Dystrophin Expression In Muscles Research Articles

Intron mutations and early transcription termination in Duchenne and Becker muscular dystrophy.

DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reactionor high-throughput RNA sequencingmethods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

From the Literature

From the Literature

Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

P.337Dystrophin restoration by exon 53 skipping in patients with Duchenne muscular dystrophy after viltolarsen treatment: phase 2 study update

Duchenne muscular dystrophy (DMD) results in the loss of motor functional ability and early mortality due to the absence of dystrophin in muscle. Restoration of an out-of-frame deletion in the dystrophin mRNA to yield a truncated, in-frame transcript can be accomplished by "exon skipping." Viltolarsen is a novel antisense oligonucleotide that promotes exon 53 skipping in the dystrophin mRNA. Viltolarsen provides the potential to treat DMD patients who have dystrophin deletions comprising exons 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, or 52 alone. A completed Phase 2 study, NS-065/NCNP-01-201 (ClinicalTrials.gov Identifier: NCT02740972), enrolled 16 DMD patients with dystrophin mutations amenable to exon 53 skipping and demonstrated safety and tolerability of viltolarsen, as previously presented. In this study, target engagement by viltolarsen was demonstrated in RNA and protein extracted from patient muscle demonstrating skipping of exon 53 with accompanying expression of a truncated dystrophin protein. For each patient, the expected truncated dystrophin protein was observed by western blot, mass spectrometry and immunofluorescence, comparing a pre-treatment baseline muscle sample to a follow up sample after 24 weeks of treatment. The mass spectrometry demonstration of muscle dystrophin expression, using an assay validated for this study, is novel. All participants from the completed study then enrolled in a Phase 2 extension study NS-065/NCNP-01-202 (ClinicalTrials.gov Identifier: NCT03167255). The present report focuses on additional exploratory investigations into safety and timed function and strength measures observed after 73 weeks of total treatment with viltolarsen.

D44 - Extracellular myomiR abundance is not clearly correlated with skeletal muscle dystrophin expression in mdx mice with skewed X-chromosome inactivation

D44 - Extracellular myomiR abundance is not clearly correlated with skeletal muscle dystrophin expression in mdx mice with skewed X-chromosome inactivation

Transcriptional and epigenetic analyses of the DMD locus reveal novel cis‑acting DNA elements that govern muscle dystrophin expression

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques.Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis‑DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA.Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.

CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy.

Muscle stem cells (MuSCs) hold great therapeutic potential for muscle genetic disorders, such as Duchenne muscular dystrophy (DMD). The CRISP/Cas9-based genome editing is a promising technology for correcting genetic alterations in mutant genes. In this study, we used fibrin-gel culture system to selectively expand MuSCs from crude skeletal muscle cells of mdx mice, a mouse model of DMD. By CRISP/Cas9-based genome editing, we corrected the dystrophin mutation in expanded MuSCs and restored the skeletal muscle dystrophin expression upon transplantation in mdx mice. Our studies established a reliable and feasible platform for gene correction in MuSCs by genome editing, thus greatly advancing tissue stem cell-based therapies for DMD and other muscle disorders.

Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.

Activation of Wnt3a signaling promotes myogenic differentiation of mesenchymal stem cells in mdx mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic muscular disorder with no effective treatment at present. Mesenchymal stem cell (MSC) transplantation has been used to treat DMD, but the efficiency is low. Our previous studies show that activation of Wnt3a signaling promotes myogenic differentiation of MSCs in vitro. Here we report an effective MSC transplantation therapy in mdx mice by activation of Wnt3a signaling. MSCs were isolated from mouse bone marrow, and pretreated with Wnt3a-conditioned medium (Wnt3a-CM), then transplanted into mdx mice. The recipient mice were euthanized at 4, 8, 12, 16 weeks after the transplantation, and muscle pathological changes were examined. The expression of dystrophin in muscle was detected using immunofluorescence staining, RT-PCR and Western blotting. Sixteen weeks later, transplantation of Wnt3a-pretreated MSCs in mdx mice improved the characteristics of dystrophic muscles evidenced by significant reductions in centrally nucleated myofibers, the variability range of cross-sectional area (CSA) and the connective tissue area of myofibers. Furthermore, transplantation of Wnt3a-pretreated MSCs in mdx mice gradually and markedly increased the expression of dystrophin in muscle, and improved the efficiency of myogenic differentiation. Transplantation of Wnt3a-pretreated MSCs in mdx mice results in long-term amelioration of the dystrophic phenotype and restores dystrophin expression in muscle. The results suggest that Wnt3a may be a promising candidate for the treatment of DMD.

