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.     

Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessivedisorder with a high spontaneous mutation rate and no effectivetreatment, hence development of genetic based therapies is animportant goal. We report that expression of a recombinant humanminidystrophin cDNA, compatible with current viral vectors,can significantly reduce the myopathic phenotype in transgenicmdx mice, even when expressed at only 20–30% of endogenousdystrophin levels at the sarcolemma. To the extent that dataobtained in mouse studies are applicable to DMD, the virtualelimination of morphological and biochemical abnormalities inthe mdx mouse supports the use of this cDNA in somatic genetherapy protocols for DMD.     

Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells

Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of approximately 8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (approximately 14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus packaging signals, a beta-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1 + E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (> 10(8)/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues.      

Contribution of oxidative stress to pathology in diaphragm and limb muscles with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease that makes walking and breathing difficult. DMD is caused by an X-linked (Xp21) mutation in the dystrophin gene. Dystrophin is a scaffolding protein located in the sarcolemmal cytoskeleton, important in maintaining structural integrity and regulating muscle cell (muscle fiber) growth and repair. Dystrophin deficiency in mouse models (e.g., mdx mouse) destabilizes the interface between muscle fibers and the extracellular matrix, resulting in profound damage, inflammation, and weakness in diaphragm and limb muscles. While the link between dystrophin deficiency with inflammation and pathology is multi-factorial, elevated oxidative stress has been proposed as a central mediator. Unfortunately, the use of non-specific antioxidant scavengers in mouse and human studies has led to inconsistent results, obscuring our understanding of the importance of redox signaling in pathology of muscular dystrophy. However, recent studies with more mechanistic approaches in mdx mice suggest that NAD(P)H oxidase and nuclear factor-kappaB are important in amplifying dystrophin-deficient muscle pathology. Therefore, more targeted antioxidant therapeutics may ameliorate damage and weakness in human population, thus promoting better muscle function and quality of life. This review will focus upon the pathobiology of dystrophin deficiency in diaphragm and limb muscle primarily in mouse models, with a rationale for development of targeted therapeutic antioxidants in DMD patients.     

Gene replacement therapies for duchenne muscular dystrophy using adeno-associated viral vectors

The muscular dystrophies collectively represent a major health challenge, as few significant treatment options currently exist for any of these disorders. Recent years have witnessed a proliferation of novel approaches to therapy, spanning increased testing of existing and new pharmaceuticals, DNA delivery (both anti-sense oligonucleotides and plasmid DNA), gene therapies and stem cell technologies. While none of these has reached the point of being used in clinical practice, all show promise for being able to impact different types of muscular dystrophies. Our group has focused on developing direct gene replacement strategies to treat recessively inherited forms of muscular dystrophy, particularly Duchenne and Becker muscular dystrophy (DMD/BMD). Both forms of dystrophy are caused by mutations in the dystrophin gene and all cases can in theory be treated by gene replacement using synthetic forms of the dystrophin gene. The major challenges for success of this approach are the development of a suitable gene delivery shuttle, generating a suitable gene expression cassette able to be carried by such a shuttle, and achieving safe and effective delivery without elicitation of a destructive immune response. This review summarizes the current state of the art in terms of using adeno-associated viral vectors to deliver synthetic dystrophin genes for the purpose of developing gene therapy for DMD.     

A novel dystrophin isoform is required for normal retinal electrophysiology

Dystrophin is present in the outer plexiform layer of the retinaand is required for normal retinal function as measured by electroretinography.We describe the identification of a novel isoform of dystrophln(Dp260) present in the mouse retina. The unIque 5' terminusof the mRNA originates from a newly identified exon and is splicedin frame to exon 30 of the Duchenne muscular dystrophy (DMD)gene. The retinal isoform of dystrophln has 13 novel amino acidsas its N-terminus followed by most of the dystrophin rod domainand the cysteine-rich C-terminal domains. Analysis of mousetissues indicated this isoform of dystrophin Is expressed inretina, brain and cardiac tissue. Comparison of retinal electrophysiologyin mdx and mdx^cv3 mouse suggests that Dp260 is required fornormal retinal function.     

Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost‐effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide–cyclic guanosine monophosphate (NO–cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14‐week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra^®, Revatio^®) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase‐13 (MMP‐13) expression. Sildenafil also normalized the expression of the pro‐fibrotic (and pro‐inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil‐treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil‐mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO–cGMP signalling with PDE5 inhibitors in dystrophin‐deficient muscle. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans,mice, and zebrafish

The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.     

mdx(⁵cv) mice manifest more severe muscle dysfunction and diaphragm force deficits than do mdx Mice

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle dysfunction leading to premature death by the third decade of life. The mdx mouse, the most widely used animal model of DMD, has been extremely useful to study disease mechanisms and to screen new therapeutics. However, unlike patients with DMD, mdx mice have a very mild motor function deficit, posing significant limitations for its use as a platform to assess the impact of treatments on motor function. It has been suggested that an mdx variant, the mdx^5cv mouse, might be more severely affected. Here, we compared the motor activity, histopathology, and individual muscle force measurements of mdx and mdx^5cv mice. Our study revealed that mdx^5cv mice showed more severe exercise-induced fatigue, Rotarod performance deficits, and gait anomalies than mdx mice and that these deficits began at a younger age. Muscle force studies showed more severe strength deficits in the diaphragm of mdx^5cv mice compared to mdx mice, but similar force generation in the extensor digitorum longus. Muscle histology was similar between the two strains. Differences in genetic background (genetic modifiers) probably account for these functional differences between mdx strains. Overall, our findings indicate that the mdx and mdx^5cv mouse models of DMD are not interchangeable and identify the mdx^5cv mouse as a valuable platform for preclinical studies that require assessment of muscle function in live animals.Duchenne muscular dystrophy (DMD), the most prevalent muscular dystrophy, is an X-linked recessive disorder affecting 1 in 3500 male births. DMD is typically diagnosed around 3 years of age with a rapid progression of muscle weakness that results in wheelchair dependence by 12 years of age and death by the third decade of life.¹ Molecularly, DMD stems from mutations in the dystrophin gene that typically result in a lack of dystrophin protein expression in skeletal and cardiac muscles.^2,3 Loss of dystrophin compromises the muscle fiber membrane, leading to cycles of muscle fiber degeneration and regeneration, chronic inflammation, and accumulation of fibrotic tissue.^4–6 Currently, there is no definitive treatment for DMD, but several gene-, cell-, and drug-based therapeutic approaches are being evaluated.^7–12 Assessing and comparing the efficacy of these treatments require an easily available, well-characterized animal model with measurable and reproducible motor deficits, histologic pathology, and physiological alterations of muscle function.The mdx mouse is the most widely studied animal model of DMD and has been extensively used in preclinical studies over the past 20 years. It arose from a spontaneous nonsense mutation in exon 23 of the dystrophin gene in an inbred C57BL/10 background.¹³ The mdx muscles lack dystrophin expression and show histopathological features similar to DMD.^13–16 However, contrary to DMD patients, mdx mice have a milder phenotype, with minimal fibrosis in all muscles with the exception of the diaphragm,¹⁷ a near normal life span,¹⁸ and only minimal motor deficits.^13,19–23 In particular, treatment effect on overall motor function has been difficult to assess, posing a challenge for preclinical studies.^24,25As a result, mdx variants were generated with the hope of obtaining a more severe phenotype.^26–28 However, these alternative DMD mouse models had histopathological features and mild functional impairment similar to the mdx mouse. Among these, mdx^5cv mice harbor a mutation affecting exon 10 that selectively disrupts expression of full-length dystrophin such as in mdx mice.²⁹ However, mdx^5cv mice have recently been preferred to mdx mice because of their very low level of dystrophin-expressing revertant fibers,³⁰ allowing a more accurate quantification of the efficiency of gene and cell therapy in restoring dystrophin expression. In addition, mdx^5cv mice are on the C57BL/6 background that allows rapid transfer of the dystrophin mutation onto transgenic and knock-out mice to dissect molecular aspects of disease.Although mdx and mdx^5cv mice have been generally regarded as similar, a recent study found significant differences in gene expression profiles in all muscle groups examined, and a qualitative histologic examination suggested a more severe pathology in mdx^5cv mice.³¹ We therefore sought to determine whether mdx^5cv mice have more severe motor function deficits that render them distinct from mdx mice and better suited to study the effect of treatments on motor function and endurance.     

