726. Prevention of Muscular Dystrophy by an AAV Vector Encoding a Nonimmunogenic Protein Based on the Evolution of Utrophin and Dystrophin

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions in the dystrophin gene. Expression of recombinant dystrophin in DMD therefore risks chronic immune recognition of the “non-self” protein. Early developmental expression of the paralogous protein utrophin in the thymus may confer central immunological tolerance to its peptide sequence. Here we address previously unanswered questions about the therapeutic efficacy and immunogenicity of “µutrophins” encoded by synthetic minigenes suitable for use in AAV. First we used a comparative phylogenomic approach to address a deceptively simple question with critical implications for this field: “which came first-dystrophin or the sarcomere”. This analysis provided evidence that the long rod-like domain of utrophin and dystrophin was “coopted” from a higher molecular weight protein in dynein-propelled animals lacking striated muscle. When considered in light of recent crystalographic studies, these results suggest that the full functionality of native dystrophin may be conferred by recombinant mini-utrophins with internally deleted rod domains that preserve inter-repeat folding and hence maximal tensile strength. We maximized recombinant µUtrophin (µU) expression from synthetic open reading frames by using the codon bias of striated myosin. Our blinded studies of mdx mice injected with a µU transgene are the first to show COMPLETE recovery of peripheral myofiber nucleation following a single systemic injection of an AAV vector. Sarcoglycan expression, as measured by western blot in the µU-treated mice, are restored to control levels throughout growth to skeletal maturity. To investigate the correlation between our histological findings and functionality, we validated a novel open field cage and attached running wheel system. Our blinded data show remarkable functional rescue in µU-treated mdx mice using this non-invasive test with relevance to the clinical assessment of disease progression in DMD. These data also strongly correlate with those derived from in vivo force grip and ex vivo force transducer measurements. Remarkably, a majority of mdx mice show complete normalization of serum creatine kinase (CK), a first for single-dose treatment by any clinically translatable modality. In dystrophic puppies, intravenous injection of a 30-fold lower relative dose of AAV9U fully restored sarcoglycan levels and normalized the myofiber size-distribution following a threefold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. These findings suggest a rationale for neonatal gene therapy using utrophin as an internally deleted “self” protein in DMD to minimize the risk of chronic immunotoxicity. We outline a rigorous translational approach using escalating vector doses in GSHPMD dogs harboring a newly defined 5 megabase deletion encompassing the dystrophin ORF.