Structure-Activity Relationship Studies of 3-epi-Deoxynegamycin Derivatives as Potent Readthrough Drug Candidates.

(+)-Negamycin (1), a natural dipeptidic antibiotic bearing a hydrazide structure, exhibits a readthrough activity toward the nonsense mutation of the dystrophin gene and restores dystrophin expression in muscles of Duchenne muscular dystrophy model mdx mice. Herein to develop more potent readthrough compounds, we performed a structure-activity relationship (SAR) study of 3-epi-deoxynegamycin (2), which is also another natural readthrough compound with little antimicrobial activity, focusing on the main carbon chain length. We found that one carbon atom shorter derivative 9b shows a higher readthrough activity than 1 and 2. Further derivatization at the carboxylic acid part of 9b demonstrates that its meta-chlorobenzyl ester derivative 17e, which has a higher ClogP value, exhibits a more potent readthrough activity than 9b. Interestingly, in the cell-free protein expression system, the readthrough activity of 17e drastically decreases compared to that in the cell-based assay. These results suggest that benzyl ester-type derivatives enhance the hydrophobicity and function as prodrugs to produce active compound 9b in living cell systems.

P.11.1 Peptide-PMO induced exon-skipping restores muscle physiology in the mdx mouse

Antisense directed exon-skipping has been shown to be a powerful method to induce expression of dystrophin in dystrophic muscle for a range of species including man. Dystrophin expression can be induced in the mdx mouse by oligonucleotides targeting exon 23 which removes the premature stop mutation in the murine dystrophin gene. We had previously shown improved resistance to eccentric exercise (10% length change of the active muscle) following intramuscular delivery of a phosphorodiamidate morpholino oligomer (PMO) targeting exon 23 in the mdx mouse (Sharp et al., 2011). Little improvement was seen in specific force which is commonly only 60% of wild-type in the mdx mouse. We now report similar physiology results following a single intravenous treatment with a peptide-PMO (PPMO) using the Pip6a peptide. Importantly, repeated intravenous PPMO treatment every two weeks starting at 12 weeks old and continuing for 10 doses, completely prevented the force drop associated with eccentric exercise and significantly improved the specific force, albeit not quite to wild-type levels. Both the acute and chronic dosing results correlated with the increased expression of dystrophin in muscle fibres and decreased levels of TIMP-1 in the serum. The chronic dosing study also showed a reduction in inflammation, restoration of a more normal fibre type pattern and reduction in fibre size variability. These studies show the potential of the PPMO formulation to significantly improve the results of exon-skipping in clinical trials.

T.I.2 Exon skipping and PRO044 in Duchenne muscular dystrophy: Extending the program

Patients with Duchenne muscular dystrophy (DMD) suffer from progressive muscle degeneration due to DMD gene mutations and resulting absence of dystrophin. Antisense oligonucleotides (AONs) have potential to modulate premRNA splicing so that a specific exon is skipped and the disrupted dystrophin reading frame restored. This allows production of internally deleted, partially functional dystrophins like those produced in Becker muscular dystrophy, which may lead to improvements in DMD skeletal muscle function through improved membrane stability and cell signaling. The first AON, currently in late phase clinical trials, is drisapersen (PRO051; GSK), a 2′-O-methyl phosphorothioate (2OMePS) RNA molecule inducing specific exon 51 skipping. Recently, a second AON was developed to target a different DMD patient population. PRO044, also a 2OMePS RNA molecule, applies to 6% of the DMD population and induces specific exon 44 skipping in dystrophin premRNA and dystrophin expression in cultured muscle cells of DMD patients. A Phase 1/2 open-label study of systemic PRO044 in 18 boys with DMD (5–15 years) is close to completion, exploring safety pharmacokinetics and molecular efficacy at different dose levels with five once-weekly sc administrations (0.5, 1.5, 5, 8, 12 mg/kg). Nine of these boys have also received 5 additional weekly doses at different dose levels by iv infusion (1.5, 5, 12 mg/kg). Preliminary data are encouraging. No clinical response was expected after 5 weeks’ treatment; however, the safety profile is consistent with the class of compound and a molecular response has been observed. Due to high degrees of spontaneous skipping, giving rise to revertant fibers in this DMD population, a new and robust, reproducible dystrophin detection methodology has been developed. Data will be presented to show effects on safety and plasma/tissue drug levels, putative biomarkers and muscle dystrophin expression, which will be used to support subsequent long-term PRO044 studies.

