Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy.

We delivered plasmid DNA encoding therapeutic genes to the muscles of mouse models of limb girdle muscular dystrophy (LGMD) 2A, 2B, and 2D, deficient in calpain3, dysferlin, and alpha-sarcoglycan, respectively. We also delivered the human follistatin gene, which has the potential to increase therapeutic benefit. After intramuscular injection of DNA, electroporation was applied to enhance delivery to muscle fibers. When plasmids encoding the human calpain3 or dysferlin cDNA sequences were injected into quadriceps muscles of LGMD2A and LGMD2B mouse models, respectively, in 3-month studies, robust levels of calpain3 and dysferlin proteins were detected. We observed a statistically significant decrease in Evans blue dye penetration in LGMD2B mouse muscles after delivery of the dysferlin gene, consistent with repair of the muscle membrane defect in these mice. The therapeutic value of delivery of the genes for alpha-sarcoglycan and follistatin was documented by significant drops in Evans blue dye penetration in gastrocnemius muscles of LGMD2D mice. These results indicated for the first time that a combined gene therapy involving both alpha-sarcoglycan and follistatin would be valuable for LGMD2D patients. We suggest that this non-viral gene delivery method should be explored for its translational potential in patients.

DNA-Mediated Gene Therapy in a Mouse Model of Limb Girdle Muscular Dystrophy 2B.

Mutations in the gene for dysferlin cause a degenerative disorder of skeletal muscle known as limb girdle muscular dystrophy 2B. To achieve gene delivery of plasmids encoding dysferlin to hind limb muscles of dysferlin knockout mice, we used a vascular injection method that perfused naked plasmid DNA into all major muscle groups of the hind limb. We monitored delivery by luciferase live imaging and western blot, confirming strong dysferlin expression that persisted over the 3-month time course of the experiment. Co-delivery of the follistatin gene, which may promote muscle growth, was monitored by ELISA. Immunohistochemistry documented the presence of dysferlin in muscle fibers in treated limbs, and PCR confirmed the presence of plasmid DNA. Because dysferlin is involved in repair of the sarcolemmal membrane, dysferlin loss leads to fragile sarcolemmal membranes that can be detected by permeability to Evan's blue dye. We showed that after gene therapy with a plasmid encoding both dysferlin and follistatin, statistically significant reduction in Evan's blue dye permeability was present in hamstring muscles. These results suggest that vascular delivery of plasmids carrying these therapeutic genes may lead to simple and effective approaches for improving the clinical condition of limb girdle muscular dystrophy 2B.

Decrease of myofiber branching via muscle-specific expression of the olfactory receptor mOR23 in dystrophic muscle leads to protection against mechanical stress.

BACKGROUND: Abnormal branched myofibers within skeletal muscles are commonly found in diverse animal models of muscular dystrophy as well as in patients. Branched myofibers from dystrophic mice are more susceptible to break than unbranched myofibers suggesting that muscles containing a high percentage of these myofibers are more prone to injury. Previous studies showed ubiquitous over-expression of mouse olfactory receptor 23 (mOR23), a G protein-coupled receptor, in wild type mice decreased myofiber branching. Whether mOR23 over-expression specifically in skeletal muscle cells is sufficient to mitigate myofiber branching in dystrophic muscle is unknown. METHODS: We created a novel transgenic mouse over-expressing mOR23 specifically in muscle cells and then bred with dystrophic (mdx) mice. Myofiber branching was analyzed in these two transgenic mice and membrane integrity was assessed by Evans blue dye fluorescence. RESULTS: mOR23 over-expression in muscle led to a decrease of myofiber branching after muscle regeneration in non-dystrophic mouse muscles and reduced the severity of myofiber branching in mdx mouse muscles. Muscles from mdx mouse over-expressing mOR23 significantly exhibited less damage to eccentric contractions than control mdx muscles. CONCLUSIONS: The decrease of myofiber branching in mdx mouse muscles over-expressing mOR23 reduced the amount of membrane damage induced by mechanical stress. These results suggest that modifying myofiber branching in dystrophic patients, while not preventing degeneration, could be beneficial for mitigating some of the effects of the disease process.

