552. AAV9-Utrophin Restores Muscle Function in Dystrophic Mice
Authors: Shira T. Rosenblum, Hansell H. Stedman
Journal: Molecular Therapy (2016)

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions, complicating therapy with recombinant dystrophin because of the potential for chronic immune recognition of the "non-self" protein. The paralogous protein utrophin is ubiquitously expressed at levels insufficient to prevent myonecrosis in animal models for DMD, but may confer central immunological tolerance through early developmental expression in the thymus. Here we show for a first time histological evidence for the complete prevention of myonecrosis in dystrophin-deficient striated muscles following systemic administration of an AAV9 vector carrying a 3.5 kb synthetic utrophin transgene (AAVµU). The cDNA was miniaturized by removal of domains least conserved in a comprehensive evolutionary comparison, and further optimized for maximal expression in striated muscle by using the codon bias of mammalian genes encoding contractile proteins. Administration of 1015 AAVµU vector genomes (vg) per kg to neonatal mice prevented centronucleation and saturated global recovery of the sarcoglycan complex, despite a subsequent tenfold increase in striated muscle mass with growth. In neonatal dystrophic dogs, intravenous injection of 1013.5 AAVµU vg/kg without immunosuppression restored sarcoglycan levels and normalized the myofiber size-distribution following a fourfold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. Here we present for the first time results of a complex approach for the evaluation of functional rescue in mdx mice using non-invasive tests relevant to the clinically relevant symptoms of DMD through an open field cage system. The comparison between congenic wild type (C57Bl10) and mdx mice indicates similarity in parameters like rest time and the number of times entering and initiating use of the running wheel per day, but very significant differences with regards to running wheel associated locomotor activity characteristics including velocity, time, distance per run, and total distance per day. These data correlate with the differences in testing results between the two mouse strains observed in the force grip evaluation and serum levels of CK. Finally, we have shown significant enhancement of physiological performance using an open field running wheel system, and normalization of serum creatine kinase (CK) for the first time ever in treated dystrophic mice. These mice also demonstrated, improvements in both in vivo and ex vivo muscle strength, when compared with untreated mdx. The combination of these findings provide a rationale for high dose, neonatal gene therapy using utrophin as a "self" protein to forestall disability and mortality in DMD, while minimizing the risk of chronic immunotoxicity. These results also support the use of AAVµU as an experimental therapeutic for DMD based on a high level of functional reversal of muscular dystrophy in mdx mice. They demonstrate strong evidence in support of the running wheel open field system as a reliable and reproducible approach to assess the therapeutic efficacy in the mouse model of DMD. This approach also provides a platform for dissecting the physiological roles of dystrophin in supporting precisely measurable volitional activities in unrestricted animals, thereby offering potential improvements in the predictive power of preclinical studies of therapeutic efficacy to inform clinical trials.