophilic polymer into preformed liposomes, such as by exposing preformed liposomes to micelles composed of lipid-grafted polymers, at lipid concentrations corresponding to the final mole percent of derivatized lipid which is desired in the liposome. Liposomes containing a hydrophilic polymer can also be formed by homogenization, lipid-field hydration, or extrusion techniques, as are known in the art. 
     In another exemplary formulation procedure, the active agent is first dispersed by sonication in a lysophosphatidylcholine or other low CMC surfactant (including polymer grafted lipids) that readily solubilizes hydrophobic molecules. The resulting micellar suspension of active agent is then used to rehydrate a dried lipid sample that contains a suitable mole percent of polymer-grafted lipid, or cholesterol. The lipid and active agent suspension is then formed into liposomes using extrusion techniques as are known in the art, and the resulting liposomes separated from the unencapsulated solution by standard column separation. 
     In one aspect of the present invention, the liposomes are prepared to have substantially homogeneous sizes in a selected size range. One effective sizing method involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membrane will correspond roughly with the largest sizes of liposomes produced by extrusion through that membrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988). In certain embodiments, reagents such as DharmaFECT® and Lipofectamine® may be utilized to introduce polynucleotides or proteins into cells. 
     The release characteristics of a formulation of the present invention depend on the encapsulating material, the concentration of encapsulated drug, and the presence of release modifiers. For example, release can be manipulated to be pH dependent, for example, using a pH sensitive coating that releases only at a low pH, as in the stomach, or a higher pH, as in the intestine. An enteric coating can be used to prevent release from occurring until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach, followed by later release in the intestine. Release can also be manipulated by inclusion of salts or pore forming agents, which can increase water uptake or release of drug by diffusion from the capsule. Excipients which modify the solubility of the drug can also be used to control the release rate. Agents which enhance degradation of the matrix or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as particulates), or can be co-dissolved in the polymer phase depending on the compound. In most cases the amount should be between 0.1 and thirty percent (w/w polymer). Types of degradation enhancers include inorganic salts such as ammonium sulfate and ammonium chloride, organic adds such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine and surfactants such as Tween® and Pluronic®. Pore forming agents which add microstructure to the matrices (i.e., water soluble compounds such as inorganic salts and sugars) are added as particulates. The range is typically between one and thirty percent (w/w polymer). 
     Uptake can also be manipulated by altering residence time of the particles in the gut. This can be achieved, for example, by coating the particle with, or selecting as the encapsulating material, a mucosal adhesive polymer. Examples include most polymers with free carboxyl groups, such as chitosan, celluloses, and especially polyacrylates (as used herein, polyacrylates refers to polymers including acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates). 
     An oligomer may be formulated to be contained within, or, adapted to release by a surgical or medical device or implant. In certain aspects, an implant may be coated or otherwise treated with an oligomer. For example, hydrogels, or other polymers, such as biocompatible and/or biodegradable polymers, may be used to coat an implant with the compositions of the present invention (i.e., the composition may be adapted for use with a medical device by using a hydrogel or other polymer). Polymers and copolymers for coating medical devices with an agent are well-known in the art. Examples of implants include, but are not limited to, stents, drug-eluting stents, sutures, prosthesis, vascular catheters, dialysis catheters, vascular grafts, prosthetic heart valves, cardiac pacemakers, implantable cardioverter defibrillators, IV needles, devices for bone setting and formation, such as pins, screws, plates, and other devices, and artificial tissue matrices for wound healing. 
     In addition to the methods provided herein, the oligomers for use according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals. The antisense oligomers and their corresponding formulations may be administered alone or in combination with other therapeutic strategies in the treatment of muscular dystrophy, such as myoblast transplantation, stem cell therapies, administration of aminoglycoside antibiotics, proteasome inhibitors, and up-regulation therapies (e.g., upregulation of utrophin, an autosomal paralogue of dystrophin). 
     All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. 
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. 
     REFERENCES 
     
         
         Aartsma-Rus, A., A. A. Janson, et al. (2004). “Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense.” Am J Hum Genet 74(1): 83-92. 
         Dunckley, M. G., I. C. Eperon, et al. (1997). “Modulation of splicing in the DMD gene by antisense oligoribonucleotides.” Nucleosides &amp; Nucleotides 16(7-9): 1665-1668. 
         Dunckley, M. G., M. Manoharan, et al. (1998). “Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides.” Hum Mol Genet 7(7): 1083-90. 
         Errington, S. J., C. J. Mann, et al. (2003), “Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene.” J Gene Med. 5 (6): 518-27. 
         Jearawiriyapaisarn, N., H. M. Moulton, et al. (2008). “Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice.” Mol Ther. 
         Lu, Q. L., C. J. Mann, et al, (2003). “Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse.” Nat Med 9(8): 1009-14. 
         Mann, C. J., K. Honeyman, et al. (2002), “Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy.” J Gene Med 4(6): 644-54. 
         Marshall, N. B., S. K. Oda, et al, (2007). “Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing.” Journal of Immunological Methods 325(1-2): 114-126. 
         Matsuo, M., T. Masumura, et al. (1991). “Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe.” J Clin Invest 87(6): 2127-31. 
         Monaco, A. P., C. J. Bertelson, et al. (1988). “An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus.” Genomics 2(1): 90-5. 
         Pramono, 7. A., Y. Takeshima, et al. (1996). “Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence.” Biochem Biophys Res Commun 226(2): 445-9. 
         Sazani, P., R. Kole, et al. (2007). Splice switching oligomers for the TNF superfamily receptors and their use in treatment of disease. PCT WO2007058894, University of North Carolina 
         Sierakowska, H., M. J. Sambade, et al. (1996). “Repair of thalassemic human beta-globin mRNA in mammalian cells by antisense oligonucleotides.” Proc Natl Acad Sci USA 93(23): 12840-4. 
         Summerton, J. and D. Weller (1997). “Morpholino antisense oligomers: design, preparation, and properties.” Antisense Nucleic Acid Drug Dev 7(3): 187-95. 
         Takeshima, Y., H. Nishio, et al. (1995). “Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.” J Clin Invest 95(2): 515-20. 
         van Deutekom, J. C., M. Bremmer-Bout, et al, (2001). “Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells.” Hum Mol Genet 10(15): 1547-54. 
         van Deutekom, J. C., A. A. Janson, et al. (2007). “Local dystrophin restoration with antisense oligonucleotide PROO51.” N Engl J Med 357(26): 2677-86. 
         Wilton, S. D., A. M. Fail, et al. (2007). “Antisense oligonucleotide-induced exon skipping across the human dystrophin gene transcript.” Mol Ther 15(7): 1288-96. 
         Wilton, S. D., F. Lloyd, et al. (1999). “Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides.” Neuromuscul Disord 9(5): 330-8, 
         Wu, B., H. M. Moulton, et al. (2008). “Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer.” Proc Natl Acad Sci USA 105(39): 14814-9. 
         Yin, H., H. M. Moulton, et al. (2008). “Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function.” Hum Mol Genet 17(24): 3909-18. 
       
    
     EXAMPLES 
     Materials and Methods 
     Cells and Tissue Culture Treatment Conditions 
     Human Rhabdomyosarcoma cells (ATCC, CCL-136; RD cells) preserved in a 5% DMSO solution (Sigma) at a low passage number were thawed in a 37° C. water bath until the ice sliver was no longer visible. Cells were seeded into tissue culture-treated T75 flasks (Nunc) at 1.5×10 6  cells/flask in 24 mL of warmed DMEM with L-Glutamine (HyClone), 10% fetal bovine serum, and 1% Penicillin-Streptomycin antibiotic solution (CelGro); after 24 hours, media was aspirated, cells were washed once in warmed PBS, and fresh media was added. Cells were grown to 80% confluence in a 37° C. incubator at 5.0% CO2. 
     Media was aspirated from T75 flasks; cells were washed once in warmed PBS and aspirated. 3 mL of Trypsin/EDTA, warmed in a 37° C. water bath, was added to each T75. Cells were incubated at 37° C. 5 2-5 minutes until, with gentle agitation, they released from the flask. Cell suspension was transferred to a 15.0 mL conical tube; flasks were rinsed with 1.0 mL of Trypsin/EDTA solution to gather remaining cells. Cells were counted with a Vi-Cell XR cell counter (Beckman Coulter). Cells were seeded into tissue culture-treated 12-well plates (Falcon) at 2.0×10 5  viable cells per well in 1.0 mL media. Cells were incubated overnight in a 37° C. incubator at 5.0% CO 2 . 
     Twelve-well seeded plates were examined for even cellular distribution and plate adherence. Lyophilized peptide conjugated phosphorodiamidate morpholino oligomers (PPMOs) were re-suspended at 2.0 mM in nuclease-free water (Ambion), and kept on ice during cell treatment; to verify molarity, PPMOs were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Immediately prior to PPMO treatment, media was aspirated, and cells were rinsed in warmed PBS. PPMOs were diluted in warmed media to the desired molarity; cells were treated in a total of 1.0 mL PPMO per well. PPMOs were tested in triplicate. For no-treatment controls, fresh, warmed media was added in 1.0 mL total volume. Cells were incubated for 48 hours in a 37*C incubator at 5.0% CO2. 
     RNA Extraction 
     Media was aspirated, and cells were rinsed in warmed PBS. RNA was extracted with the QuickGene-Mini80 system, QuickGene RNA cultured cell HC kit 5, and MagNAlyser with ceramic bead homogenization using the manufacturers&#39; recommended protocols. Briefly, cells were lysed in treatment plates with 350 uL LRP (10 uL β-Mercaptoethanol added per 100 uL LRP) lysis buffer; homogenate was gently triturated to ensure full lysis, and transferred to MagNAlyser tubes. Tubes were spun at 2800 rpm for 30 seconds in the MagNAlyser to ensure full homogenization, and iced briefly. 50 uL SRP solubilization buffer was added and homogenate was vortexed for 15 seconds. 170 uL&gt;99% ethanol was added to each tube, and homogenate was vortexed for 60 seconds. Homogenate was flash-spun and transferred to Mini80 RNA cartridges, samples were pressurized and flow-through was discarded. Cartridges were washed in 750 uL WRP wash buffer and pressurized. 40 uL of DNase solution (1.25 uL Qiagen DNaseI, 35 uL RDD Buffer, 3.75 uL nuclease-free water) was added directly to the cartridge membrane; cartridges were incubated four minutes at room temperature. Cartridges were washed twice with 750 uL WRP, pressurizing after each wash. Cartridges were placed over nuclease-free tubes. 50 uL CRP elution buffer was added to each membrane; membranes were incubated for five minutes at room-temperature. Cartridges were pressurized and eluate was collected. RNA was stored at −80° C. pending quantification. RNA was quantified using the NanoDrop™ 2000 spectrophotometer. 
     Nested RT-PCR 
     Primer-specific, exon-specific, optimized nested RT-PCR amplification was performed using the primer pair sets for each dystrophin exon as shown below in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Primer pair sets used to PCR amplify 
               
