Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Four single-exon skipping PMOs are approved for DMD but treat only 8 to 14% of patients each, and some exhibit poor efficacy. Alternatively, exons 45 to 55 skipping could treat 40 to 47% of all patients and is associated with improved clinical outcomes. Here, we report the development of peptide-conjugated PMOs for exons 45 to 55 skipping. Experiments with immortalized patient myotubes revealed that exons 45 to 55 could be skipped by targeting as few as five exons. We also found that conjugating DG9, a cell-penetrating peptide, to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45 to 55-skipping DG9-PMO mixture restored dystrophin production. This study provides proof of concept toward the development of a more economical and effective exons 45 to 55-skipping DMD therapy.

Therapeutic Exon Skipping Through a CRISPR-Guided Cytidine Deaminase Rescues Dystrophic Cardiomyopathy in Vivo.

BACKGROUND: Loss of dystrophin protein causes Duchenne muscular dystrophy (DMD), characterized by progressive degeneration of cardiac and skeletal muscles, and mortality in adolescence or young adulthood. Although cardiac failure has risen as the leading cause of mortality in patients with DMD, effective therapeutic interventions remain underdeveloped, in part, because of the lack of a suitable preclinical model. METHODS: We analyzed a novel murine model of DMD created by introducing a 4-bp deletion into exon 4, one of the exons encoding the actin-binding domain 1 of dystrophin (referred to as DmdE4* mice). Echocardiography, microcomputed tomography, muscle force measurement, and histological analysis were performed to determine cardiac and skeletal muscle defects in these mice. Using this model, we examined the feasibility of using a cytidine base editor to install exon skipping and rescue dystrophic cardiomyopathy in vivo. AAV9-based CRISPR/Cas9-AID (eTAM) together with AAV9-sgRNA was injected into neonatal DmdE4* mice, which were analyzed 2 or 12 months after treatment to evaluate the extent of exon skipping, dystrophin restoration, and phenotypic improvements of cardiac and skeletal muscles. RESULTS: DmdE4* mice recapitulated many aspects of human DMD, including shortened life span (by ≈50%), progressive cardiomyopathy, kyphosis, profound loss of muscle strength, and myocyte degeneration. A single-dose administration of AAV9-eTAM instituted >50% targeted exon skipping in the Dmd transcripts and restored up to 90% dystrophin in the heart. As a result, early ventricular remodeling was prevented and cardiac and skeletal muscle functions were improved, leading to an increased life span of the DmdE4* mice. Despite gradual decline of AAV vector and base editor expression, dystrophin restoration and pathophysiological rescue of muscular dystrophy were long lasted for at least 1 year. CONCLUSIONS: Our study demonstrates the feasibility and efficacy to institute exon skipping through an enhanced TAM (eTAM) for therapeutic application(s).

High-Throughput Digital Image Analysis Reveals Distinct Patterns of Dystrophin Expression in Dystrophinopathy Patients.

Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy (BMD). In the last decade several antisense oligonucleotides drugs have been developed to induce a partially functional internally deleted dystrophin, similar to that produced in BMD, and expected to ameliorate the disease course. The pattern of dystrophin expression and functionality in dystrophinopathy patients is variable due to multiple factors, such as molecular functionality of the dystrophin and its distribution. To benchmark the success of therapeutic intervention, a clear understanding of dystrophin expression patterns in dystrophinopathy patients is vital. Recently, several groups have used innovative techniques to quantify dystrophin in muscle biopsies of children but not in patients with milder BMD. This study reports on dystrophin expression using both Western blotting and an automated, high-throughput, image analysis platform in DMD, BMD, and intermediate DMD/BMD skeletal muscle biopsies. Our results found a significant correlation between Western blot and immunofluorescent quantification indicating consistency between the different methodologies. However, we identified significant inter- and intradisease heterogeneity of patterns of dystrophin expression in patients irrespective of the amount detected on blot, due to variability in both fluorescence intensity and dystrophin sarcolemmal circumference coverage. Our data highlight the heterogeneity of the pattern of dystrophin expression in BMD, which will assist the assessment of dystrophin restoration therapies.

