Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice.

Duchenne and the less severe Becker form of muscular dystrophy (DMD,BMD) result from genetic deficiency in the level and/or activity of the protein dystrophin. The recent availability of cDNA based minigenes encoding recombinant dystrophin polypeptides has raised the possibility of somatic gene transfer as a therapeutic approach to treat dystrophin deficiency. In this respect, the mdx mouse provides a useful model of DMD exhibiting features characteristic of both the early myopathic and later fibrotic phases of the human disease. Using a mutated human cDNA, compatible in size with virus-based somatic gene transfer vectors, the pathophysiological consequences of restoring dystrophin expression have been examined in transgenic mdx mice. Transgene expression was correlated with a marked reduction of the skeletal myofibre necrosis and regeneration which is a major feature of the dystrophin-deficient phenotype in young mdx mice. The cDNA construct which is based on a very mild BMD phenotype thus encodes a highly functional dystrophin molecule whose reduced size renders it an attractive candidate for development as a therapeutic gene transfer reagent.     

Smooth muscle-specific dystrophin expression improves aberrant vasoregulation in mdx mice

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Therapeutic options for DMD are limited because the pathogenetic mechanism by which dystrophin deficiency produces the clinical phenotype remains obscure. Recent reports of abnormal alpha-adrenergic vasoregulation in the exercising muscles of DMD patients and in the mdx mouse, an animal model of DMD, prompted us to hypothesize that the dystrophin-deficient smooth muscle contributes to the vascular and dystrophic phenotypes of DMD. To test this, we generated transgenic mdx mice that express dystrophin only in smooth muscle (SMTg/mdx). We found that alpha-adrenergic vasoconstriction was markedly attenuated in the contracting hindlimbs of C57BL/10 wild-type mice, an effect that was mediated by nitric oxide (NO) and was severely impaired in the mdx mice. SMTg/mdx mice showed an intermediate phenotype, with partial restoration of the NO-dependent modulation of alpha-adrenergic vasoconstriction in active muscle. In addition, the elevated serum creatine kinase levels observed in mdx mice were significantly reduced in SMTg/mdx mice. This is the first report of a functional role of dystrophin in vascular smooth muscle.     

Full-length dystrophin expression in half of the heart cells ameliorates beta-isoproterenol-induced cardiomyopathy in mdx mice

Gene therapy holds great promise for curing Duchenne muscular dystrophy (DMD), the most common fatal inherited childhood muscle disease. Success of DMD gene therapy depends upon functional improvement in both skeletal and cardiac muscle. Numerous gene transfer studies have been performed to correct skeletal muscle pathology, yet little is known about cardiomyopathy gene therapy. Since complete transduction of the entire heart is an impractical goal, it becomes critical to determine the minimal level of correction needed for successful DMD cardiomyopathy gene therapy. To address this question, we generated heterozygous mice that persistently expressed the full-length dystrophin gene in 50% of the cardiomyocytes of mdx mice, a model for DMD. We questioned whether dystrophin expression in half of the heart cells was sufficient to prevent stress-induced cardiomyopathy. Heart function of mdx mouse is normal in the absence of external stress. To determine the therapeutic effect, we challenged 3-month-old mice with beta-isoproterenol. Cardiomyocyte sarcolemma integrity was significantly impaired in mdx but not in heterozygous and C57Bl/10 mice. Importantly, in vivo closed-chest hemodynamic assays revealed normal left ventricular function in beta-isoproterenol-stimulated heterozygous mice. Since the expression profile in the heterozygous mice mimicked viral transduction, we conclude that gene therapy correction in 50% of the heart cells may be sufficient to treat cardiomyopathy in mdx mice. This finding may also apply to the gene therapy of other inherited cardiomyopathies.     

Prolonged dystrophin expression and functional correction of mdx mouse muscle following gene transfer with a helper-dependent (gutted) adenovirus-encoding murine dystrophin

Dystrophin gene transfer using helper-dependent adenoviruses (HDAd), which are deleted of all viral genes, is a promising option to treat muscles in Duchenne muscular dystrophy. We investigated the benefits of this approach by injecting the tibialis anterior (TA) muscle of neonatal and juvenile (4-6-week-old) dystrophin-deficient (mdx) mice with a fully deleted HDAd (HDCBDysM). This vector encoded two full-length murine dystrophin cDNAs regulated by the powerful cytomegalovirus enhancer/beta-actin promoter. At 10 days post-injection of neonatal muscles, 712 fibers (42% of the total number of TA fibers) were dystrophin-positive (dys+), a value that did not decrease for 6 months (the study duration). In treated juveniles, maximal transduction occurred at 30 days post-injection (414 dys+ fibers, 24% of the total number of TA fibers), but decreased by 51% after 6 months. All studied aspects of the pathology were improved in neonatally treated muscles: the percentage of dys+ fibers with centrally localized myonuclei remained low, localization of the dystrophin associated protein complex was restored at the plasma membrane, muscle hypertrophy was reduced, and maximal force-generating capacity and resistance to contraction-induced injuries were increased. The same pathological aspects were improved in the treated juveniles, except for reduction of muscle hypertrophy and maximal force-generating capacity. We demonstrated a strong humoral response against murine dystrophin in both animal groups, but mild inflammatory response occurred only in the treated juveniles. HDCBDysM is thus one of the most promising and efficient vectors for treating DMD by gene therapy.     