               
                 human dystrophin mRNA to detect exon-skipping. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 SEQ 
               
               
                 Name 
                 F/R 
                 I/0 
                 Sequence (5′-3′) 
                 Exon 
                 Purpose 
                 ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 PS170 
                 F 
                 O 
                 CCAGAGCTTTACCTGAGAAACAAG 
                 48 
                 Detection 
                 640 
               
               
                 PS172 
                 F 
                 I 
                 CCAGCCACTCAGCCAGTGAAG 
                 49 
                 of Exon 50 
                 641 
               
               
                 PS174 
                 R 
                 I 
                 CGATCCGTAATGATTGTTCTAGCC 
                 52 
                 and 51 Skipping in 
                 642 
               
               
                 PS176 
                 R 
                 O 
                 CATTTCATTCAACTGTTGCCTCCG 
                 53 
                 Human Dystrophin 
                 643 
               
               
                   
               
               
                 PS186 
                 F 
                 O 
                 CAATGCTCCTGACCTCTGTGC 
                 42 
                 Detection 
                 644 
               
               
                 PS187 
                 F 
                 I 
                 GTCTACAACAAAGCTCAGGTCG 
                 43 
                 of Exon 44 
                 645 
               
               
                 PS189 
                 F 
                 I 
                 GCAATGTTATCTGCTTCCTCCAACC 
                 46 
                 and 45 Skipping in 
                 646 
               
               
                 PS190 
                 R 
                 O 
                 GCTCTTTTCCAGGTTCAAGTGG 
                 46 
                 Human Dystrophin 
                 647 
               
               
                   
               
               
                 PS192 
                 F 
                 O 
                 CTTGGACAGAACTTACCGACTGG 
                 51 
                 Detection 
                 648 
               
               
                 PS193 
                 F 
                 I 
                 GCAGGATTTGGAACAGAGGCG 
                 52 
                 of Exon 53 
                 649 
               
               
                 PS195 
                 R 
                 I 
                 CATCTACATTTGTCTGCCACTGG 
                 54 
                 Skipping in 
                 650 
               
               
                 PS197 
                 R 
                 O 
                 GTTTCTTCCAAAGCAGCCTCTCG 
                 55 
                 Human Dystrophin 
                 651 
               
               
                   
               
            
           
         
       
     
     The indicated primer pairs are shown as either forward or reverse (F/R) and either outer or inner primer pairs (I/O) corresponding to primary or secondary amplifications, respectively. The location of the primer target is indicated in the Exon column and the Purpose indicates the exon-skipping events can be detected. For example, PS170 and PS176 primers amplify a region from exon 48 to 53 in the primary amplification. Primers PS172 and PS174 then amplify a region from exon 49 to 52 in the secondary amplication. This nested PCR reaction will detect exon skipping of both exons 50 and/or exon 51. The specific nested RT-PCR reaction conditions are provided below. 
     RNA extracted from treated cells (described above) was diluted to 20 ng/ul for all samples. 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Reaction setup for RT-PCR and primary amplification (50 μl reaction): 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 2× Reaction mix 
                  25 μl 
               
               
                 PS XXX Forward Primer (30 μM) 
                 0.5 μl 
               
               
                 (see Table 1) 
                   
               
               
                 PS XXX Reverse Primer (30 μM) 
                 0.5 μl 
               
               
                 (see Table 1) 
                   
               
               
                 Superscript III Platinum Taq mix 
                   2 μl 
               
               
                 Template RNA (20 ng/μl) 
                  10 μl 
               
               
                 Nuclease-Free Water (50 μl total 
                  12 μl 
               
               
                 volume) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 RT-PCR and primary amplification program: 
               
            
           
           
               
               
               
               
            
               
                   
                 Temperature 
                 Time 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Reverse 
                 55° C. 
                 30 minutes 
                 8 Cycles 
               
               
                 Transcription 
                   
                   
                   
               
               
                 RT Inactivation 
                 94° C. 
                  2 minutes 
                   
               
               
                 Denaturing 
                 94° C. 
                  1 minute 
                   
               
               
                 Annealing 
                 59° C. 
                  1 minute 
                   
               
               
                 Extension 
                 68° C. 
                  1 minute 
                   
               
               
                   
                  4° C. 
                 ∞ 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Reaction setup for nested secondary  
               
               
                 amplification 150 μl reaction: 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 10 × PCR Buffer 
                 5  
                 μl 
               
               
                   
                 dNTP solution (10 mM) 
                 0.5  
                 μl 
               
               
                   
                 50 mM MgCl 
                 1.5  
                 μl 
               
               
                   
                 PS XXX Forward Primer (30 μM) 
                 0.33  
                 μl 
               
               
                   
                 (see Table 1) 
                   
                   
               
               
                   
                 PS XXX Reverse Primer (30 μM) 
                 0.33  
                 μl 
               
               
                   
                 (see Table 1) 
                   
                   
               
               
                   
                 Platinum Taq DNA polymerase 
                 0.2  
                 μl 
               
               
                   
                 0.1 mM Cy5-dCTP 
                 1  
                 μl 
               
               
                   
                 RT-PCR product (from Step 1) 
                 1  
                 μl 
               
               
                   
                 Nuclease-Free Water (50 μl total 
                 40.15  
                 μl 
               
            
           
           
               
               
               
            
               
                   
                 volume) 
                   
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Nested secondary amplification program: 
               
            
           
           
               
               
               
               
            
               
                   
                 Temperature 
                 Time 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Primary 
                 94° C. 
                  3 minutes 
                 28-30 
               
               
                 Denature 
                   
                   
                 Cycles 
               
               
                 Denaturing 
                 94° C. 
                 45 seconds 
                   
               
               
                 Annealing 
                 59° C. 
                 30 seconds 
                   
               
               
                 Extension 
                 68° C. 
                  1 minute 
                   
               
               
                   
                  4° C. 
                 ∞ 
               
               
                   
               
            
           
         
       
     