Creatine kinase B is necessary to limit myoblast fusion during myogenesis.

Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.

Incidence and severity of myofiber branching with regeneration and aging.

BACKGROUND: Myofibers with an abnormal branching cytoarchitecture are commonly found in muscular dystrophy and in regenerated or aged nondystrophic muscles. Such branched myofibers from dystrophic mice are more susceptible to damage than unbranched myofibers in vitro, suggesting that muscles containing a high percentage of these myofibers are more prone to injury. Little is known about the regulation of myofiber branching. METHODS: To gain insights into the formation and fate of branched myofibers, we performed in-depth analyses of single myofibers isolated from dystrophic and nondystrophic (myotoxin-injured or aged) mouse muscles. The proportion of branched myofibers, the number of branches per myofiber and the morphology of the branches were assessed. RESULTS: Aged dystrophic mice exhibited the most severe myofiber branching as defined by the incidence of branched myofibers and the number of branches per myofiber, followed by myotoxin-injured, wild-type muscles and then aged wild-type muscles. In addition, the morphology of the branched myofibers differed among the various models. In response to either induced or ongoing muscle degeneration, branching was restricted to regenerated myofibers containing central nuclei. In myotoxin-injured muscles, the amount of branched myofibers remained stable over time. CONCLUSION: We suggest that myofiber branching is a consequence of myofiber remodeling during muscle regeneration. Our present study lays valuable groundwork for identifying the molecular pathways leading to myofiber branching in dystrophy, trauma and aging. Decreasing myofiber branching in dystrophic patients may improve muscle resistance to mechanical stress.

Generation of lentiviral vectors for use in skeletal muscle research.

Gene therapy is a promising approach for the treatment of a variety of disorders including genetic diseases and cancer. Among the viral vectors used in gene therapy, the lentiviral vector, based on HIV-1, is the only integrative vector able to transduce nondividing cells. The first generation of lentiviral vector was -established in 1996. Since then, other generations of lentiviral vector packaging systems were developed to improve this first vector. In this chapter, we describe these different packaging systems, the generation of lentiviral vector from productive cells, the 293T cell line, and the transduction of myogenic cells with a lentiviral vector as well.

Intramuscular Transplantation of Muscle Precursor Cells over-expressing MMP-9 improves Transplantation Success.

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin in muscles. A therapeutic approach to restore dystrophin expression in DMD patient's muscles is the transplantation of muscle precursor cells (MPCs). However, this transplantation is limited by the low MPC capacity to migrate beyond the injection trajectory. Matrix metalloproteases (MMPs) are key regulatory molecules in the remodeling of extracellular matrix (ECM) components. MPCs over-expressing MMP-9 were tested by zymography, migration and invasion assays in vitro and by transplantation in mouse muscle. In vitro, MPCs over-expressing MMP-9 have a better invasion capacity than control MPCs. When these cells are transplanted in mouse muscles, the transplantation success is increased by more than 50% and their dispersion is higher than normal cells. MMP-9 over-expression could thus be an approach to improve cell transplantation in DMD patients by increasing the dispersion capacity of transplanted cells.

Current status of pharmaceutical and genetic therapeutic approaches to treat DMD.

Duchenne muscular dystrophy (DMD) is a genetic disease affecting about one in every 3,500 boys. This X-linked pathology is due to the absence of dystrophin in muscle fibers. This lack of dystrophin leads to the progressive muscle degeneration that is often responsible for the death of the DMD patients during the third decade of their life. There are currently no curative treatments for this disease but different therapeutic approaches are being studied. Gene therapy consists of introducing a transgene coding for full-length or a truncated version of dystrophin complementary DNA (cDNA) in muscles, whereas pharmaceutical therapy includes the use of chemical/biochemical substances to restore dystrophin expression or alleviate the DMD phenotype. Over the past years, many potential drugs were explored. This led to several clinical trials for gentamicin and ataluren (PTC124) allowing stop codon read-through. An alternative approach is to induce the expression of an internally deleted, partially functional dystrophin protein through exon skipping. The vectors and the methods used in gene therapy have been continually improving in order to obtain greater encapsidation capacity and better transduction efficiency. The most promising experimental approaches using pharmaceutical and gene therapies are reviewed in this article.