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.     

Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice

Duchenne and Becker muscular dystrophy are caused by defectsin the dystrophin gene, and are candidates for treatment bygene therapy. We have shown previously that overexpression ofa full-length dystrophin cDNA prevents the development of dystrophicsymptoms in mdx mice. We show here that this functional correctioncan be achieved by expressing the full-length muscle isoformat a lower level than is present in control animals. Gene therapyfor DMD may necessitate the use of truncated dystrophin mini-genesto accommodate the limited cloning capacity of current-generationviral delivery vectors. We have constructed both murine andhuman mini-genes deleted for exons 17–48, and have demonstratedthat expression of either mini-gene can almost completely preventthe development of dystrophic symptoms in transgenic mdx mice.These results suggest that viral-mediated expression of moderatelevels of a truncated dystrophin could be an effective treatmentfor DMD.     

Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice

Duchenne muscular dystrophy (DMD) is caused by the absence of a functional dystrophin protein and is modeled by the mdx mouse. The mdx mouse suffers an early necrotic bout in the hind limb muscles lasting from approximately 4 to 7 weeks. The purpose of this investigation was to determine the extent to which dystrophin deficiency changed the proteome very early in the disease process. In order to accomplish this, proteins from gastrocnemius from 6-week-old C57 (n = 6) and mdx (n = 6) mice were labeled with fluorescent dye and subjected to two-dimensional differential in-gel electrophoresis (2D-DIGE). Resulting differentially expressed spots were excised and protein identity determined via MALDI-TOF followed by database searching using MASCOT. Proteins of the immediate energy system and glycolysis were generally down-regulated in mdx mice compared to C57 mice. Conversely, expression of proteins involved in the Kreb’s cycle and electron transport chain were increased in dystrophin-deficient muscle compared to control. Expression of cytoskeletal components, including tubulins, vimentin, and collagen, were increased in mdx mice compared to C57 mice. Importantly, these changes are occurring at only 6 weeks of age and are caused by acute dystrophin deficiency rather than more chronic injury. These data may provide insight regarding early pathologic changes occurring in dystrophin-deficient skeletal muscle.     

Increased oxidative stress in dystrophin deficient (mdx) mice masticatory muscles

Background

It has been suggested that increased oxidative stress and the glutathione antioxidant system play an important role in the pathogenesis of Duchenne muscular dystrophy. However, there is still a lack of data about the oxidative status in dystrophic masticatory muscles.

Methods

In the masticatory muscles of the mouse model of Duchenne muscular dystrophy (mdx and controls; 100 days old, n = 8-10 each group) we examined the GSH and GSSG content (glutathione reduced/oxidized form) and the level of lipid peroxidation (LPO) as measured by the thiobarbituric acid-reaction.

Results

In the mdx mice masticatory muscles we found increased oxidative stress as compared to the controls. The GSH values in mdx muscles were decreased (mean ± SEM; masseter 339.8 ± 37.6 μg/g vs. 523.1 ± 36.1 μg/g, temporal 304.1 ± 49.6 μg/g vs.512.6 ± 60.6 μg/g, tongue muscle 243.3 ± 28.8 μg/g vs. 474.9 ± 40.1 μg/g; Fig. 1) as compared to normal mice. The GSH/GSSG ratio in mdx mice was consequently decreased. No significant differences in GSSG content and LPO levels were found between mdx and control mice.

Conclusions

The results imply that oxidative stress is present in all three studied mdx mouse masticatory muscles.     

Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded ‘revertant’ dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a truncated dystrophin

Myotendinous strain injury is the most common injury of human skeletal muscles because the majority of muscle forces are transmitted through this region. Although the immediate response to strain injury is well characterized, the chronic response to myotendinous strain injury is less clear. Here we examined the molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a microdystrophin transgene (microdystrophin(DeltaR4-R23)). We found that muscles with myotendinous strain injury had an increased expression of utrophin and alpha7-integrin together with the dramatic restructuring of peripheral myofibrils into concentric rings. The sarcolemma of the microdystrophin(DeltaR4-R23)/mdx gastrocnemius muscles was highly protected from experimental lengthening contractions, better than wild-type muscles. We also found a positive correlation between myotendinous strain injury and ringed fibers in the HSA(LR) (human skeletal actin, long repeat) mouse model of myotonic dystrophy. We suggest that changes in protein expression and the formation of rings are adaptations to myotendinous strain injury that help to prevent muscle necrosis and retain the function of necessary muscles during injury, ageing and disease.     

Lack of dystrophin but normal calcium homeostasis in smooth muscle from dystrophic mdx mice

Summary The free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the dystrophin-lacking smooth muscle from mdx mice was studied to gain new insights into the relation between dystrophin and cytoplasmic Ca²⁺ hoemostasis, which was reported to be impaired in the mdx skeletal muscle. We observed that [Ca²⁺]_i, as measured with the fluorescent Ca²⁺ indicator fura-2, was not elevated in resting smooth muscle of the vas deferens from mdx mice, in comparison with control C57 mice. Changes of the external Ca²⁺ concentration evoked similar changes of [Ca²⁺]_i in mdx and control vas deferens. During contraction, cytosolic Ca²⁺ transients were identical, both in amplitude and in kinetics, whether or not dystrophin was present. Stretches evoked similar Ca²⁺ increases in muscles from both strains. Intracellular Ca²⁺ homeostasis appears to be unimpaired in mdx smooth muscle. Thus, the lack of dystrophin per se does not automatically induce a perturbation of Ca metabolism in muscle cells.     

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.     

Direct retroviral-mediated transfer of a dystrophin minigene into mdx mouse muscle in vivo

At the cellular level, the primary pathology in Duchenne musculardystrophy (DMD) is caused by deficiency of the sarcolemmal-associatedprotein, dystrophin, in the striated musculature. Here we describethe somatic transfer and longterm expression of a human dystrophinminigene corresponding to a mild Becker muscular dystrophy (BMD)phenotype in skeletal muscle tissues of the dystrophin-deficientmdx mouse by direct retroviral transduction. Following a singleintramuscular injection of recombinant retrovirus, sarcolemmalexpression of dystrophin was observed in an average of     

Three-dimensional cytoarchitecture of complex branched fibers in soleus muscle from mdx mutant mice

Three-dimensional cytoarchitecture and types and features of muscle fibers were examined in soleus muscles from mdx mutant mice at different stages of development. In the 2-week-old mice, no abnormal muscle fibers were observed light microscopically, whereas in the 4-week-old animals, disrupted fibers were frequent in light microscopy and scanning electron microscopy. Muscle fibers fused with several short fiber branches appeared at the sixth week after birth and increased in number until the tenth week. In the 1-year-old mice, approximately ten or more muscle fibers were seen fused together. They had many complex branches forming an “anastomosing syncytial reticulum.” Muscle fibers with irregular diameters and aggregations of the same type fibers were also observed. Our results demonstrated that these complex branched fibers might be formed by long term repetition of the degeneration and regeneration cycle during the development of soleus muscles, indicating that the characteristic features of muscle fibers with irregular diameters and aggregations of the same type fibers are certainly dependent on the existence of the complex branched fibers. © 1993 Wiley-Liss, Inc.     

Increased catalase expression improves muscle function in mdx mice

It has been well established that oxidative stress contributes to pathology associated with Duchenne muscular dystrophy (DMD). I hypothesized that overexpression of the antioxidant enzyme catalase would improve muscle function in the mdx mouse, the mouse model of DMD. To test this hypothesis, neonatal mdx mice were injected with a recombinant adeno-associated virus driving the catalase transgene. Animals were killed 4 or 6 weeks or 6 months following injection. Muscle function was generally improved by catalase overexpression. Four weeks following injection, extensor digitorum longus specific tension was improved twofold, while soleus was similar between groups. Resistance to contraction-induced injury was similar between groups; however, resistance to fatigue was increased 25% in catalase-treated soleus compared with control muscle. Six weeks following injection, extensor digitorum longus specific tension was increased 15%, while soleus specific tension was similar between treated and untreated limbs. Catalase overexpression reduced contraction-induced injury by 30-45% and fatigue by 20% compared with control limbs. Six months following injection, diaphragm specific tension was similar between groups, but resistance to contraction-induced injury was improved by 35% and fatigue by 25%. Taken together, these data indicate that catalase can improve a subset of parameters of muscle function in dystrophin-deficient skeletal muscle.     

Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors

Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction.     

Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.