     Gel Electrophoresis Analysis 
     Ten microliters of 5× Ficoll loading dye was added to each 50 microliter nested RT-PCR reaction. Fifteen microliters of PCR/dye mixture was run on a 10% TBE gel at 300 volts for 30 minutes. After electrophoresis, the gel was washed in diH2O for at least one hour, changing the water every 30 minutes. The gel was then scanned on a Typhoon Trio Variable Mode Imager (GE Healthcare). For exon 44 skipping, the nested RT-PCR product from full-length dystrophin transcript is 571 bp, and 423 by from Exon 44-skipped mRNA (exon 44 is 148 bp). For exon 45, the nested RT-PCR product from full-length dystrophin transcript is 571 bp, and 395 bp from Exon 45-skipped mRNA (exon 45 is 176 bp). For exon 53, the PCR product from full-length dystrophin transcript is 365 bp, and 153 bp from exon 53-skipped mRNA (exon 53 is 212 bp). 
     The gel images were subjected to quantitative analysis by measuring the band intensities of the full-length PCR product compared to the exon-skipped product. In some cases, the percent skipping at a fixed PPMO concentration (e.g., 3 micromolar) was used to determine the relative activity of a series of PPMO to induce exon skipping of a given exon. In other situations, a PPMO dose-range was used to treat cells (e.g., 0.1, 0.3, 1.0, 3.0 and 10 micromolar) and an EC 50  was calculated based on the percent skipping induced at each concentration. 
     Example 1 
     Exon 51 Scan 
     A series of overlapping antisense PPMOs that target human dystrophin exon 51 were designed, synthesized and used to treat either human rhabdomyosarcoma cells (RD cells) or primary human skeletal muscle cells. This strategy is termed an “exon scan” and was used similarly for several other dystrophin exons as described below. All the PPMOs were synthesized as peptide-conjugated PMO (PPMO) using the CP06062 peptide (SEQ ID NO: 578) and a 3′ terminal PMO linkage. For exon 51, a series of 26 PPMOs, each 26 bases in length, were made (SEQ ID NOS: 309-311, 314, 316, 317, 319, 321, 323, 324, 326, 327, 329-331, 333, 335, 336, 338-345) as shown in  FIG. 2A . The PPMOs were evaluated for exon skipping efficacy by treating RD cells at various concentrations as described above in the Materials and Methods. Three PPMOs (SEQ ID NOS: 324, 326 and 327) were identified as effective in inducing exon-skipping and selected for additional evaluation. Dose-ranging experiments in RD cells and primary human skeletal muscle cells were used to confirm the relative efficacy of these three PPMO sequences, SEQ ID NO: 327 was shown to be most effective at inducing exon 51 skipping as shown in  FIGS. 2B and 2C . 
     A comparison of the relative effectiveness of SEQ ID NO: 327 to other exon 51-targeted antisense sequences was performed in RD cells and primary human skeletal muscle cells, as described above. AU the evaluated sequences were made as peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO: 578). This allowed direct comparison of the relative effectiveness of the antisense sequences without regard to antisense chemistry or cell delivery. The relative location of the certain exon 51-targeted oligos compared to SEQ ID NO: 327 is shown in  FIG. 2D . As shown in  FIG. 20 , there is a ranked hierarchy of exon-skipping effectiveness, with SEQ ID NO: 327 being the most effective by at least a factor of several-fold compared to other sequences. 
     Example 2 
     Exon 50 Scan 
     A series of overlapping antisense PPMOs that target human dystrophin exon 50 were designed and synthesized. For exon 50, a series of 17 PPMOs, each 25 bases in length, were made (SEQ ID NOS:267, 269, 271, 273, 275, 277, 279, 280, 282 and 284-291) as shown in  FIG. 3A . The PPMOs were evaluated for exon skipping efficacy by treating RD cells at various concentrations as described above in the Materials and Methods. Four PPMOs (SEQ ID NOS: 277, 287, 290 and 291) were identified as effective in inducing exon-skipping and selected for additional evaluation. Dose-ranging experiments in RD cells were used to confirm the relative efficacy of these four PMO sequences. SEQ ID NOs: 584 (AVI-5656) and 287 (AVI-5038) were shown to be most effective at inducing exon 50 skipping as shown in  FIG. 3B . The EC 50  values were derived from the dose-ranging experiments and represent the calculated concentration where 50% of the PCR product is from the mRNA lacking exon 50 relative to the PCR product produced from the mRNA containing exon 50. Compared to other sequences (see, e.g., SEQ ID NOs: 584 and 585 correspond to SEQ ID NOs: 173 and 175 in WO20061000057, respectively) AVI-5038 (SEQ ID NO: 287) is equivalent or better at inducing exon-skipping activity in the RD cell assay as shown in  FIG. 3B . 
     Example 3 
     Exon 53 Scan 
     A series of overlapping antisense PPMOs that target human dystrophin exon 53 were designed and synthesized. For exon 53, a series of 24 PPMOs, each 25 bases in length, were made (SEQ ID NOS:416, 418, 420, 422, 424, 426, 428, 429, 431, 433, 434, 436, 438-440 and 443-451) as shown in  FIG. 4A . The PPMOs were evaluated for exon skipping efficacy by treating RD cells and primary human skeletal muscle cells at various concentrations as described above in the Materials and Methods. Three PPMOs (SEQ ID NOS: 428, 429 and 431) were identified as effective in inducing exon-skipping and selected for additional evaluation. Dose-ranging experiments in RD cells were used to confirm the relative efficacy of these three PMO sequences. SEQ ID NO: 429 was shown to be most effective at inducing exon 53 skipping as shown in  FIGS. 4B-F . However, when compared to other exon 53 antisense sequences, SEQ ID NO: 429 proved identical to H53A(+23+47) which is listed as SEQ ID NO: 195 in WO2006/000057 and SEQ ID NO: 609 in the present application. Other sequences were compared to SEQ ID NO: 429 including H53A(+39+69) and H53A(−12+10) (listed as SEQ ID NOs:193 and 199 in WO2006/000057, respectively) and h53AON1 (listed as SEQ ID NO:39 in U.S. application Ser. No. 11/233,507) and listed as SEQ ID NOs: 608, 611 and 610, respectively, in the present application. All the evaluated sequences were made as peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO: 578). This allowed direct comparison of the relative effectiveness of the antisense sequences without regard to antisense chemistry or cell delivery. As shown in  FIGS. 4I and 4G -H, SEQ ID NO: 429 was shown to be superior to each of these four sequences. 
     Example 4 
     Exon 44 Scan 
     A series of overlapping antisense PPMOs that target human dystrophin exon 44 were designed and synthesized. For exon 44, a series of PPMOs, each 25 bases in length, were made (SEQ ID NOS:1-20) as shown in  FIG. 5A . The PPMOs were evaluated for exon skipping efficacy by treating RD cells at various concentrations as described above in the Materials and Methods. Five PPMOs (SEQ ID NOS:4, 8, 11, 12 and 13) were identified as effective in inducing exon-skipping and selected for additional evaluation. Dose-ranging experiments in RD cells were used to confirm the relative efficacy of these five PPMO sequences as shown in  FIGS. 5C to 5H . SEQ ID NOs: 8, 11 and 12 were shown to be most effective at inducing exon 44 skipping as shown in  FIG. 5H  with SEQ ID NO:12 proving the most efficacious. 
     Comparison of SEQ ID NO: 12 to other exon 44 antisense sequences was done in both RD cells and human primary skeletal muscle cells. All the evaluated sequences were made as peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO: 578). This allowed direct comparison of the relative effectiveness of the antisense sequences without regard to antisense chemistry or cell delivery. 
     The alignment of the sequences (SEQ ID NOS: 600, 601, 602 and 603) with SEQ ID NOS: 4, 8, 11 and 12 is shown in  FIG. 5B . SEQ D NOS: 601 and 603 are listed as SEQ ID NOS: 165 and 167 in WO2006/000057. SEQ ID NO:602 is listed in WO2004/083446 and as SEQ ID NO: 21 in U.S. application Ser. No. 11/233,507. SEQ ID NO:600 was published in 2007 (Wilton, Fall et al. 2007). The comparison in RD cells showed that both SEQ ID NOS: 602 and 603 were superior to SEQ ID NO:12 ( FIG. 5I ). However, as shown in  FIG. 5J , in human primary skeletal muscle cells SEQ ID NO:12 was superior (8.86% exon skipping) to SEQ ID NO:602 (6.42%). Similar experiments are performed with SEQ ID NO:603. 
     Example 5 
     Exon 45 Scan 
     A series of overlapping antisense PPMOs that target human dystrophin exon 45 were designed and synthesized. For exon 45, a series of 22 PPMOs, each 25 bases in length, were made (SEQ ID NOS: 21, 23, 25, 27, 29, 31, 32, 34, 35, 37, 39, 41, 43 and 45-53) as shown in  FIG. 6A . The PPMOs were evaluated for exon skipping efficacy by treating RD cells and human primary skeletal muscle cells at various concentrations as described above in the Materials and Methods. Five PPMOs (SEQ ID NOS:27, 29, 34, and 39) were identified as effective in inducing exon-skipping and selected for additional evaluation. Dose-ranging experiments in RD cells were used to confirm the relative efficacy of these four PMO sequences as shown in  FIGS. 6C-G  and summarized in  FIG. 6H . SEQ ID NO: 49 was used as a negative control in these experiments. SEQ ID NOs: 29 and 34 were shown to be most effective at inducing exon 45 skipping as shown in  FIG. 6H . 
     Comparison of SEQ ID NO: 34 to other exon 45 antisense sequences was done in both RD cells and human primary skeletal muscle cells. All the evaluated sequences were made as peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO: 578). This allowed direct comparison of the relative effectiveness of the antisense sequences without regard to antisense chemistry or cell delivery. The alignment of the sequences (SEQ ID NOS: 604, 605, 606 and 607) with SEQ ID NOS: 27, 29, 34 and 39 is shown in  FIG. 6B . SEQ ID NOS: 604 and 607 are listed as SEQ ID NOS: 211 and 207 in WO2006/000057, respectively. SEQ ID NOS:605 and 606 are listed in U.S. application Ser. No. 11/233,507 as SEQ ID NOS: 23 and 1, respectively. The comparison in RD cells showed that SEQ ID NO: 34 was superior to all four sequences evaluated as shown in  FIG. 6I . Testing of these compounds in different populations of human primary skeletal muscle cells is performed as described above. 
     SEQUENCE ID LISTING 
     Sequences are shown using the nucleotide base symbols common for DNA: A, G, C and T. Other antisense chemistries such as 2′-O-methyl use U in place of T. Any of the bases may be substituted with inosine (I) especially in stretches of three or more G residues. 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                 Sequences 
                   
               
               
                   
                 Oligomer Targeting 
                   
               
               
                 Name 
                 Sequences (5′ to 3′): 
                 SEQ ID NO. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Hu.DMD.Exon44.25.001 
                 CTGCAGGTAAAAGCATATGGATCAA 
                 1 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.002 
                 ATCGCCTGCAGGTAAAAGCATATGG 
                 2 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.003 
                 GTCAAATCGCCTGCAGGTAAAAGCA 
                 3 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.004 
                 GATCTGTCAAATCGCCTGCAGGTAA 
                 4 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.005 
                 CAACAGATCTGTCAAATCGCCTGCA 
                 5 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.006 
                 TTTCTCAACAGATCTGTCAAATCGC 
                 6 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.007 
                 CCATTTCTCAACAGATCTGTCAAAT 
                 7 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.008 
                 ATAATGAAAACGCCGCCATTTCTCA 
                 8 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.009 
                 AAATATCTTTATATCATAATGAAAA 
                 9 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.010 
                 TGTTAGCCACTGATTAAATATCTTT 
                 10 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.011 
                 AAACTGTTCAGCTTCTGTTAGCCAC 
                 11 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.012 
                 TTGTGTCTTTCTGAGAAACTGTTCA 
                 12 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.013 
                 CCAATTCTCAGGAATTTGTGTCTTT 
                 13 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.014 
                 GTATTTAGCATGTTCCCAATTCTCA 
                 14 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.015 
                 CTTAAGATACCATTTGTATTTAGCA 
                 15 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.016 
                 CTTACCTTAAGATACCATTTGTATT 
                 16 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.017 
                 AAAGACTTACCTTAAGATACCATTT 
                 17 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.018 
                 AAATCAAAGACTTACCTTAAGATAC 
                 18 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.019 
                 AAAACAAATCAAAGACTTACCTTAA 
                 19 
               
               
                   
               
               
                 Hu.DMD.Exon44.25.020 
                 TCGAAAAAACAAATCAAAGACTTAC 
                 20 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.001 
                 CTGTAAGATACCAAAAAGGCAAAAC 
                 21 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.002 
                 CCTGTAAGATACCAAAAAGGCAAAA 
                 22 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.002.2 
                 AGTTCCTGTAAGATACCAAAAAGGC 
                 23 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.003 
                 GAGTTCCTGTAAGATACCAAAAAGG 
                 24 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.003.2 
                 CCTGGAGTTCCTGTAAGATACCAAA 
                 25 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.004 
                 TCCTGGAGTTCCTGTAAGATACCAA 
                 26 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.004.2 
                 GCCATCCTGGAGTTCCTGTAAGATA 
                 27 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.005 
                 TGCCATCCTGGAGTTCCTGTAAGAT 
                 28 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.005.2 
                 CCAATGCCATCCTGGAGTTCCTGTA 
                 29 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.006 
                 CCCAATGCCATCCTGGAGTTCCTGT 
                 30 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.006.2 
                 GCTGCCCAATGCCATCCTGGAGTTC 
                 31 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.007 
                 CGCTGCCCAATGCCATCCTGGAGTT 
                 32 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.008 
                 AACAGTTTGCCGCTGCCCAATGCCA 
                 33 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.008.2 
                 CTGACAACAGTTTGCCGCTGCCCAA 
                 34 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.009 
                 GTTGCATTCAATGTTCTGACAACAG 
                 35 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.010 
                 GCTGAATTATTTCTTCCCCAGTTGC 
                 36 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.010.2 
                 ATTATTTCTTCCCCAGTTGCATTCA 
                 37 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.011 
                 GGCATCTGTTTTTGAGGATTGCTGA 
                 38 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.011.2 
                 TTTGAGGATTGCTGAATTATTTCTT 
                 39 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.012 
                 AATTTTTCCTGTAGAATACTGGCAT 
                 40 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.012.2 
                 ATACTGGCATCTGTTTTTGAGGATT 
                 41 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.013 
                 ACCGCAGATTCAGGCTTCCCAATTT 
                 42 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.013.2 
                 AATTTTTCCTGTAGAATACTGGCAT 
                 43 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.014 
                 CTGTTTGCAGACCTCCTGCCACCGC 
                 44 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.014.2 
                 AGATTCAGGCTTCCCAATTTTTCCT 
                 45 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.015 
                 CTCTTTTTTCTGTCTGACAGCTGTT 
                 46 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.015.2 
                 ACCTCCTGCCACCGCAGATTCAGGC 
                 47 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.016 
                 CCTACCTCTTTTTTCTGTCTGACAG 
                 48 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.016.2 
                 GACAGCTGTTTGCAGACCTCCTGCC 
                 49 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.017 
                 GTCGCCCTACCTCTTTTTTCTGTCT 
                 50 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.018 
                 GATCTGTCGCCCTACCTCTTTTTTC 
                 51 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.019 
                 TATTAGATCTGTCGCCCTACCTCTT 
                 52 
               
               
                   
               
               
