4 research outputs found

    Gene therapy for muscular dystrophy using secondary modifiers of the dystrophic phenotype

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    The absence of dystrophin in Duchenne muscular dystrophy causes sarcolemmal instability and renders muscle fibers susceptible to contraction-induced injury. Muscle repair mechanisms cannot compensate for progressive muscle degeneration, leading to a gradual replacement of muscle with adipose and connective tissue. Viral vectors have been successfully used to deliver dystrophin cDNA to dystrophin-deficient mdx mice, leading to a partial functional and morphological recovery of dystrophic muscle. However, a complete rescue of the dystrophic phenotype may require the co-delivery of additional factors to treat secondary pathological changes of dystrophic muscle. Since several malfunctions such as mechanical, signaling and regeneration may contribute to muscular dystrophy, we sought to study a number of treatments and their relative and combined potential for reversing the dystrophic pathology of the mdx mouse. The first part of the work centers around the characterization of ARC, apoptosis repressor interacting with caspase-recruitment domain, in normal and dystrophic mdx muscle and its potential to ameliorate muscular dystrophy. ARC was shown previously to interact selectively with caspases and to prevent hypoxia-induced release of cytochrome c from mitochondria, both features that may play a role in dystrophic pathology. We examined ARC protein expression in striated muscles and found that ARC displayed fiber-type restricted expression patterns and co-localized with the mitochondrial marker cytochrome oxidase. ARC expression in normal muscle showed a distinct regular pattern of ARC positive and negative fibers, while ARC expression in dystrophic mdx muscle appeared as a less distinct, irregular pattern. To gain further insights in the role of ARC in dystrophic mdx muscle, we generated transgenic mdx mice that over-expressed ARC under a tissue-specific promoter. These mice demonstrated high expression levels of transgenic ARC in all, oxidative and glycolytic, muscle fibers. Despite the over-expression of ARC in mdx skeletal muscle, these mice developed a dystrophic phenotype. We evaluated muscle morphology in ARC transgenic/mdx in comparison with mdx animals and did not observe an amelioration of the dystrophic pathology in ARC transgenic/mdx mice in various muscles at different ages. In summary, these studies suggested that the apoptotic pathways regulated by ARC do not significantly contribute to myofiber death in muscular dystrophy. The second part of this work focuses on the delivery of insulin-like growth factor (Igf-I) to mdx muscle without and in conjunction with gene replacement therapy. We wanted to assess if the beneficial effect of Igf-I is synergistic with the protective effect of dystrophin in restoring muscle strength and function in muscular dystrophy. In contrast to ARC, the effects of Igf-I have been widely studied in various cell types and tissues. In particular, overexpression of Igf-I in transgenic mdx muscles has been shown to protect the animals from the loss of muscle mass and function and to enhance muscle repair mechanisms. We have cloned and characterized the isoforms of Igf-I that are expressed in normal and dystrophic mouse muscle. Based on this information, we have tested virally mediated overexpression of Igf-I in vitro and in vivo in dystrophic muscle and established quantitative PCR and morphological and functional assays to assess expression and effects of Igf-I. Then we delivered dystrophin (AAV-mdys) and the muscle-specific Igf-I isoform (AAV-Igf-I) to dystrophic mdx muscles via recombinant adeno-associated viruses and analyzed their relative and combined potential in ameliorating the dystrophic phenotype. Functional measurements demonstrated that AAV-mdys injected animals were partially protected from contraction-induced injury, whereas animals injected with AAV-Igf-I alone were as susceptible as mdx animals to muscle damage. AAV-Igf-I treated animals, on the other hand, showed an increase in muscle mass, which was not seen after AAV-mdys only treatment. In contrast, co-injection of AAV-Igf-I and AAV-mdys resulted in increased muscle mass and muscle strength, and in protection from contraction-induced injury. These results suggest that the combination of AAV-Igf-I and AAV-mdys acted synergistically in ameliorating the dystrophic phenotype
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