Short-term treatment of golden retriever muscular dystrophy (GRMD) dogs with rAAVrh74.MHCK7.GALGT2 induces muscle glycosylation and utrophin expression but has no significant effect on muscle strength.

We have examined the effects of intravenous (IV) delivery of rAAVrh74.MHCK7.GALGT2 in the golden retriever muscular dystrophy (GRMD) model of Duchenne Muscular Dystrophy (DMD). After baseline testing, GRMD dogs were treated at 3 months of age and reassessed at 6 months. This 3-6 month age range is a period of rapid disease progression, thus offering a relatively short window to establish treatment efficacy. Measures analyzed included muscle AAV transduction, GALGT2 transgene expression, GALGT2-induced glycosylation, muscle pathology, and muscle function. A total of five dogs were treated, 4 at 2x1014vg/kg and one at 6x1014vgkg. The 2x1014vg/kg dose led to transduction of regions of the heart with 1-3 vector genomes (vg) per nucleus, while most skeletal muscles were transduced with 0.25-0.5vg/nucleus. GALGT2-induced glycosylation paralleled levels of myofiber vg transduction, with about 90% of cardiomyocytes having increased glycosylation versus 20-35% of all myofibers across the skeletal muscles tested. Conclusions from phenotypic testing were limited by the small number of dogs. Treated dogs had less pronounced fibrosis and overall lesion severity when compared to control groups, but surprisingly no significant changes in limb muscle function measures. GALGT2-treated skeletal muscle and heart had elevated levels of utrophin protein expression and GALGT2-induced expression of glycosylated α dystroglycan, providing further evidence of a treatment effect. Serum chemistry, hematology, and cardiac function measures were largely unchanged by treatment. Cumulatively, these data show that short-term intravenous treatment of GRMD dogs with rAAVrh74.MHCK7.GALGT2 at high doses can induce muscle glycosylation and utrophin expression and may be safe over a short 3-month interval, but that such treatments had only modest effects on muscle pathology and did not significantly improve muscle strength.

Prediction of Premature Termination Codon Suppressing Compounds for Treatment of Duchenne Muscular Dystrophy Using Machine Learning.

A significant percentage of Duchenne muscular dystrophy (DMD) cases are caused by premature termination codon (PTC) mutations in the dystrophin gene, leading to the production of a truncated, non-functional dystrophin polypeptide. PTC-suppressing compounds (PTCSC) have been developed in order to restore protein translation by allowing the incorporation of an amino acid in place of a stop codon. However, limitations exist in terms of efficacy and toxicity. To identify new compounds that have PTC-suppressing ability, we selected and clustered existing PTCSC, allowing for the construction of a common pharmacophore model. Machine learning (ML) and deep learning (DL) models were developed for prediction of new PTCSC based on known compounds. We conducted a search of the NCI compounds database using the pharmacophore-based model and a search of the DrugBank database using pharmacophore-based, ML and DL models. Sixteen drug compounds were selected as a consensus of pharmacophore-based, ML, and DL searches. Our results suggest notable correspondence of the pharmacophore-based, ML, and DL models in prediction of new PTC-suppressing compounds.

Characterization of the expression of dystrophins and dystrophin-associated proteins during embryonic neural stem/progenitor cell differentiation.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene. Dystrophin is required for the organization of a complex consisting of dystroglycans, sarcoglycans, dystrobrevins and syntrophins, known as the dystrophin-associated proteins complex (DAPC). In addition to muscle degeneration, cognitive impairment has been reported in DMD patients. To characterize a suitable model for studying the embryonic cerebral functions of dystrophin, we analyzed the expression patterns of dystrophins/DAPC in undifferentiated and differentiated embryonic neural stem/progenitor cells (NSPC). We found that NSPC express mRNAs for dystrophins Dp427, Dp140, Dp71 and Dp40; ß-dystroglycan; α- and ß-dystrobrevin; α1-, ß1-, ß2- and γ2-syntrophin; and ß-, γ-, δ- and ε-sarcoglycan. Some of these were differentially regulated during neuronal or astrocytic differentiation. Interestingly, the protein expression levels of Dp140, ß-dystroglycan and α2-dystrobrevin were also differentially regulated. Additionally, we found that proliferating NSPC and differentiated neurons and astrocytes show immuno-positive staining for dystrophins and ß-dystroglycan. Our results show that dystrophins and DAPC components are expressed and regulated during the neuronal or astrocytic differentiation of NSPC, suggesting that these proteins may have different roles in the brain development.

