Duchenne muscular dystrophies (DMD) and Limb-girdle muscular dystrophies (LGMD) are common inherited degenerative muscle diseases caused by mutations in genes coding for memberance associated proteins in muscle cells. DMD and LGMD often manifest themselves in young age and lead to severe morbidity and fatality, with no currently available effective treatment. In addition, the diseases are usually genotypically recessive, which makes them suitable for gene replacement therapy with vectors. Recombinant adeno-associated virus (rAAV) is one such vector based on defective human parvoviruses. rAAV system has attracted attention due to its non-pathogenicity, genomic integration, transduction of quiescent cells, and apparent lack of cellular immune reactions. In contrast to other viral vectors, rAAV is capable of efficiently bypassing the myofiber basal lamina and tranducing mature muscle cells. We have demonstrated that rAAV vectors harboring a foreign gene can achieve highly efficient and sustained gene transfer in mature muscle of immunocompetent animals for more than 1.5 years without detectable toxicity. Recently, significant improvement in vector production methodology has made it possible to generate high titer and high quality rAAV vectors completely free of helper adenovirus contamination. However, no experiments using rAAV vectors to restore the functional deficits in muscle tissue itself have been reported to date. In this proposal, we will use delta-SG as the target disease gene, the delta-SG deficient hamster as the LGDM animal model, and rAAV as the gene delivery vector to test our general hypothesis that safe, efficient and sustained functional rescue of muscle deficiency can be achieved by genetic complementation of inherited muscular dystrophies with rAAV vectors. Specifically, we would like to achieve the following aims: 1) To study gene transfer efficiency and functional rescue in the LGMD hamster model by local intramuscular delivery of delta-SG-rAAV vectors and examine their short term ability to correlate the genetic defect in both skeletal and cardiac muscle. 2) To evaluate the gene therapy efficacy after systemic delivery of rAAV vectors through intra-artery or intra-ventricle injection. 3) To investigate the molecular kinetics and fate of rAAV vectors, especially after systemic vector delivery. 4) To develop new generation, high titer, helper-virus-free rAAV producer cells, which not only harbor vector and packaging genes, but also contain the necessary helper genes from adenovirus in a highly regulated and inducible manner.
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