DMD, an X-linked recessive disorder, is the most frequent hereditary myopathy affecting approximately 1 in 3500 boys of all races. It is caused by mutations in the gene for the myocyte structural protein dystrophin. The loss of functional dystrophin in striated muscles causes extreme myocyte fragility and damage. The skeletal muscles degenerate continuously becoming fibrotic with adipose infiltrates. Clinical symptoms first manifest in boys between 18 months and 3 years of age. Muscle wasting occurs as the disease progresses and the patients become nonambulatory between 9 to 12 years of age. Mortality results from continuous loss of skeletal muscle causing spinal deformation, breathing difficulties, and cardiomyopathies. The disease is always fatal, often in the second decade and invariably in the third decade of life. There are no cures for DMD, and the current therapeutics regimens involve steroidal anti-inflamatories to limit immune responses to muscle fiber damage and only palliative treatments. Recombinant AAV may be used to restore dystrophin expression to skeletal muscles, either using a protein replacement approach, e.g. vectors that express micro-dystrophin, or an exon-skipping approach. For either application, producing sufficient quantities of rAAV to attain meaningful endpoints in the clinically relevant canine model of DMD has been the major obstacle for clinical trials. Most of the rAAV vector we produce contain a non-structural gene expressing a modified U7 RNA that interferes with processing the primary dystrophin transcript. Using specific anti-sense elements, our collaborators determined that specific exons may be omitted from the mature mRNA. This exon-skipping approach has been validated in murine DMD models and limited trials in canine DMD models. Based on these encouraging results, studies are underway using rAAV that express reporter proteins to establish the pharmacological parameters for treating DMD. From these results, the bio-distribution, routes of administration, vector toxicity are determined. Finally, using the data obtained with reporter constructs, doses that produce phenotypic improvements in large animals DMD models can then be established.

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National Heart, Lung, and Blood Institute
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