DESCRIPTION (provided by applicant): DMD is a progressive muscle disease caused by lack of dystrophin. Gene therapy is an important potential approach to replace the missing gene using adeno-associated virus (AAV). Micro- and mini-dystrophins have been developed for gene replacement but they either lack the C-terminal or the nNOSm binding site. To circumvent these issues we generated dual vectors, each containing nearly one half of the mini-dystrophin transgene with 372bp sequence homology to facilitate homologuous recombination. Upon reconstitution, this mini-dystrophin (ΔH2-R15/ΔR18-19) has the N-terminal domain, 10 spectrin repeats including repeats 16 and 17 for nNOS binding, cysteine-rich and C-terminal domains. Our preliminary studies indicate that a full-length mini-dystrophin is reconstituted, expressed and localized to the sarcolemma in about 50% of transduced muscle fibers. The aims of this study are specifically directed at functional outcomes for this recombinant transgene to lay the foundation for bringing this to clinical trial. Aim 1 will establish proof of principle that gene expression from this dual vector approach following intramuscular delivery will restore specific force and protect against contraction induced injury at doses that can reasonably be brought to vascular delivery and ultimately to clinical translation. Thus, Aim 2 will use a vascular delivery approach to the lower extremities through isolated limb perfusion that we have used extensively in mouse and monkeys and will be bringing to clinical trial for replacement of the alpha-sarcoglycan gene in LGMD2D. Success in vascular delivery lays the foundation for a clinical trial. Outcome measures for both Aim 1 and Aim 2 include dystrophin gene expression and functional improvements in specific force measurements and protection against eccentric contraction induced damage. Mini-dystrophin reconstitution and localization will be demonstrated by RT-PCR, western immunoblotting and immunofluorescence after 12 weeks. If successful in this experimental paradigm, our next step (beyond the scope of this grant) would be to take this to the next level by further testing the recombinant transgene in the non-human primate targeting their lower limbs by isolated limb perfusion. This we have done for many other transgenes that are being brought to clinical trial (e.g., RAC meeting on March 12, 2013 for LGMD2D) and nearly completed a toxicology-biodistribution study. Histological analysis in the monkey, using a FLAG tag as a marker for transduction deficiency will exclude any adverse effects and further refine the dose if necessary. On the safety front, we will also explore any immune issues using IFN-g ELISpot assays as we have done in preparation for multiple clinical trials. We also believe that folding of this gene product will be closer to the natural dystrophin and less likely to yield any hidden epitopes that could be immunogenic.

PUBLIC HEALTH RELEVANCE: In this study, we are proposing to do gene therapy with a dystrophin (DMD) gene that provides all important domains and has a more likely chance to correct the underlying defect. Upon translation, it is expected to improve the quality of life of DMD patients.