The DMD locus harbours multiple long non-coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms.

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.

Long-Term Rescue of Dystrophin Expression and Improvement in Muscle Pathology and Function in Dystrophic mdx Mice by Peptide-Conjugated Morpholino

Exon skipping is capable of correcting frameshift and nonsense mutations in Duchenne muscular dystrophy. Phase 2 clinical trials in the United Kingdom and the Netherlands have reported induction of dystrophin expression in muscle of Duchenne muscular dystrophy patients by systemic administration of both phosphorodiamidate morpholino oligomers (PMO) and 2'-O-methyl phosphorothioate. Peptide-conjugated phosphorodiamidate morpholino offers significantly higher efficiency than phosphorodiamidate morpholino, with the ability to induce near-normal levels of dystrophin, and restores function in both skeletal and cardiac muscle. We examined 1-year systemic efficacy of peptide-conjugated phosphorodiamidate morpholino targeting exon 23 in dystrophic mdx mice. The LD(50) of peptide-conjugated phosphorodiamidate morpholino was determined to be approximately 85 mg/kg. The half-life of dystrophin expression was approximately 2 months in skeletal muscle, but shorter in cardiac muscle. Biweekly injection of 6 mg/kg peptide-conjugated phosphorodiamidate morpholino produced >20% dystrophin expression in all skeletal muscles and ≤5% in cardiac muscle, with improvement in muscle function and pathology and reduction in levels of serum creatine kinase. Monthly injections of 30 mg/kg peptide-conjugated phosphorodiamidate morpholino restored dystrophin to >50% normal levels in skeletal muscle, and 15% in cardiac muscle. This was associated with greatly reduced serum creatine kinase levels, near-normal histology, and functional improvement of skeletal muscle. Our results demonstrate for the first time that regular 1-year administration of peptide-conjugated phosphorodiamidate morpholino can be safely applied to achieve significant therapeutic effects in an animal model.

Idiopathic Intracranial Hypertension in a Child With Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is an X-linked, recessively inherited disorder characterized by progressive weakness attributable to the absence of dystrophin expression in muscle. In multiple studies, the chronic administration of corticosteroids slowed the loss of ambulation that develops in mid to late childhood. Corticosteroids, however, frequently produce unacceptable side effects, including Cushingoid appearance and weight gain. Deflazacort, an oxazoline analogue of prednisolone, produces equivalent benefits on muscle with fewer reported Cushingoid side effects. We present a 9-year-old boy with Duchenne muscular dystrophy who developed morbid obesity and subsequent idiopathic intracranial hypertension after 2 years of receiving deflazacort. Although deflazacort is typically thought to produce less obesity than prednisone, severe Cushingoid side effects may occur in some individuals. To our knowledge, this description is the first of idiopathic intracranial hypertension complicating chronic corticosteroid treatment of Duchenne muscular dystrophy.

Physiological Characterization of Muscle Strength With Variable Levels of Dystrophin Restoration in mdx Mice Following Local Antisense Therapy

Antisense-induced exon skipping can restore the open reading frame, and thus correct the dystrophin deficiency that causes Duchenne muscular dystrophy (DMD), a lethal muscle wasting condition. Successful proof-of-principle in preclinical models has led to human clinical trials. However, it is still not known what percentage of dystrophin-positive fibers and what level of expression is necessary for functional improvement. This study directly address these key questions in the mdx mouse model of DMD. To achieve a significant variation in dystrophin expression, we locally administered into tibialis anterior muscles various doses of a phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 from the mRNA of murine dystrophin. We found a highly significant correlation between the number of dystrophin-positive fibers and resistance to contraction-induced injury, with a minimum of 20% of dystrophin-positive fibers required for meaningful improvement. Furthermore, our results also indicate that a relatively low level of dystrophin expression in muscle fibers may have significant clinical benefits. In contrast, improvements in muscle force were not correlated with either the number of positive fibers or total dystrophin levels, which highlight the need to conduct appropriate functional assessments in preclinical testing using the mdx mouse.

Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity

Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600 rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900 rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle.