                 Hu.DMD.Exon45.25.020 
                 ATTCCTATTAGATCTGTCGCCCTAC 
                 53 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.001 
                 AGATACCAAAAAGGCAAAAC 
                 54 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.002 
                 AAGATACCAAAAAGGCAAAA 
                 55 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.003 
                 CCTGTAAGATACCAAAAAGG 
                 56 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.004 
                 GAGTTCCTGTAAGATACCAA 
                 57 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.005 
                 TCCTGGAGTTCCTGTAAGAT 
                 58 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.006 
                 TGCCATCCTGGAGTTCCTGT 
                 59 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.007 
                 CCCAATGCCATCCTGGAGTT 
                 60 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.008 
                 CGCTGCCCAATGCCATCCTG 
                 61 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.009 
                 CTGACAACAGTTTGCCGCTG 
                 62 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.010 
                 GTTGCATTCAATGTTCTGAC 
                 63 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.011 
                 ATTATTTCTTCCCCAGTTGC 
                 64 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.012 
                 TTTGAGGATTGCTGAATTAT 
                 65 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.013 
                 ATACTGGCATCTGTTTTTGA 
                 66 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.014 
                 AATTTTTCCTGTAGAATACT 
                 67 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.015 
                 AGATTCAGGCTTCCCAATTT 
                 68 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.016 
                 ACCTCCTGCCACCGCAGATT 
                 69 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.017 
                 GACAGCTGTTTGCAGACCTC 
                 70 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.018 
                 CTCTTTTTTCTGTCTGACAG 
                 71 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.019 
                 CCTACCTCTTTTTTCTGTCT 
                 72 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.020 
                 GTCGCCCTACCTCTTTTTTC 
                 73 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.021 
                 GATCTGTCGCCCTACCTCTT 
                 74 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.022 
                 TATTAGATCTGTCGCCCTAC 
                 75 
               
               
                   
               
               
                 Hu.DMD.Exon45.20.023 
                 ATTCCTATTAGATCTGTCGC 
                 76 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.001 
                 GGGGGATTTGAGAAAATAAAATTAC 
                 77 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.002 
                 ATTTGAGAAAATAAAATTACCTTGA 
                 78 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.002.2 
                 CTAGCCTGGAGAAAGAAGAATAAAA 
                 79 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.003 
                 AGAAAATAAAATTACCTTGACTTGC 
                 80 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.003.2 
                 TTCTTCTAGCCTGGAGAAAGAAGAA 
                 81 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.004 
                 ATAAAATTACCTTGACTTGCTCAAG 
                 82 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.004.2 
                 TTTTGTTCTTCTAGCCTGGAGAAAG 
                 83 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.005 
                 ATTACCTTGACTTGCTCAAGCTTTT 
                 84 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.005.2 
                 TATTCTTTTGTTCTTCTAGCCTGGA 
                 85 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.006 
                 CTTGACTTGCTCAAGCTTTTCTTTT 
                 86 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.006.2 
                 CAAGATATTCTTTTGTTCTTCTAGC 
                 87 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.007 
                 CTTTTAGTTGCTGCTCTTTTCCAGG 
                 88 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.008 
                 CCAGGTTCAAGTGGGATACTAGCAA 
                 89 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.008.2 
                 ATCTCTTTGAAATTCTGACAAGATA 
                 90 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.009 
                 AGCAATGTTATCTGCTTCCTCCAAC 
                 91 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.009.2 
                 AACAAATTCATTTAAATCTCTTTGA 
                 92 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.010 
                 CCAACCATAAAACAAATTCATTTAA 
                 93 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.010.2 
                 TTCCTCCAACCATAAAACAAATTCA 
                 94 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.011 
                 TTTAAATCTCTTTGAAATTCTGACA 
                 95 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.012 
                 TGACAAGATATTCTTTTGTTCTTCT 
                 96 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.012.2 
                 TTCAAGTGGGATACTAGCAATGTTA 
                 97 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.013 
                 AGATATTCTTTTGTTCTTCTAGCCT 
                 98 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.013.2 
                 CTGCTCTTTTCCAGGTTCAAGTGGG 
                 99 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.014 
                 TTCTTTTGTTCTTCTAGCCTGGAGA 
                 100 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.014.2 
                 CTTTTCTTTTAGTTGCTGCTCTTTT 
                 101 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.015 
                 TTGTTCTTCTAGCCTGGAGAAAGAA 
                 102 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.016 
                 CTTCTAGCCTGGAGAAAGAAGAATA 
                 103 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.017 
                 AGCCTGGAGAAAGAAGAATAAAATT 
                 104 
               
               
                   
               
               
                 Hu.DMD.Exon46.25.018 
                 CTGGAGAAAGAAGAATAAAATTGTT 
                 105 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.001 
                 GAAAGAAGAATAAAATTGTT 
                 106 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.002 
                 GGAGAAAGAAGAATAAAATT 
                 107 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.003 
                 AGCCTGGAGAAAGAAGAATA 
                 108 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.004 
                 CTTCTAGCCTGGAGAAAGAA 
                 109 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.005 
                 TTGTTCTTCTAGCCTGGAGA 
                 110 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.006 
                 TTCTTTTGTTCTTCTAGCCT 
                 111 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.007 
                 TGACAAGATATTCTTTTGTT 
                 112 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.008 
                 ATCTCTTTGAAATTCTGACA 
                 113 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.009 
                 AACAAATTCATTTAAATCTC 
                 114 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.010 
                 TTCCTCCAACCATAAAACAA 
                 115 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.011 
                 AGCAATGTTATCTGCTTCCT 
                 116 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.012 
                 TTCAAGTGGGATACTAGCAA 
                 117 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.013 
                 CTGCTCTTTTCCAGGTTCAA 
                 118 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.014 
                 CTTTTCTTTTAGTTGCTGCT 
                 119 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.015 
                 CTTGACTTGCTCAAGCTTTT 
                 120 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.016 
                 ATTACCTTGACTTGCTCAAG 
                 121 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.017 
                 ATAAAATTACCTTGACTTGC 
                 122 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.018 
                 AGAAAATAAAATTACCTTGA 
                 123 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.019 
                 ATTTGAGAAAATAAAATTAC 
                 124 
               
               
                   
               
               
                 Hu.DMD.Exon46.20.020 
                 GGGGGATTTGAGAAAATAAA 
                 125 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.001 
                 CTGAAACAGACAAATGCAACAACGT 
                 126 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.002 
                 AGTAACTGAAACAGACAAATGCAAC 
                 127 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.003 
                 CCACCAGTAACTGAAACAGACAAAT 
                 128 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.004 
                 CTCTTCCACCAGTAACTGAAACAGA 
                 129 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.005 
                 GGCAACTCTTCCACCAGTAACTGAA 
                 130 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.006 
                 GCAGGGGCAACTCTTCCACCAGTAA 
                 131 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.007 
                 CTGGCGCAGGGGCAACTCTTCCACC 
                 132 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.008 
                 TTTAATTGTTTGAGAATTCCCTGGC 
                 133 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.008.2 
                 TTGTTTGAGAATTCCCTGGCGCAGG 
                 134 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.009 
                 GCACGGGTCCTCCAGTTTCATTTAA 
                 135 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.009.2 
                 TCCAGTTTCATTTAATTGTTTGAGA 
                 136 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.010 
                 GCTTATGGGAGCACTTACAAGCACG 
                 137 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.010.2 
                 TACAAGCACGGGTCCTCCAGTTTCA 
                 138 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.011 
                 AGTTTATCTTGCTCTTCTGGGCTTA 
                 139 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.012 
                 TCTGCTTGAGCTTATTTTCAAGTTT 
                 140 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.012.2 
                 ATCTTGCTCTTCTGGGCTTATGGGA 
                 141 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.013 
                 CTTTATCCACTGGAGATTTGTCTGC 
                 142 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.013.2 
                 CTTATTTTCAAGTTTATCTTGCTCT 
                 143 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.014 
                 CTAACCTTTATCCACTGGAGATTTG 
                 144 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.014.2 
                 ATTTGTCTGCTTGAGCTTATTTTCA 
                 145 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.015 
                 AATGTCTAACCTTTATCCACTGGAG 
                 146 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.016 
                 TGGTTAATGTCTAACCTTTATCCAC 
                 147 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.017 
                 AGAGATGGTTAATGTCTAACCTTTA 
                 148 
               
               
                   
               
               
                 Hu.DMD.Exon47.25.018 
                 ACGGAAGAGATGGTTAATGTCTAAC 
                 149 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.001 
                 ACAGACAAATGCAACAACGT 
                 150 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.002 
                 CTGAAACAGACAAATGCAAC 
                 151 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.003 
                 AGTAACTGAAACAGACAAAT 
                 152 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.004 
                 CCACCAGTAACTGAAACAGA 
                 153 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.005 
                 CTCTTCCACCAGTAACTGAA 
                 154 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.006 
                 GGCAACTCTTCCACCAGTAA 
                 155 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.007 
                 CTGGCGCAGGGGCAACTCTT 
                 156 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.008 
                 TTGTTTGAGAATTCCCTGGC 
                 157 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.009 
                 TCCAGTTTCATTTAATTGTT 
                 158 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.010 
                 TACAAGCACGGGTCCTCCAG 
                 159 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.011 
                 GCTTATGGGAGCACTTACAA 
                 160 
               
               
                   
               
               
                 Hu.DMD.Exon47.20,012 
                 ATCTTGCTCTTCTGGGCTTA 
                 161 
               
               
                   
               
               
                 Hu.DMD.Exon47 20.013 
                 CTTATTTTCAAGTTTATCTT 
                 162 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.014 
                 ATTTGTCTGCTTGAGCTTAT 
                 163 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.015 
                 CTTTATCCACTGGAGATTTG 
                 164 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.016 
                 CTAACCTTTATCCACTGGAG 
                 165 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.017 
                 AATGTCTAACCTTTATCCAC 
                 166 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.018 
                 TGGTTAATGTCTAACCTTTA 
                 167 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.019 
                 AGAGATGGTTAATGTCTAAC 
                 168 
               
               
                   
               
               