Influence of dystrophin-gene mutation onmdx mouse behavior. I. Retention deficits at long delays in spontaneous alternation and bar-pressing tasks

X-linked Duchenne muscular dystrophy (DMD) is frequently associated with a nonprogressive, cognitive defect attributed to the absence of dystrophin in the brain of DMD patients. The mutantmdx mouse, lacking in 427-kDa dystrophin in both muscle and brain tissues, is considered to be a valuable model of human DMD. In the present study, we comparedmdx and C57BL/10 control mice and showed thatmdx mice had impaired retention in a T-maze, delayed spontaneous alternation task 24 h, but not 6 h, after acquisition.mdx mice were not impaired in acquisition of a bar-pressing task on 4 consecutive days but showed poor retention 22 days after the last training session. Mutants and controls showed similar behavioral responses in free exploration and light/dark choice situations and did not differ in spontaneous locomotor activity or motor coordination. Retention impairments at long delays inmdx mice suggest a role of dystrophin in long-term consolidation processes.     

Gastric emptying,small intestinal transit and fecal output in dystrophic (<Emphasis Type="Italic">mdx</Emphasis>) mice

Duchenne muscular dystrophy (DMD), which results from deficiency in dystrophin, a sarcolemma protein of skeletal, cardiac and smooth muscle, is characterized by progressive striated muscle degeneration, but various gastrointestinal clinical manifestations have been observed. The aim was to evaluate the possible impact of the dystrophin loss on the gastrointestinal propulsion in mdx mice (animal model for DMD). The gastric emptying of a carboxymethyl cellulose/phenol red dye non-nutrient meal was not significantly different at 20 min from gavaging between wild-type and mdx mice. The intestinal transit and the fecal output were significantly decreased in mdx versus normal animals, although the length of the intestine was similar in both animals. The present results provide evidence for motor intestinal alterations in mdx mice in in vivo conditions.     

Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice

Duchenne muscular dystrophy is a severe life-threatening X-linked recessive disorder, caused by mutations in the dystrophin gene, for which currently there is no effective treatment. Because of the large size of the dystrophin cDNA (14 kb) this precluded it from being used in early adenovirus- or retrovirus-based gene therapy vectors. However, some therapeutic success has been achieved in mdx mice using adenovirus- and retrovirus-mediated transfer of a 6.3 kb recombinant mini-dystrophin cDNA. Despite this, problems with immunogenicity and inefficient transduction of mature myofibres make these vectors less than ideal for gene transfer to skeletal muscle. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems. However, AAV vectors can only accommodate <5 kb of foreign DNA. For this reason we have produced a micro-dystrophin cDNA gene construct that is <3.8 kb. This construct, driven by a CMV promoter, was introduced into the skeletal muscle of 12-day-old nude/mdx mice using an AAV vector, resulting in specific sarcolemmal expression of micro-dystrophin in >50% of myofibres up to 20 weeks of age, and effective restoration of the dystrophin-associated protein (DAP) complex components. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. We have therefore shown that the current micro-dystrophin gene delivered in vivo using an AAV vector is not only capable of restoring sarcolemmal DAP complexes, but can also ameliorate dystrophic pathology at the cellular level.     

Human Adipose Tissue Derived Pericytes Increase Life Span in Utrn tm1Ked Dmd mdx /J Mice

Duchenne muscular dystrophy (DMD) is still an untreatable lethal X-linked disorder, which affects 1 in 3500 male births. It is caused by the absence of muscle dystrophin due to mutations in the dystrophin gene. The potential regenerative capacity as well as immune privileged properties of mesenchymal Stem Cells (MSC) has been under investigation for many years in an attempt to treat DMD. One of the questions to be addressed is whether stem cells from distinct sources have comparable clinical effects when injected in murine or canine muscular dystrophy animal models. Many studies comparing different stem cells from various sources were reported but these cells were obtained from different donors and thus with different genetic backgrounds. Here we investigated whether human pericytes obtained from 4 different tissues (muscle, adipose tissue, fallopian tube and endometrium) from the same donor have a similar clinical impact when injected in double mutant Utrn ^tm1Ked Dmd ^mdx /J mice, a clinically relevant model for DMD. After a weekly regimen of intraperitoneal injections of 10⁶ cells per 8 weeks we evaluated the motor ability as well as the life span of the treated mice as compared to controls. Our experiment showed that only adipose tissue derived pericytes are able to increase significantly (39 days on average) the life span of affected mice. Microarray analysis showed an inhibition of the interferon pathway by adipose derived pericytes. Our results suggest that the clinical benefit associated with intraperitoneal injections of these adult stem cells is related to immune modulation rather than tissue regeneration.     

Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin–deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5?×?10⁶) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27?±?12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.     

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.     

Comparative evolution of muscular dystrophy in diaphragm,gastrocnemius and masseter muscles from old male mdx mice

X chromosome-linked muscular dystrophic mdx mouse lacks the sarcolemmal protein dystrophin and represents a genetic homologue of human Duchenne muscular dystrophy (DMD). The present study analysed some aspects of pathological processes such as fibrosis, frequency of centralized nuclei, presence of degenerative or regenerative fibres, expression of utrophin and associated protein complexes, and myosin heavy chain isoforms in three muscles [diaphragm (DIA), gastrocnemius (GTC) and masseter (MAS)] from old male mdx mice. All parameters investigated comparatively in these pathological muscles provided evidence that the MAS mdx muscle presents a slight deterioration pattern in comparison to that of DIA and GTC muscles. Utrophin and associated proteins are present in many cell clusters with continuous membrane labelling in MAS muscle. Respective proportions of myosin heavy chain isoforms, measured by electrophoresis/densitometry, showed only slight change in GTC muscle, significant evolution in DIA muscle but drastic isoform conversions in MAS muscle. These results highlighted the difference in deterioration susceptibility of various muscles to muscular dystrophy. The reason why this occurs in MAS muscles is still obscure and discussed in terms of the comparative developmental origins of these muscles.     

Long‐Term Therapy With Omega‐3 Ameliorates Myonecrosis and Benefits Skeletal Muscle Regeneration in Mdx Mice

In Duchenne muscle dystrophy (DMD) and in the mdx mouse model of DMD, a lack of dystrophin leads to myonecrosis and cardiorespiratory failure. Several lines of evidence suggest a detrimental role of the inflammatory process in the dystrophic process. Previously, we demonstrated that short‐term therapy with eicosapentaenoic acid (EPA), at early stages of disease, ameliorated dystrophy progression in the mdx mouse. In the present study, we evaluated the effects of a long‐term therapy with omega‐3 later in dystrophy progression. Three‐month‐old mdx mice received omega‐3 (300 mg/kg) or vehicle by gavage for 5 months. The quadriceps and diaphragm muscles were removed and processed for histopathology and Western blot. Long‐term therapy with omega‐3 increased the regulatory protein MyoD and muscle regeneration and reduced markers of inflammation (TNF‐α and NF‐kB) in both muscles studied. The present study supports the long‐term use of omega‐3 at later stages of dystrophy as a promising option to be investigated in DMD clinical trials. Anat Rec, 298:1589–1596, 2015. © 2015 Wiley Periodicals, Inc.     

Dystrophin Expressing Chimeric (DEC) Human Cells Provide a Potential Therapy for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a progressive and lethal disease caused by mutations of the dystrophin gene. Currently no cure exists. Stem cell therapies targeting DMD are challenged by limited engraftment and rejection despite the use of immunosuppression. There is an urgent need to introduce new stem cell-based therapies that exhibit low allogenic profiles and improved cell engraftment. In this proof-of-concept study, we develop and test a new human stem cell-based approach to increase engraftment, limit rejection, and restore dystrophin expression in the mdx/scid mouse model of DMD. We introduce two Dystrophin Expressing Chimeric (DEC) cell lines created by ex vivo fusion of human myoblasts (MB) derived from two normal donors (MB^N1/MB^N2), and normal and DMD donors (MB^N/MB^DMD). The efficacy of fusion was confirmed by flow cytometry and confocal microscopy based on donor cell fluorescent labeling (PKH26/PKH67). In vitro, DEC displayed phenotype and genotype of donor parent cells, expressed dystrophin, and maintained proliferation and myogenic differentiation. In vivo, local delivery of both DEC lines (0.5?×?10⁶) restored dystrophin expression (17.27%±8.05—MB^N1/MB^N2 and 23.79%±3.82—MB^N/MB^DMD) which correlated with significant improvement of muscle force, contraction and tolerance to fatigue at 90 days after DEC transplant to the gastrocnemius muscles (GM) of dystrophin-deficient mdx/scid mice. This study establishes DEC as a potential therapy for DMD and other types of muscular dystrophies.