Correction of muscular dystrophies by CRISPR gene editing.

Muscular dystrophies are debilitating disorders that result in progressive weakness and degeneration of skeletal muscle. Although the genetic mutations and clinical abnormalities of a variety of neuromuscular diseases are well known, no curative therapies have been developed to date. The advent of genome editing technology provides new opportunities to correct the underlying mutations responsible for many monogenic neuromuscular diseases. For example, Duchenne muscular dystrophy, which is caused by mutations in the dystrophin gene, has been successfully corrected in mice, dogs, and human cells through CRISPR/Cas9 editing. In this Review, we focus on the potential for, and challenges of, correcting muscular dystrophies by editing disease-causing mutations at the genomic level. Ideally, because muscle tissues are extremely long-lived, CRISPR technology could offer a one-time treatment for muscular dystrophies by correcting the culprit genomic mutations and enabling normal expression of the repaired gene.

Severe cardiac involvement with preserved truncated dystrophin expression in Becker muscular dystrophy by +1G>A DMD splice-site mutation: a case report.

Becker muscular dystrophy (BMD) is caused by specific mutations in the DMD gene that causes progressive muscle weakness and primarily affects skeletal and cardiac muscle. Although cardiac involvement is a significant cause of mortality in BMD, the genetic-phenotype correlation for skeletal and cardiac muscles has not been elucidated. Here, we described a 39-year-old man with BMD, who presented with subtle skeletal muscle weakness in the right leg in his 20s and underwent left ventricular restoration for severe dilated cardiomyopathy at the age of 29. He had difficulty climbing stairs after the age of 35. Neither duplication nor deletion of exons was detected by multiplex ligation-dependent probe amplification. A hemizygous c.264 + 1G>A mutation in intron 4 of the DMD was identified by next-generation sequencing. Furthermore, exon 4 skipping of the DMD was confirmed in both skeletal and cardiac muscles evaluated by reverse transcriptase PCR. Endomyocardial and skeletal muscle biopsies revealed dystrophic pathology characterized by muscle fiber atrophy and hypertrophy with a mild degree of interstitial fibrosis. Interestingly, dystrophin immunohistochemistry demonstrated patchy and faint staining of the skeletal muscle membranes but almost normal staining of the cardiac muscle membranes. Western blot analysis revealed a decreased amount of truncated dystrophin in skeletal muscle but surprisingly almost normal amount in cardiac muscle. This case indicates that BMD patients may have severe cardiac dysfunction despite preserved cardiac truncated dystrophin expression.

Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy.

OBJECTIVE: To report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping. METHODS: Part 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry. RESULTS: Twelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%-4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry. CONCLUSION: Golodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization. CLINICALTRIALSGOV IDENTIFIER: NCT02310906. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.

What is the level of dystrophin expression required for effective therapy of Duchenne muscular dystrophy?

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disease. The disease is due to mutations in the DMD gene that encodes for a large intracellular protein called dystrophin. Dystrophin plays a critical role in linking the internal cytoskeleton of the striated muscle cell with the extracellular matrix as well as having cell signalling functions. In its absence muscle contraction is associated with cycles of damage, repair, inflammation and fibrosis with eventual loss of muscle and replacement with fat. Experiments in animal models of DMD have generated a number of different approaches to the induction of dystrophin including viral vector mediated delivery of a recombinant dystrophin gene, antisense oligonucleotide mediated exon-skipping to restore the open reading frame in the dystrophin mRNA, read-through of premature stop mutations, genome modification using CRISPR-Cas9 or cell based transfer of a functional dystrophin gene. In all cases, it will be important to understand how much dystrophin expression is required for a clinically effective therapy and this review examines the data from humans and animal models to estimate the percentage of endogenous dystrophin that is likely to have significant clinical benefit. While there are a number of important caveats to consider, including the appropriate outcome measures, this review suggests that approximately 20% of endogenous levels uniformly distributed within the skeletal muscles and the heart may be sufficient to largely prevent disease progression.