                 Hu.DMD.Exon47.20.020 
                 ACGGAAGAGATGGTTAATGT 
                 169 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.001 
                 CTGAAAGGAAAATACATTTTAAAAA 
                 170 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.002 
                 CCTGAAAGGAAAATACATTTTAAAA 
                 171 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.002.2 
                 GAAACCTGAAAGGAAAATACATTTT 
                 172 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.003 
                 GGAAACCTGAAAGGAAAATACATTT 
                 173 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.003.2 
                 CTCTGGAAACCTGAAAGGAAAATAC 
                 174 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.004 
                 GCTCTGGAAACCTGAAAGGAAAATA 
                 175 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.004.2 
                 TAAAGCTCTGGAAACCTGAAAGGAA 
                 634 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.005 
                 GTAAAGCTCTGGAAACCTGAAAGGA 
                 176 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.005.2 
                 TCAGGTAAAGCTCTGGAAACCTGAA 
                 177 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.006 
                 CTCAGGTAAAGCTCTGGAAACCTGA 
                 178 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.006.2 
                 GTTTCTCAGGTAAAGCTCTGGAAAC 
                 179 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.007 
                 TGTTTCTCAGGTAAAGCTCTGGAAA 
                 180 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.007.2 
                 AATTTCTCCTTGTTTCTCAGGTAAA 
                 181 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.008 
                 TTTGAGCTTCAATTTCTCCTTGTTT 
                 182 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.008 
                 TTTTATTTGAGCTTCAATTTCTCCT 
                 183 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.009 
                 AAGCTGCCCAAGGTCTTTTATTTGA 
                 184 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.010 
                 AGGTCTTCAAGCTTTTTTTCAAGCT 
                 185 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.010.2 
                 TTCAAGCTTTTTTTCAAGCTGCCCA 
                 186 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.011 
                 GATGATTTAACTGCTCTTCAAGGTC 
                 187 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.011.2 
                 CTGCTCTTCAAGGTCTTCAAGCTTT 
                 188 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.012 
                 AGGAGATAACCACAGCAGCAGATGA 
                 189 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.012.2 
                 CAGCAGATGATTTAACTGCTCTTCA 
                 190 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.013 
                 ATTTCCAACTGATTCCTAATAGGAG 
                 191 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.014 
                 CTTGGTTTGGTTGGTTATAAATTTC 
                 192 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.014.2 
                 CAACTGATTCCTAATAGGAGATAAC 
                 193 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.015 
                 CTTAACGTCAAATGGTCCTTCTTGG 
                 194 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.015.2 
                 TTGGTTATAAATTTCCAACTGATTC 
                 195 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.016 
                 CCTACCTTAACGTCAAATGGTCCTT 
                 196 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.016.2 
                 TCCTTCTTGGTTTGGTTGGTTATAA 
                 197 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.017 
                 AGTTCCCTACCTTAACGTCAAATGG 
                 198 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.018 
                 CAAAAAGTTCCCTACCTTAACGTCA 
                 199 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.019 
                 TAAAGCAAAAAGTTCCCTACCTTAA 
                 200 
               
               
                   
               
               
                 Hu.DMD.Exon48.25.020 
                 ATATTTAAAGCAAAAAGTTCCCTAC 
                 201 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.001 
                 AGGAAAATACATTTTAAAAA 
                 202 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.002 
                 AAGGAAAATACATTTTAAAA 
                 203 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.003 
                 CCTGAAAGGAAAATACATTT 
                 204 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.004 
                 GGAAACCTGAAAGGAAAATA 
                 205 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.005 
                 GCTCTGGAAACCTGAAAGGA 
                 206 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.006 
                 GTAAAGCTCTGGAAACCTGA 
                 207 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.007 
                 CTCAGGTAAAGCTCTGGAAA 
                 208 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.008 
                 AATTTCTCCTTGTTTCTCAG 
                 209 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.009 
                 TTTTATTTGAGCTTCAATTT 
                 210 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.010  
                 AAGCTGCCCAAGGTCTTTTA 
                 211 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.011  
                 TTCAAGCTTTTTTTCAAGCT 
                 212 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.012 
                 CTGCTCTTCAAGGTCTTCAA 
                 213 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.013 
                 CAGCAGATGATTTAACTGCT 
                 214 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.014 
                 AGGAGATAACCACAGCAGCA 
                 215 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.015 
                 CAACTGATTCCTAATAGGAG 
                 216 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.016 
                 TTGGTTATAAATTTCCAACT 
                 217 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.017 
                 TCCTTCTTGGTTTGGTTGGT 
                 218 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.018 
                 CTTAACGTCAAATGGTCCTT 
                 219 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.019 
                 CCTACCTTAACGTCAAATGG 
                 220 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.020 
                 AGTTCCCTACCTTAACGTCA 
                 221 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.021  
                 CAAAAAGTTCCCTACCTTAA 
                 222 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.022 
                 TAAAGCAAAAAGTTCCCTAC 
                 223 
               
               
                   
               
               
                 Hu.DMD.Exon48.20.023  
                 ATATTTAAAGCAAAAAGTTC 
                 224 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.001 
                 CTGGGGAAAAGAACCCATATAGTGC 
                 225 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.002  
                 TCCTGGGGAAAAGAACCCATATAGT 
                 226 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.002.2 
                 GTTTCCTGGGGAAAAGAACCCATAT 
                 227 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.003  
                 CAGTTTCCTGGGGAAAAGAACCCAT 
                 228 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.003.2 
                 TTTCAGTTTCCTGGGGAAAAGAACC 
                 229 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.004 
                 TATTTCAGTTTCCTGGGGAAAAGAA 
                 230 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.004.2 
                 TGCTATTTCAGTTTCCTGGGGAAAA 
                 231 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.005 
                 ACTGCTATTTCAGTTTCCTGGGGAA 
                 232 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.005.2 
                 TGAACTGCTATTTCAGTTTCCTGGG 
                 233 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.006 
                 CTTGAACTGCTATTTCAGTTTCCTG 
                 234 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.006.2 
                 TAGCTTGAACTGCTATTTCAGTTTC 
                 235 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.007  
                 TTTAGCTTGAACTGCTATTTCAGTT 
                 236 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.008 
                 TTCCACATCCGGTTGTTTAGCTTGA 
                 237 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.009 
                 TGCCCTTTAGACAAAATCTCTTCCA 
                 238 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.009.2 
                 TTTAGACAAAATCTCTTCCACATCC 
                 239 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.010  
                 GTTTTTCCTTGTACAAATGCTGCCC 
                 240 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.010.2 
                 GTACAAATGCTGCCCTTTAGACAAA 
                 241 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.011 
                 CTTCACTGGCTGAGTGGCTGGTTTT 
                 242 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.011.2  
                 GGCTGGTTTTTCCTTGTACAAATGC 
                 243 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.012 
                 ATTACCTTCACTGGCTGAGTGGCTG 
                 244 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.013 
                 GCTTCATTACCTTCACTGGCTGAGT 
                 245 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.014 
                 AGGTTGCTTCATTACCTTCACTGGC 
                 246 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.015 
                 GCTAGAGGTTGCTTCATTACCTTCA 
                 247 
               
               
                   
               
               
                 Hu.DMD.Exon49.25.016 
                 ATATTGCTAGAGGTTGCTTCATTAC 
                 248 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.001 
                 GAAAAGAACCCATATAGTGC 
                 249 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.002 
                 GGGAAAAGAACCCATATAGT 
                 250 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.003 
                 TCCTGGGGAAAAGAACCCAT 
                 251 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.004  
                 CAGTTTCCTGGGGAAAAGAA 
                 252 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.005  
                 TATTTCAGTTTCCTGGGGAA 
                 253 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.006  
                 ACTGCTATTTCAGTTTCCTG 
                 254 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.007 
                 CTTGAACTGCTATTTCAGTT 
                 255 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.008 
                 TTTAGCTTGAACTGCTATTT 
                 256 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.009  
                 TTCCACATCCGGTTGTTTAG 
                 257 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.010 
                 TTTAGACAAAATCTCTTCCA 
                 258 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.011  
                 GTACAAATGCTGCCCTTTAG 
                 259 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.012  
                 GGCTGGTTTTTCCTTGTACA 
                 260 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.013 
                 CTTCACTGGCTGAGTGGCTG 
                 261 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.014 
                 ATTACCTTCACTGGCTGAGT 
                 262 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.015  
                 GCTTCATTACCTTCACTGGC 
                 263 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.016 
                 AGGTTGCTTCATTACCTTCA 
                 264 
               
               
                   
               
               
                 Hu.DMD.Exon49.20,017 
                 GCTAGAGGTTGCTTCATTAC 
                 265 
               
               
                   
               
               
                 Hu.DMD.Exon49.20.018 
                 ATATTGCTAGAGGTTGCTTC 
                 266 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.001 
                 CTTTAACAGAAAAGCATACACATTA 
                 267 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.002 
                 TCCTCTTTAACAGAAAAGCATACAC 
                 268 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.002.2 
                 TTCCTCTTTAACAGAAAAGCATACA 
                 269 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.003 
                 TAACTTCCTCTTTAACAGAAAAGCA 
                 270 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.003.2 
                 CTAACTTCCTCTTTAACAGAAAAGC 
                 271 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.004  
                 TCTTCTAACTTCCTCTTTAACAGAA 
                 272 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.004.2 
                 ATCTTCTAACTTCCTCTTTAACAGA 
                 273 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.005 
                 TCAGATCTTCTAACTTCCTCTTTAA 
                 274 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.005.2 
                 CTCAGATCTTCTAACTTCCTCTTTA 
                 275 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.006 
                 AGAGCTCAGATCTTCTAACTTCCTC 
                 276 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.006.2 
                 CAGAGCTCAGATCTTCTAACTTCCT 
                 277 
               
               
                   
               
               
                 NG-08-0731 
                   
                   
               
               
                 Hu.DMD.Exon50.25.007 
                 CACTCAGAGCTCAGATCTTCTACT 
                 278 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.007.2 
                 CCTTCCACTCAGAGCTCAGATCTTC 
                 279 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.008  
                 GTAAACGGTTTACCGCCTTCCACTC 
                 280 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.009 
                 CTTTGCCCTCAGCTCTTGAAGTAAA 
                 281 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.009.2 
                 CCCTCAGCTCTTGAAGTAAACGGTT 
                 282 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.010 
                 CCAGGAGCTAGGTCAGGCTGCTTTG 
                 283 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.010.2 
                 GGTCAGGCTGCTTTGCCCTCAGCTC 
                 284 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.011 
                 AGGCTCCAATAGTGGTCAGTCCAGG 
                 285 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.011.2 
                 TCAGTCCAGGAGCTAGGTCAGGCTG 
                 286 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.012 
                 CTTACAGGCTCCAATAGTGGTCAGT 
                 287 
               
               
                   
               
               
                 AVI-5038 
                   
                   
               
               
                 Hu.DMD.Exon50.25.013 
                 GTATACTTACAGGCTCCAATAGTGG 
                 288 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.014 
                 ATCCAGTATACTTACAGGCTCCAAT 
                 289 
               
               
                   
               
               
                 Hu.DMD.Exon50.25.015 
                 ATGGGATCCAGTATACTTACAGGCT 
                 290 
               
               
                   
               
               
                 NG-08-0741 
                   
                   
               
               
                 Hu.DMD.Exon50.25.016 
                 AGAGAATGGGATCCAGTATACTTAC 
                 291 
               
               
                   
               
               
                 NG-08-0742 
                   
                   
               
               
                 Hu.DMD.Exon50.20.001  
                 ACAGAAAAGCATACACATTA 
                 292 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.002  
                 TTTAACAGAAAAGCATACAC 
                 293 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.003 
                 TCCTCTTTAACAGAAAAGCA 
                 294 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.004 
                 TAACTTCCTCTTTAACAGAA 
                 295 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.005  
                 TCTTCTAACTTCCTCTTTAA 
                 296 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.006 
                 TCAGATCTTCTAACTTCCTC 
                 297 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.007 
                 CCTTCCACTCAGAGCTCAGA 
                 298 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.008 
                 GTAAACGGTTTACCGCCTTC 
                 299 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.009 
                 CCCTCAGCTCTTGAAGTAAA 
                 300 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.010 
                 GGTCAGGCTGCTTTGCCCTC 
                 301 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.011 
                 TCAGTCCAGGAGCTAGGTCA 
                 302 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.012 
                 AGGCTCCAATAGTGGTCAGT 
                 303 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.013  
                 CTTACAGGCTCCAATAGTGG 
                 304 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.014  
                 GTATACTTACAGGCTCCAAT 
                 305 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.015 
                 ATCCAGTATACTTACAGGCT 
                 306 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.016  
                 ATGGGATCCAGTATACTTAC 
                 307 
               