Splice-switching as a treament for duchenne muscular dystrophy

Antisense oligomer manipulation of pre-mRNA splicing can be used to remove exons carrying premature stop codons or to restore the reading frame around frame-shifting mutations. Phase 1 clinical trials have demonstrated that antisense oligomer-mediated splicing manipulation can restore dystro-phin expression in muscle in a subset of Duchenne muscular dystrophy (DMD) patients. The complexities of dystrophin gene expression and widespread distribution of the protein have presented major challenges to gene and cell therapies for DMD. However, the size of the dystrophin gene, and the fact that not all of the 79 exons are necessary to encode a protein with at least partial function, make DMD amenable to splice switching intervention. While dystrophin deletions are clustered in two hotspots, non-deletion mutations are spread throughout the gene. Although 10 oligomers will be required to restore the reading-frame around the more common deletions, numerous compounds are required to by-pass the many different mutations that cause DMD. We have developed compounds to remove each of the dystrophin exons and are currently optimising preparations to remove in-frame blocks of exons to optimise induced dystrophin isoform function. The next challenge will be to extend the application of antisense oligomers to other conditions that may benefit from splice switching.

Central Tolerance to Myogenic Cell Transplants Does Not Include Muscle Neoantigens

Duchenne muscular dystrophy is a fatal genetic disease caused by lack of dystrophin. Myogenic cell transplantation (MT), a potential therapy for Duchenne muscular dystrophy, can restore dystrophin expression in muscles. Because allogeneic MT is highly resistant to peripheral tolerance, we proposed to induce central tolerance. However, given its immunogenicity, we asked whether central tolerance to donor major histocompatibility complex would allow long-term expression of dystrophin, a tissue-specific neoantigen in dystrophic recipients. Central tolerance was induced in C57BL/10J mdx (dystrophic) mice by allogeneic bone marrow transplantation (BMT) after conditioning with either lethal total body irradiation (TBI) or an established nonmyeloablative protocol (anti-CD154, anti-CD8 mAbs, and low-dose TBI). Recipients subsequently received donor-strain MT or skin grafts. Long-term hematopoietic chimeras generated using either lethal TBI or the nonmyeloablative regimen were tolerant to donor skin grafts and both primary and secondary donor MT (>90 days). Myogenic cell transplantation survival was decreased when chimerism was transient, which was most common with nonmyeloablative conditioning and fully rather than haplo-mismatched donors. Interestingly, regardless of conditioning, MT was associated with localized muscle infiltration with Foxp3CD4, CD25CD4, and PerforinCD8 cells, whereas skin grafts lacked infiltration. Central tolerance achieved using regimens that eliminate nearly all endogenous peripheral lymphocytes (i.e., lethal irradiation) or a nonmyeloablative protocol that depleted peripheral CD8 cells, results in lymphocytic infiltration in muscles that received MT but not in skin allografts. This suggests that muscle-specific infiltration may result from lack of negative selection for peripheral neoantigens in the thymus after BMT and that tolerance after MT may rely on peripheral regulatory mechanisms.

Transduction of Full-length Dystrophin to Multiple Skeletal Muscles Improves Motor Performance and Life Span in Utrophin/Dystrophin Double Knockout Mice

Duchenne muscular dystrophy (DMD) is a fatal, progressive, muscle-wasting disease caused by defects in the dystrophin. No viral vector except the helper-dependent adenovirus vector (HDAdv) can package 14-kilobase (kb) full-length dystrophin complementary DNA (cDNA), and HDAdv is considerably safer than old-generation adenovirus vectors because of the large-size deletion in its genome. We have generated HDAdv that carries myc-tagged murine full-length dystrophin cDNA (HDAdv-myc-mFLdys). We injected it into multiple proximal muscles of 7-day-old utrophin/dystrophin double knockout mice (dko mice) (which typically show symptoms quite similar to human DMD) because the proximal muscles are affected in DMD patients. Eight weeks after the injections, the transduced dystrophin was widely expressed, and we found a significant reduction in centrally nucleated myofibers and the restoration of the dystrophin-associated proteins, beta-dystroglycan (beta-DG) and alpha-sarcoglycan (alpha-SG), as well as neuronal nitric oxide synthase (nNOS). The injected dko mice also showed an increase in body weight, an improvement in motor performance, and a prolongation of life span. Using HDAdv, we could treat DMD model mice even by transferring the therapeutic gene into multiple skeletal muscles. Our results suggest that multiple intramuscular administrations of HDAdv carrying full-length dystrophin cDNA may reduce symptoms and compensate for lost functions in DMD patients.