The expression of the distal dystrophin isoforms Dp140 and Dp71 in the human epileptic hippocampus in relation to cognitive functioning.

Dystrophin is an important protein within the central nervous system. The absence of dystrophin, characterizing Duchenne muscular dystrophy (DMD), is associated with brain related comorbidities such as neurodevelopmental (e.g., cognitive and behavioural) deficits and epilepsy. Especially mutations in the downstream part of the DMD gene affecting the dystrophin isoforms Dp140 and Dp71 are found to be associated with cognitive deficits. However, the function of Dp140 is currently not well understood and its expression pattern has previously been implicated to be developmentally regulated. Therefore, we evaluated Dp140 and Dp71 expression in human hippocampi in relation to cognitive functioning in patients with drug-resistant temporal lobe epilepsy (TLE) and post-mortem controls. Hippocampal samples obtained as part of epilepsy surgery were quantitatively analyzed by Western blot and correlations with neuropsychological test results (i.e., memory and intelligence) were examined. First, we demonstrated that the expression of Dp140 does not appear to differ across different ages throughout adulthood. Second, we identified an inverse correlation between memory loss (i.e., verbal and visual memory), but not intelligence (i.e., neither verbal nor performance), and hippocampal Dp140 expression. Finally, patients with TLE appeared to have similar Dp140 expression levels compared to post-mortem controls without neurological disease. Dp140 may thus have a function in normal cognitive (i.e., episodic memory) processes.

Monitoring disease activity noninvasively in the mdx model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a rare, muscle degenerative disease resulting from the absence of the dystrophin protein. DMD is characterized by progressive loss of muscle fibers, muscle weakness, and eventually loss of ambulation and premature death. Currently, there is no cure for DMD and improved methods of disease monitoring are crucial for the development of novel treatments. In this study, we describe a new method of assessing disease progression noninvasively in the mdx model of DMD. The reporter mice, which we term the dystrophic Degeneration Reporter strains, contain an inducible CRE-responsive luciferase reporter active in mature myofibers. In these mice, muscle degeneration is reflected in changes in the level of luciferase expression, which can be monitored using noninvasive, bioluminescence imaging. We monitored the natural history and disease progression in these dystrophic report mice and found that decreases in luciferase signals directly correlated with muscle degeneration. We further demonstrated that this reporter strain, as well as a previously reported Regeneration Reporter strain, successfully reveals the effectiveness of a gene therapy treatment following systemic administration of a recombinant adeno-associated virus-6 (rAAV-6) encoding a microdystrophin construct. Our data demonstrate the value of these noninvasive imaging modalities for monitoring disease progression and response to therapy in mouse models of muscular dystrophy.

Eteplirsen treatment for Duchenne muscular dystrophy: Exon skipping and dystrophin production.

OBJECTIVE: To describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen. METHODS: Clinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen. RESULTS: Reverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001). CONCLUSION: Taken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

Cardiac-Specific Expression of ΔH2-R15 Mini-Dystrophin Normalized All Electrocardiogram Abnormalities and the End-Diastolic Volume in a 23-Month-Old Mouse Model of Duchenne Dilated Cardiomyopathy.

Heart disease is a major health threat for Duchenne/Becker muscular dystrophy patients and carriers. Expression of a 6-8 kb mini-dystrophin gene in the heart holds promise to change the disease course dramatically. However, the mini-dystrophin gene cannot be easily studied with adeno-associated virus (AAV) gene delivery because the size of the minigene exceeds AAV packaging capacity. Cardiac protection of the ΔH2-R19 minigene was previously studied using the cardiac-specific transgenic approach. Although this minigene fully normalized skeletal muscle force, it only partially corrected electrocardiogram and heart hemodynamics in dystrophin-null mdx mice that had moderate cardiomyopathy. This study evaluated the ΔH2-R15 minigene using the same transgenic approach in mdx mice that had more severe cardiomyopathy. In contrast to the ΔH2-R19 minigene, the ΔH2-R15 minigene carries dystrophin spectrin-like repeats 16 to 19 (R16-19), a region that has been suggested to protect the heart in clinical studies. Cardiac expression of the ΔH2-R15 minigene normalized all aberrant electrocardiogram changes and improved hemodynamics. Importantly, it corrected the end-diastolic volume, an important diastolic parameter not rescued by ΔH2-R19 mini-dystrophin. It is concluded that that ΔH2-R15 mini-dystrophin is a superior candidate gene for heart protection. This finding has important implications in the design of the mini/micro-dystrophin gene for Duchenne cardiomyopathy therapy.