               
                   
               
               
                 Hu.DMD.Exon50.20.017 
                 AGAGAATGGGATCCAGTATA 
                 308 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.001-44  
                 CTAAAATATTTTGGGTTTTTGCAAAA 
                 309 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.002-45 
                 GCTAAAATATTTTGGGTTTTTGCAAA 
                 310 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.002.2-46 
                 TAGGAGCTAAAATATTTTGGGTTTTT 
                 311 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.003 
                 AGTAGGAGCTAAAATATTTTGGGTT 
                 312 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.003.2 
                 TGAGTAGGAGCTAAAATATTTTGGG 
                 313 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.004  
                 CTGAGTAGGAGCTAAAATATTTTGGG 
                 314 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.004.2 
                 CAGTCTGAGTAGGAGCTAAAATATT 
                 315 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.005  
                 ACAGTCTGAGTAGGAGCTAAAATATT 
                 316 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.005.2 
                 GAGTAACAGTCTGAGTAGGAGCTAAA 
                 317 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.006 
                 CAGAGTAACAGTCTGAGTAGGAGCT 
                 318 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.006.2 
                 CACCAGAGTAACAGTCTGAGTAGGAG 
                 319 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.007 
                 GTCACCAGAGTAACAGTCTGAGTAG 
                 320 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.007.2  
                 AACCACAGGTTGTGTCACCAGAGTAA 
                 321 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.008  
                 GTTGTGTCACCAGAGTAACAGTCTG 
                 322 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.009 
                 TGGCAGTTTCCTTAGTAACCACAGGT 
                 323 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.010  
                 ATTTCTAGTTTGGAGATGGCAGTTTC 
                 324 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.010.2 
                 GGAAGATGGCATTTCTAGTTTGGAG 
                 325 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.011  
                 CATCAAGGAAGATGGCATTTCTAGTT 
                 326 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.011.2 
                 GAGCAGGTACCTCCAACATCAAGGAA 
                 327 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.012 
                 ATCTGCCAGAGCAGGTACCTCCAAC 
                 328 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.013 
                 AAGTTCTGTCCAAGCCCGGTTGAAAT 
                 329 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.013.2 
                 CGGTTGAAATCTGCCAGAGCAGGTAC 
                 330 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.014 
                 GAGAAAGCCAGTCGGTAAGTTCTGTC 
                 331 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.014.2 
                 GTCGGTAAGTTCTGTCCAAGCCCGG 
                 332 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.015 
                 ATAACTTGATCAAGCAGAGAAAGCCA 
                 333 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.015.2  
                 AAGCAGAGAAAGCCAGTCGGTAAGT 
                 334 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.016  
                 CACCCTCTGTGATTTTATAACTTGAT 
                 335 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.017  
                 CAAGGTCACCCACCATCACCCTCTGT 
                 336 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.017.2 
                 CATCACCCTCTGTGATTTTATAACT 
                 337 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.018 
                 CTTCTGCTTGATGATCATCTCGTTGA 
                 338 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.019  
                 CCTTCTGCTTGATGATCATCTCGTTG 
                 339 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.019.2 
                 ATCTCGTTGATATCCTCAAGGTCACC 
                 340 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.020 
                 TCATACCTTCTGCTTGATGATCATCT 
                 341 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.020.2 
                 TCATTTTTTCTCATACCTTCTGCTTG 
                 342 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.021 
                 TTTTCTCATACCTTCTGCTTGATGAT 
                 343 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.022 
                 TTTTATCATTTTTTCTCATACCTTCT 
                 344 
               
               
                   
               
               
                 Hu.DMD.Exon51.25.023  
                 CCAACTTTTATCATTTTTTCTCATAC 
                 345 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.001 
                 ATATTTTGGGTTTTTGCAAA 
                 346 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.002 
                 AAAATATTTTGGGTTTTTGC 
                 347 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.003 
                 GAGCTAAAATATTTTGGGTT 
                 348 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.004 
                 AGTAGGAGCTAAAATATTTT 
                 349 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.005 
                 GTCTGAGTAGGAGCTAAAAT 
                 350 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.006 
                 TAACAGTCTGAGTAGGAGCT 
                 351 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.007 
                 CAGAGTAACAGTCTGAGTAG 
                 352 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.008 
                 CACAGGTTGTGTCACCAGAG 
                 353 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.009 
                 AGTTTCCTTAGTAACCACAG 
                 354 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.010 
                 TAGTTTGGAGATGGCAGTTT 
                 355 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.011 
                 GGAAGATGGCATTTCTAGTT 
                 356 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.012 
                 TACCTCCAACATCAAGGAAG 
                 357 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.013 
                 ATCTGCCAGAGCAGGTACCT 
                 358 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.014 
                 CCAAGCCCGGTTGAAATCTG 
                 359 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.015 
                 GTCGGTAAGTTCTGTCCAAG 
                 360 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.016 
                 AAGCAGAGAAAGCCAGTCGG 
                 361 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.017 
                 TTTTATAACTTGATCAAGCA 
                 362 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.018 
                 CATCACCCTCTGTGATTTTA 
                 363 
               
               
                   
               
               
                 Hu.DMD.Exon51 20.019 
                 CTCAAGGTCACCCACCATCA 
                 364 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.020 
                 CATCTCGTTGATATCCTCAA 
                 365 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.021 
                 CTTCTGCTTGATGATCATCT 
                 366 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.022 
                 CATACCTTCTGCTTGATGAT 
                 367 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.023 
                 TTTCTCATACCTTCTGCTTG 
                 368 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.024 
                 CATTTTTTCTCATACCTTCT 
                 369 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.025 
                 TTTATCATTTTTTCTCATAC 
                 370 
               
               
                   
               
               
                 Hu.DMD.Exon51.20.026 
                 CAACTMATCATTTTTTCT 
                 371 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.001  
                 CTGTAAGAACAAATATCCCTTAGTA 
                 372 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.002 
                 TGCCTGTAAGAACAAATATCCCTTA 
                 373 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.002.2 
                 GTTGCCTGTAAGAACAAATATCCCT 
                 374 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.003 
                 ATTGTTGCCTGTAAGAACAAATATC 
                 375 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.003.2 
                 GCATTGTTGCCTGTAAGAACAAATA 
                 376 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.004 
                 CCTGCATTGTTGCCTGTAAGAACAA 
                 377 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.004.2 
                 ATCCTGCATTGTTGCCTGTAAGAAC 
                 378 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.005 
                 CAAATCCTGCATTGTTGCCTGTAAG 
                 379 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.005.2 
                 TCCAAATCCTGCATTGTTGCCTGTA 
                 380 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.006 
                 TGTTCCAAATCCTGCATTGTTGCCT 
                 381 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.006.2 
                 TCTGTTCCAAATCCTGCATTGTTGC 
                 382 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.007 
                 AACTGGGGACGCCTCTGTTCCAAAT 
                 383 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.007.2 
                 GCCTCTGTTCCAAATCCTGCATTGT 
                 384 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.008 
                 CAGCGGTAATGAGTTCTTCCAACTG 
                 385 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.008.2 
                 CTTCCAACTGGGGACGCCTCTGTTC 
                 386 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.009 
                 CTTGTTTTTCAAATTTTGGGCAGCG 
                 387 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.010 
                 CTAGCCTCTTGATTGCTGGTCTTGT 
                 388 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.010.2 
                 TTTTCAAATTTTGGGCAGCGGTAAT 
                 389 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.011 
                 TTCGATCCGTAATGATTGTTCTAGC 
                 390 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.011.2 
                 GATTGCTGGTCTTGTTTTTCAAATT 
                 391 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.012 
                 CTTACTTCGATCCGTAATGATTGTT 
                 392 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.012.2 
                 TTGTTCTAGCCTCTTGATTGCTGGT 
                 393 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.013 
                 AAAAACTTACTTCGATCCGTAATGA 
                 394 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.014 
                 TGTTAAAAAACTTACTTCGATCCGT 
                 395 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.015 
                 ATGCTTGTTAAAAAACTTACTTCGA 
                 396 
               
               
                   
               
               
                 Hu.DMD.Exon52.25.016 
                 GTCCCATGCTTGTTAAAAAACTTAC 
                 397 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.001 
                 AGAACAAATATCCCTTAGTA 
                 398 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.002 
                 GTAAGAACAAATATCCCTTA 
                 399 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.003 
                 TGCCTGTAAGAACAAATATC 
                 400 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.004 
                 ATTGTTGCCTGTAAGAACAA 
                 401 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.005 
                 CCTGCATTGTTGCCTGTAAG 
                 402 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.006 
                 CAAATCCTGCATTGTTGCCT 
                 403 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.007 
                 GCCTCTGTTCCAAATCCTGC 
                 404 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.008 
                 CTTCCAACTGGGGACGCCTC 
                 405 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.009 
                 CAGCGGTAATGAGTTCTTCC 
                 406 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.010 
                 TTTTCAAATTTTGGGCAGCG 
                 407 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.011 
                 GATTGCTGGTCTTGTTTTTC 
                 408 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.012 
                 TTGTTCTAGCCTCTTGATTG 
                 409 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.013  
                 TTCGATCCGTAATGATTGTT 
                 410 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.014 
                 CTTACTTCGATCCGTAATGA 
                 411 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.015 
                 AAAAACTTACTTCGATCCGT 
                 412 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.016 
                 TGTTAAAAAACTTACTTCGA 
                 413 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.017 
                 ATGCTTGTTAAAAAACTTAC 
                 414 
               
               
                   
               
               