Electrotransfer of the full-length dog dystrophin into mouse and dystrophic dog muscles.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by the absence of dystrophin (427 kDa). An approach to eventually restore this protein in patients with DMD is to introduce into their muscles a plasmid encoding dystrophin cDNA. Because the phenotype of the dystrophic dog is closer to the human phenotype than is the mdx mouse phenotype, we have studied the electrotransfer of a plasmid carrying the full-length dog dystrophin (FLDYS(dog)) in dystrophic dog muscle. To achieve this nonviral delivery, the FLDYS(dog) cDNA was cloned in two plasmids containing either a cytomegalovirus or a muscle creatine kinase promoter. In both cases, our results showed that the electrotransfer of these large plasmids (∼17 kb) into mouse muscle allowed FLDYS(dog) expression in the treated muscle. The electrotransfer of pCMV.FLDYS(dog) in a dystrophic dog muscle also led to the expression of dystrophin. In conclusion, introduction of the full-length dog dystrophin cDNA by electrotransfer into dystrophic dog muscle is a potential approach to restore dystrophin in patients with DMD. However, the electrotransfer procedure should be improved before applying it to humans.

Expression of dog microdystrophin in mouse and dog muscles by gene therapy.

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. Several previous studies demonstrated the feasibility of delivering microdystrophin complementary DNA (cDNA) into mouse and normal nonhuman primate muscles by ex vivo gene therapy. However, these animal models do not reproduce completely the human DMD phenotype, while the dystrophic dog model does. To progress toward the use of the best animal model of DMD, a dog microdystrophin was transduced into human and dystrophic dog muscle precursor cells (MPCs) with a lentivirus before their transplantation into mouse muscles. One month following MPC transplantation, myofibers expressing the dog microdystrophin were observed. We also used another approach to introduce this transgene into myofibers, i.e., the electrotransfer of a plasmid coding for the dog microdystrophin. The plasmid was injected into mouse and dog muscles, and brief electric pulses were applied in the region of injection. Two weeks later, the transgene was detected in both animals. Therefore, ex vivo gene therapy and electrotransfer are two possible methods to introduce a truncated version of dystrophin into myofibers of animal models and eventually into myofibers of DMD patients.

Induction of tolerance across fully mismatched barriers by a nonmyeloablative treatment excluding antibodies or irradiation use.

A mixed-chimerism approach is a major goal to circumvent sustained immunosuppression, but most of the proposed protocols need antibody treatment or host irradiation. Another promising experience involves busulfan combined with cyclophosphamide treatment. Additionally, recent publications demonstrated that, differing from busulfan, treosulfan administration does not present severe organ or hemato toxicities. Currently, Duchenne muscular dystrophy (DMD) patients are treated with chronic immunosuppression for muscle precursor cell transplantation (MT). We have developed a safe tolerance approach within this cellular allotransplantation therapy background. Thus, we have conditioned, prior to a donor BALB/c MT, the dystrophic mouse model C57Bl10J mdx/mdx, with our treatment based on a donor-specific transfusion, then a treosulfan treatment combined with single cyclophosphamide dose, and finally a donor bone marrow transplantation (TTCB). A first MT was performed in all mixed chimeric mice resulting from the TTCB treatment in the left tibialis anterior (TA) muscles. A second MT from the same donor strain was performed 100 days later in the right TA without any additional therapy. Results show that all treated mice developed permanent mixed chimerism. Long-lasting donor-positive fibers were present in both TAs of the mice, which received MT after the TTCB treatment. Only a basal level of infiltration was observed around donor fibers and mixed chimeric mice rejected third-party haplotype skin grafts. Thus, mixed chimerism development with this TTCB conditioning regimen promotes donor-specific stable tolerance, avoiding costimulatory blockade antibodies or irradiation use and side effects of sustained immunosuppressive treatments. This protocol could be eventually applied for MT to DMD patients or others tissue transplantations.