Short (16-mer) locked nucleic acid splice-switching oligonucleotides restore dystrophin production in Duchenne Muscular Dystrophy myotubes.

Splice-switching antisense oligonucleotides (SSOs) offer great potential for RNA-targeting therapies, and two SSO drugs have been recently approved for treating Duchenne Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA). Despite promising results, new developments are still needed for more efficient chemistries and delivery systems. Locked nucleic acid (LNA) is a chemically modified nucleic acid that presents several attractive properties, such as high melting temperature when bound to RNA, potent biological activity, high stability and low toxicity in vivo. Here, we designed a series of LNA-based SSOs complementary to two sequences of the human dystrophin exon 51 that are most evolutionary conserved and evaluated their ability to induce exon skipping upon transfection into myoblasts derived from a DMD patient. We show that 16-mers with 60% of LNA modification efficiently induce exon skipping and restore synthesis of a truncated dystrophin isoform that localizes to the plasma membrane of patient-derived myotubes differentiated in culture. In sum, this study underscores the value of short LNA-modified SSOs for therapeutic applications.

Normal and altered pre-mRNA processing in the DMD gene.

Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression controlled by multiple post- and co-transcriptional mechanisms. The large Duchenne muscular dystrophy gene encoding the protein dystrophin provides a striking example of the complexity of human pre-mRNAs. In this review, we summarize the current state of knowledge about canonical and non-canonical splicing in the DMD pre-mRNA, with a focus on mechanisms that take place in the full-length transcript isoform expressed in human skeletal muscle. In particular, we highlight recent work demonstrating that multi-step events are required for long DMD intron removal. The role of temporary intron retention in the occurrence of alternative splicing events is also discussed. Even though the proportion of splicing mutations is lower than reported in other genes, a great diversity of splicing defects linked to point mutations, but also large genomic rearrangements are observed in the DMD gene. We provide an overview of the molecular mechanisms underlying aberrant splicing in patients with Duchenne or Becker muscular dystrophy, and we also detail how alternative splicing can serve as a disease modifier in patients by changing the outcome of the primary defect.

Adenoviral vectors encoding CRISPR/Cas9 multiplexes rescue dystrophin synthesis in unselected populations of DMD muscle cells.

Mutations disrupting the reading frame of the ~2.4 Mb dystrophin-encoding DMD gene cause a fatal X-linked muscle-wasting disorder called Duchenne muscular dystrophy (DMD). Genome editing based on paired RNA-guided nucleases (RGNs) from CRISPR/Cas9 systems has been proposed for permanently repairing faulty DMD loci. However, such multiplexing strategies require the development and testing of delivery systems capable of introducing the various gene editing tools into target cells. Here, we investigated the suitability of adenoviral vectors (AdVs) for multiplexed DMD editing by packaging in single vector particles expression units encoding the Streptococcus pyogenes Cas9 nuclease and sequence-specific gRNA pairs. These RGN components were customized to trigger short- and long-range intragenic DMD excisions encompassing reading frame-disrupting exons in patient-derived muscle progenitor cells. By allowing synchronous and stoichiometric expression of the various RGN components, we demonstrate that dual RGN-encoding AdVs can correct over 10% of target DMD alleles, readily leading to the detection of Becker-like dystrophin proteins in unselected muscle cell populations. Moreover, we report that AdV-based gene editing can be tailored for removing mutations located within the over 500-kb major DMD mutational hotspot. Hence, this single DMD editing strategy can in principle tackle a broad spectrum of mutations present in more than 60% of patients with DMD.

Characterization of a Dmd (EGFP) reporter mouse as a tool to investigate dystrophin expression.

BACKGROUND: Dystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining. RESULTS: We generated a Dmd (EGFP) reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes. CONCLUSIONS: The novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.