                 Hu.DMD.Exon52.20.018 
                 GTCCCATGCTTGTTAAAAAA 
                 415 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.001 
                 CTAGAATAAAAGGAAAAATAAATAT 
                 416 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.002 
                 AACTAGAATAAAAGGAAAAATAAAT 
                 417 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.002.2 
                 TTCAACTAGAATAAAAGGAAAAATA 
                 418 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.003 
                 CTTTCAACTAGAATAAAAGGAAAAA 
                 419 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.003.2 
                 ATTCTTTCAACTAGAATAAAAGGAA 
                 420 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.004 
                 GAATTCTTTCAACTAGAATAAAAGG 
                 421 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.004.2 
                 TCTGAATTCTTTCAACTAGAATAAA 
                 422 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.005 
                 ATTCTGAATTCTTTCAACTAGAATA 
                 423 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.005.2  
                 CTGATTCTGAATTCTTTCAACTAGA 
                 424 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.006 
                 CACTGATTCTGAATTCTTTCAACTA 
                 425 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.006.2 
                 TCCCACTGATTCTGAATTCTTTCAA 
                 426 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.007  
                 CATCCCACTGATTCTGAATTCTTTC 
                 427 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.008 
                 TACTTCATCCCACTGATTCTGAATT 
                 428 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.008.2 
                 CTGAAGGTGTTCTTGTACTTCATCC 
                 429 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.009 
                 CGGTTCTGAAGGTGTTCTTGTACT 
                 430 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.009.2  
                 CTGTTGCCTCCGGTTCTGAAGGTGT 
                 431 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.010 
                 TTTCATTCAACTGTTGCCTCCGGTT 
                 432 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.010.2 
                 TAACATTTCATTCAACTGTTGCCTC 
                 433 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.011 
                 TTGTGTTGAATCCTTTAACATTTCA 
                 434 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.012  
                 TCTTCCTTAGCTTCCAGCCATTGTG 
                 435 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.012.2 
                 CTTAGCTTCCAGCCATTGTGTTGAA 
                 436 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.013 
                 GTCCTAAGACCTGCTCAGCTTCTTC 
                 437 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.013.2 
                 CTGCTCAGCTTCTTCCTTAGCTTCC 
                 438 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.014 
                 CTCAAGCTTG GCTCTGGCCTGTCCT 
                 439 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.014.2 
                 GGCCTGTCCTAAGACCTGCTCAGCT 
                 440 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.015 
                 TAGGGACCCTCCTTCCATGACTCAA 
                 441 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.016  
                 TTTGGATTGCATCTACTGTATAGGG 
                 442 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.016.2 
                 ACCCTCCTTCCATGACTCAAGCTTG 
                 443 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.017 
                 CTTGGTTTCTGTGATTTTCTTTTGG 
                 444 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.017.2 
                 ATCTACTGTATAGGGACCCTCCTTC 
                 445 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.018 
                 CTAACCTTGGTTTCTGTGATTTTCT 
                 446 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.018.2 
                 TTTCTTTTGGATTGCATCTACTGTA 
                 447 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.019  
                 TGATACTAACCTTGGTTTCTGTGAT 
                 448 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.020 
                 ATCTTTGATACTAACCTTGGTTTCT 
                 449 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.021 
                 AAGGTATCTTTGATACTAACCTTGG 
                 450 
               
               
                   
               
               
                 Hu.DMD.Exon53.25.022 
                 TTAAAAAGGTATCTTTGATACTAAC 
                 451 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.001 
                 ATAAAAGGAAAAATAAATAT 
                 452 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.002 
                 GAATAAAAGGAAAAATAAAT 
                 453 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.003 
                 AACTAGAATAAAAGGAAAAA 
                 454 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.004  
                 CTTTCAACTAGAATAAAAGG 
                 455 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.005 
                 GAATTCTTTCAACTAGAATA 
                 456 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.006 
                 ATTCTGAATTCTTTCAACTA 
                 457 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.007 
                 TACTTCATCCCACTGATTCT 
                 458 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.008 
                 CTGAAGGTGTTCTTGTACT 
                 459 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.009  
                 CTGTTGCCTCCGGTTCTGAA 
                 460 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.010 
                 TAACATTTCATTCAACTGTT 
                 461 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.011 
                 TTGTGTTGAATCCTTTAACA 
                 462 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.012 
                 CTTAGCTTCCAGCCATTGTG 
                 463 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.013 
                 CTGCTCAGCTTCTTCCTTAG 
                 464 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.014 
                 GGCCTGTCCTAAGACCTGCT 
                 465 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.015  
                 CTCAAGCTTGGCTCTGGCCT 
                 466 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.016 
                 ACCCTCCTTCCATGACTCAA 
                 467 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.017 
                 ATCTACTGTATAGGGACCCT 
                 468 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.018 
                 TTTCTTTTGGATTGCATCTA 
                 469 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.019 
                 CTTGGTTTCTGTGATTTTCT 
                 470 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.020 
                 CTAACCTTGGTTTCTGTGAT 
                 471 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.021 
                 TGATACTAACCTTGGTTTCT 
                 472 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.022 
                 ATCTTTGATACTAACCTTGG 
                 473 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.023 
                 AAGGTATCTTTGATACTAAC 
                 474 
               
               
                   
               
               
                 Hu.DMD.Exon53.20.024 
                 TTAAAAAGGTATCTTTGATA 
                 475 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.001 
                 CTATAGATTTTTATGAGAAAGAGA 
                 476 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.002 
                 AACTGCTATAGATTTTTATGAGAAA 
                 477 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.003 
                 TGGCCAACTGCTATAGATTTTTATG 
                 478 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.004 
                 GTCTTTGGCCAACTGCTATAGATTT 
                 479 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.005 
                 CGGAGGTCTTTGGCCAACTGCTATA 
                 480 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.006 
                 ACTGGCGGAGGTCTTTGGCCAACTG 
                 481 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.007 
                 TTTGTCTGCCACTGGCGGAGGTCTT 
                 482 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.008 
                 AGTCATTTGCCACATCTACATTTGT 
                 483 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.008.2 
                 TTTGCCACATCTACATTTGTCTGCC 
                 484 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.009 
                 CCGGAGAAGTTTCAGGGCCAAGTCA 
                 485 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.010 
                 GTATCATCTGCAGAATAATCCCGGA 
                 486 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.010.2 
                 TAATCCCGGAGAAGTTTCAGGGCCA 
                 487 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.011 
                 TTATCATGTGGACTTTTCTGGTATC 
                 488 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.012 
                 AGAGGCATTGATATTCTCTGTTATC 
                 489 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.012.2 
                 ATGTGGACTTTTCTGGTATCATCTG 
                 490 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.013  
                 CTTTTATGAATGCTTCTCCAAGAGG 
                 491 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.013.2 
                 ATATTCTCTGTTATCATGTGGACTT 
                 492 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.014 
                 CATACCTTTTATGAATGCTTCTCCA 
                 493 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.014.2 
                 CTCCAAGAGGCATTGATATTCTCTG 
                 494 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.015 
                 TAATTCATACCTTTTATGAATGCTT 
                 495 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.015.2 
                 CTTTTATGAATGCTTCTCCAAGAGG 
                 496 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.016 
                 TAATGTAATTCATACCTTTTATGAA 
                 497 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.017 
                 AGAAATAATGTAATTCATACCTTTT 
                 498 
               
               
                   
               
               
                 Hu.DMD.Exon54.25.018 
                 GTTTTAGAAATAATGTAATTCATAC 
                 499 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.001 
                 GATTTTTATGAGAAAGAGA 
                 500 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.002 
                 CTATAGATTTTTATGAGAAA 
                 501 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.003 
                 AACTGCTATAGATTTTTATG 
                 502 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.004  
                 TGGCCAACTGCTATAGATTT 
                 503 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.005 
                 GTCTTTGGCCAACTGCTATA 
                 504 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.006 
                 CGGAGGTCTTTGGCCAACTG 
                 505 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.007 
                 TTTGTCTGCCACTGGCGGAG 
                 506 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.008 
                 TTTGCCACATCTACATTTGT 
                 507 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.009 
                 TTCAGGGCCAAGTCATTTGC 
                 508 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.010  
                 TAATCCCGGAGAAGTTTCAG 
                 509 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.011 
                 GTATCATCTGCAGAATAATC 
                 510 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.012 
                 ATGTGGACTTTTCTGGTATC 
                 511 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.013  
                 ATATTCTCTGTTATCATGTG 
                 512 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.014 
                 CTCCAAGAGGCATTGATATT 
                 513 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.015  
                 CTTTTATGAATGCTTCTCCA 
                 514 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.016 
                 CATACCTTTTATGAATGCTT 
                 515 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.017 
                 TAATTCATACCTTTTATGAA 
                 516 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.018 
                 TAATGTAATTCATACCTTTT 
                 517 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.019 
                 AGAAATAATGTAATTCATAC 
                 518 
               
               
                   
               
               
                 Hu.DMD.Exon54.20.020 
                 GTTTTAGAAATAATGTAATT 
                 519 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.001  
                 CTGCAAAGGACCAAATGTTCAGATG 
                 520 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.002 
                 TCACCCTGCAAAGGACCAAATGTTC 
                 521 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.003  
                 CTCACTCACCCTGCAAAGGACCAAA 
                 522 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.004 
                 TCTCGCTCACTCACCCTGCAAAGGA 
                 523 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.005 
                 CAGCCTCTCGCTCACTCACCCTGCA 
                 524 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.006  
                 CAAAGCAGCCTCTCGCTCACTCACC 
                 525 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.007 
                 TCTTCCAAAGCAGCCTCTCGCTCAC 
                 526 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.007.2 
                 TCTATGAGTTTCTTCCAAAGCAGCC 
                 527 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.008  
                 GTTGCAGTAATCTATGAGTTTCTTC 
                 528 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.008.2 
                 GAACTGTTGCAGTAATCTATGAGTT 
                 529 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.009 
                 TTCCAGGTCCAGGGGGAACTGTTGC 
                 530 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.010 
                 GTAAGCCAGGCAAGAAACTTTTCCA 
                 531 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.010.2 
                 CCAGGCAAGAAACTTTTCCAGGTCC 
                 532 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.011  
                 TGGCAGTTGTTTCAGCTTCTGTAAG 
                 533 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.011.2 
                 TTCAGCTTCTGTAAGCCAGGCAAGA 
                 635 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.012  
                 GGTAGCATCCTGTAGGACATTGGCA 
                 534 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.012.2 
                 GACATTGGCAGTTGTTTCAGCTTCT 
                 535 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.013 
                 TCTAGGAGCCTTTCCTTACGGGTAG 
                 536 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.014 
                 CTTTTACTCCCTTGGAGTCTTCTAG 
                 537 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.014.2 
                 GAGCCTTTCCTTACGGGTAGCATCC 
                 538 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.015  
                 TTGCCATTGTTTCATCAGCTCTTTT 
                 539 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.015.2 
                 CTTGGAGTCTTCTAGGAGCCTTTCC 
                 540 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.016 
                 CTTACTTGCCATTGTTTCATCAGCT 
                 541 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.016.2 
                 CAGCTCTTTTACTCCCTTGGAGTCT 
                 542 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.017 
                 CCTGACTTACTTGCCATTGTTTCAT 
                 543 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.018 
                 AAATGCCTGACTTACTTGCCATTGT 
                 544 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.019 
                 AGCGGAAATGCCTGACTTACTTGCC 
                 545 
               
               
                   
               
               
