Safety and Efficacy of Systemic Gene Therapy in Informative Models for DMD

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 DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is caused by inherited deficiency of the protein dystrophin. The overall aim of this project is to build towards rational clinical trials of gene therapy in DMD, following recent studies showing unprecedented protection of muscle fibers by a single dose of a novel vector in dystrophic mice and dogs. To minimize the risk of an immunological reaction against the transgene product, we have substituted utrophin for dystrophin. A systematically optimized, synthetic utrophin minigene is used with a muscle-targeting vector, AAV9, to achieve systemic gene transfer in newborn animals. In mdx mice following subsequent growth to maturity, the histological appearance of the treated muscle is restored to normal; in Aim 1 we now test whether this level of muscle fiber protection will correct muscle function in a wide range of clinically relevant assays. We use appropriate methods to study locomotor activity of the mice, types of movement, voluntary running times and distances, as well as forelimb and hindlimb muscle strength in grip tests. We assay for normalization of the CPK following exercise. Finally, we establish the function of the recombinant utrophin in isolated muscles, including the diaphragm and the heart, post mortem. To address the major safety issue facing potential subjects in clinical trials, in Aim 2 we turn ou attention to the immune response, using a unique disease model afforded by a naturally occurring deletion in the dog. The index mutation was identified in a German Short Haired Pointer (GSHPMD), and was shown by our lab to result from a homologous recombination event between ferritin-like pseudogenes flanking the entire canine dystrophin gene. We systematically investigate the immunological response to recombinant dystrophin and utrophin in this model to rigorously test the hypothesis that in the absence of central (thymic) immunological tolerance, host T cells will invade AAV-transduced muscle fibers, thereby eliminating recombinant dystrophin expression and causing a clinically severe myositis that exacerbates the clinical course of GSHPMD. We further hypothesize that central immunological tolerance will protect muscle fibers transduced with recombinant utrophin, thereby facilitating therapeutic efficacy. Finally, in Aim 3 we conduct a dose-finding study of systemic vector delivery in the dystrophic dog to estimate the appropriate dose for later use in a preclinical tria of efficacy. This will require the use of unprecedented titers of AAV in individual dogs, based on our studies thus far in mice, and will include screening tests for vector toxicity.