                 Hu.DMD.Exon55.25.020 
                 GCTAAAGCGGAAATGCCTGACTTAC 
                 546 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.001  
                 AAGGACCAAATGTTCAGATG 
                 547 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.002 
                 CTGCAAAGGACCAAATGTTC 
                 548 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.003 
                 TCACCCTGCAAAGGACCAAA 
                 549 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.004 
                 CTCACTCACCCTGCAAAGGA 
                 550 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.005  
                 TCTCGCTCACTCACCCTGCA 
                 551 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.006 
                 CAGCCTCTCGCTCACTCACC 
                 552 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.007  
                 CAAAGCAGCCTCTCGCTCAC 
                 553 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.008 
                 TCTATGAGTTTCTTCCAAAG 
                 554 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.009 
                 GAACTGTTGCAGTAATCTAT 
                 555 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.010 
                 TTCCAGGTCCAGGGGGAACT 
                 556 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.011 
                 CCAGGCAAGAAACTTTTCCA 
                 557 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.012 
                 TTCAGCTTCTGTAAGCCAGG 
                 558 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.013 
                 GACATTGGCAGTTGTTTCAG 
                 559 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.014 
                 GGTAGCATCCTGTAGGACAT 
                 560 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.015  
                 GAGCCTTTCCTTACGGGTAG 
                 511 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.016  
                 CTTGGAGTCTTCTAGGAGCC 
                 562 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.017 
                 CAGCTCTTTTACTCCCTTGG 
                 563 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.018  
                 TTGCCATTGTTTCATCAGCT 
                 564 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.019 
                 CTTACTTGCCATTGTTTCAT 
                 565 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.020 
                 CCTGACTTACTTGCCATTGT 
                 566 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.021 
                 AAATGCCTGACTTACTTGCC 
                 567 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.022 
                 AGCGGAAATGCCTGACTTAC 
                 568 
               
               
                   
               
               
                 Hu.DMD.Exon55.20.023 
                 GCTAAAGCGGAAATGCCTGA 
                 569 
               
               
                   
               
               
                 H50A(+02+30)-AVI-5656  
                 CCACTCAGAGCTCAGATCTTCTAACTTCC 
                 584 
               
               
                   
               
               
                 H50D(+07−18)-AVI-5915 
                 GGGATCCAGTATACTTACAGGCTCC 
                 585 
               
               
                   
               
               
                 H50A(+07+33) 
                 CTTCCACTCAGAGCTCAGATCTTCTAA 
                 586 
               
               
                   
               
               
                 H51A(+61+90)-AVI-4657 
                 ACATCAAGGAAGATGGCATTTCTAGTTTGG 
                 587 
               
               
                   
               
               
                 H51A(+66+95)-AVI-4658 
                 CTCCAACATCAAGGAAGATGGCATTTCTAG 
                 588 
               
               
                   
               
               
                 H51A(+111+134) 
                 TTCTGTCCAAGCCCGGTTGAAATC 
                 589 
               
               
                   
               
               
                 H51A(+175+195) 
                 CACCCACCATCACCCTCYGTG 
                 590 
               
               
                   
               
               
                 H51A(+199+220) 
                 ATCATCTCGTTGATATCCTCAA 
                 591 
               
               
                   
               
               
                 H51A(+66+90) 
                 ACATCAAGGAAGATGGCATTTCTAG 
                 592 
               
               
                   
               
               
                 H51A(−01+25) 
                 ACCAGAGTAACAGTCTGAGTAGGAGC 
                 593 
               
               
                   
               
               
                 h51AON1 
                 TCAAGGAAGATGGCATTTCT 
                 594 
               
               
                   
               
               
                 h51AON2 
                 CCTCTGTGATTTTATAACTTGAT 
                 595 
               
               
                   
               
               
                 H51D(+08−17) 
                 ATCATTTTTTCTCATACCTTCTGCT 
                 596 
               
               
                   
               
               
                 H51D(+16−07) 
                 CTCATACCTTCTGCTTGATGATC 
                 597 
               
               
                   
               
               
                 hAON#23 
                 TGGCATTTCTAGTTTGG 
                 598 
               
               
                   
               
               
                 hAON#24 
                 CCAGAGCAGGTACCTCCAACATC 
                 599 
               
               
                   
               
               
                 H44A(+61+84) 
                 TGTTCAGCTTCTGTTAGCCACTGA 
                 600 
               
               
                   
               
               
                 H44A(+85+104) 
                 TTTGTGTCTTTCTGAGAAAC 
                 601 
               
               
                   
               
               
                 h44AON1 
                 CGCCGCCATTTCTCAACAG 
                 602 
               
               
                   
               
               
                 H44A(−06+14) 
                 ATCTGTCAAATCGCCTGCAG 
                 603 
               
               
                   
               
               
                 H45A(+71+90) 
                 TGTTTTTGAGGATTGCTGAA 
                 604 
               
               
                   
               
               
                 h45AON1 
                 GCTGAATTATTTCTTCCCC 
                 605 
               
               
                   
               
               
                 h45AON5 
                 GCCCAATGCCATCCTGG 
                 606 
               
               
                   
               
               
                 H45A(−06+20) 
                 CCAATGCCATCCTGGAGTTCCTGTAA 
                 607 
               
               
                   
               
               
                 H53A(+39+69) 
                 CATTCAACTGTTGCCTCCGGTTCTGAAGGTG 
                 608 
               
               
                   
               
               
                 H53A(+23+47) 
                 CTGAAGGTGTTCTTGTACTTCATCC 
                 609 
               
               
                   
               
               
                 h53AON1 
                 CTGTTGCCTCCGGTTCTG 
                 610 
               
               
                   
               
               
                 H53A(−12+10) 
                 ATTCTTTCAACTAGAATAAAAG 
                 611 
               
               
                   
               
               
                 huEx45.30.66 
                 GCCATCCTGGAGTTCCTGTAAGATACCAAA 
                 612 
               
               
                   
               
               
                 huEx45.30.71 
                 CCAATGCCATCCTGGAGTTCCTGTAAGATA 
                 613 
               
               
                   
               
               
                 huEx45.30.79 
                 GCCGCTGCCCAATGCCATCCTGGAGTTCCT 
                 614 
               
               
                   
               
               
                 huEx45.30.83 
                 GTTTGCCGCTGCCCAATGCCATCCTGGAGT 
                 615 
               
               
                   
               
               
                 huEx45.30.88 
                 CAACAGTTTGCCGCTGCCCAATGCCATCCT 
                 616 
               
               
                   
               
               
                 huEx45.30.92 
                 CTGACAACAGTTTGCCGCTGCCCAATGCCA 
                 617 
               
               
                   
               
               
                 huEx45.30.96 
                 TGTTCTGACAACAGTTTGCCGCTGCCCAAT 
                 618 
               
               
                   
               
               
                 huEx45.30.99 
                 CAATGTTCTGACAACAGTTTGCCGCTGCCC 
                 619 
               
               
                   
               
               
                 huEx45.30.103 
                 CATTCAATGTTCTGACAACAGTTTGCCGCT 
                 620 
               
               
                   
               
               
                 huEx45.30.120 
                 TATTTCTTCCCCAGTTGCATTCAATGTTCT 
                 621 
               
               
                   
               
               
                 huEx45.30.127 
                 GCTGAATTATTTCTTCCCCAGTTGCATTCA 
                 622 
               
               
                   
               
               
                 huEx45.30.132 
                 GGATTGCTGAATTATTTCTTCCCCAGTTGC 
                 623 
               
               
                   
               
               
                 huEx45.30.137 
                 TTTGAGGATTGCTGAATTATTTCTTCCCCA 
                 624 
               
               
                   
               
               
                 huEx53.30.84 
                 GTACTTCATCCCACTGATTCTGAATTCTTT 
                 625 
               
               
                   
               
               
                 huEx53.30.88 
                 TCTTGTACTTCATCCCACTGATTCTGAATT 
                 626 
               
               
                   
               
               
                 huEx53.30.91 
                 TGTTCTTGTACTTCATCCCACTGATTCTGA 
                 627 
               
               
                   
               
               
                 huEx53.30.103 
                 CGGTTCTGAAGGTGTTCTTGTACTTCATCC 
                 628 
               
               
                   
               
               
                 huEx53.30.106 
                 CTCCGGTTCTGAAGGTGTTCTTGTACTTCA 
                 629 
               
               
                   
               
               
                 huEx53.30.109 
                 TGCCTCCGGTTCTGAAGGTGTTCTTGTACT 
                 630 
               
               
                   
               
               
                 huEx53.30.112 
                 TGTTGCCTCCGGTTCTGAAGGTGTTCTTGT 
                 631 
               
               
                   
               
               
                 huEx53.30.115 
                 AACTGTTGCCTCCGGTTCTGAAGGTGTTCT 
                 632 
               
               
                   
               
               
                 huEx53.30.118 
                 TTCAACTGTTGCCTCCGGTTCTGAAGGTGT 
                 633 
               
               
                   
               
               
                 h50AON1 
                   
                   
               
               
                   
               
               
                 h50AON2 
                   
                   
               
            
           
           
               
            
               
                 Peptide Transporters (NH 2  to COOH)*: 
               
            
           
           
               
               
               
            
               
                 rTAT 
                 RRRQRRKKRC 
                 570 
               
               
                   
               
               
                 R 9 F 2   
                 RRRRRRRRRFFC 
                 571 
               
               
                   
               
               
                 (RRAhx) 4 B 
                 RRAhxRRAhxRRAhxRRAhxB 
                 572 
               
               
                   
               
               
                 (RAhxR) 4 AhxB; (P007) 
                 RAhxRRAhxRRAhxRRAhxRAhxB 
                 573 
               
               
                   
               
               
                 (AhxRR) 4 hxB 
                 AhxRRAhxRRAhxRRAhxRRAhxB 
                 574 
               
               
                   
               
               
                 (RAhx) 6 B 
                 RAhxRAhxRAhxRAhxRAhxRAhxB 
                 575 
               
               
                   
               
               
                 (RAhx) 8 B 
                 RAhxRAhxRAhxRAhxRAhxRAhxRAhxRAhxB 
                 576 
               
               
                   
               
               
                 (RAhxR) 5 AhxB 
                 RAhxRRAhxRRAhxRRAhxRRAhxRAhxB 
                 577 
               
               
                   
               
               
                 (RAhxRRBR) 2 AhxB; 
                 RAhxRRBRRAhxRRBRAhxB 
                 578 
               
               
                 (CPO6062 
                   
                   
               
               
                 MSP 
                 ASSLNIA 
                 579 
               
            
           
           
               
            
               
                 Cell Penetrating Peptide/Homing Peptide/PMO Conjugates 
               
               
                 (NH 2  to COOH and 5′ to 3′) 
               
            
           
           
               
               
               
            
               
                 MSP-PMO 
                 ASSLNIA-XB- 
                 580 
               
               
                   
                 GGCCAAACCTCGGCTTACCTGAAAT 
                 636 
               
               
                   
               
               
                 CP06062-MSP-PMO 
                 RXRRBRRXRRBR-XB-ASSLNIA-X- 
                 581 
               
               
                   
                 GGCCAAACCTCGGCTTACCTGAAAT 
                 636 
               
               
                   
               
               
                 MSP-CP06062-PMO 
                 ASSLN1A-X-RXRRBRRXRRBR-B- 
                 582 
               
               
                   
                 GGCCAAACCTCGGCTTACCTGAAAT 
                 636 
               
               
                   
               
               
                 CP06062-PMO 
                 RXRRBRRXRRBR-XB- 
                 583 
               
               
                   
                 GGCCAAACCTCGGCTTACCTGAAAT 
                 636 
               
               
                   
               
               
                 *Ahx is 6-amindhexandic acid and B is beta-alanine.