380. Therapeutic Capacity of AAV-Delivered Muscle-Specific-Expressed Micro-Utrophin (ΔR4-R21/ΔCT) in mdX4cv Skeletal Muscles

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by dystrophin mutations. Utrophin is a dystrophin paralogue that can prevent necrosis in the mdx mouse DMD model. We designed an expression plasmid containing a miniaturized M-creatine kinase regulatory cassette (CK8e) and a rationally designed micro-utrophinΔR4-21/ΔCT (µUtrn) to accommodate the limited capacity of recombinant adeno-associated virus (rAAV). Both intramuscular (IM) and intravascular (IV) delivery of rAAV6-CK8-µUtrn at 2 weeks of age profoundly mitigated the dystrophic phenotype in skeletal muscles when examined at 3-6 months of age. These improvements were observed in many but not all skeletal muscle histological and functional parameters. At a pathological level µUtrn production was associated with significant reductions in centrally nucleated muscle fibers and with a greatly reduced prevalence of muscle regions containing inflammatory cells. µUtrn production also restored dystrophin-glycoprotein-complex (DGC) components β-Dystroglycan, Δ-Sarcoglycan, α-Dystrobrevin-2, and α1-Syntrophin to the sarcolemma. As expected, nNOS was not restored, presumably because CK8-µUtrn lacks direct nNOS binding sites. Analysis of µUtrn immunostaining intensities 3-months post-treatment suggested lower µUtrn levels in many of the 1, 2a and 2d/x fibers than in the 2b fibers. These differences would be consistent with the relative transcriptional activities of the CK8e regulatory cassette in different adult mouse fiber types: 2b > 2d/x > 2a > 1. Given the important structural role of costameres in connecting the sarcolemma to sarcomeres, we examined utrophin and µUtrn localization patterns and found endogenous utrophin localized in a costameric pattern adjacent to Z-line end points while abutting sarcolemma junctions. However, while µUtrn was also associated with a costameric lattice, its striations were unexpectedly only ~0.8 µm apart compared to ~2.2 µm for endogenous utrophin in untreated mdx4cv muscles. How the presence of two different costameric lattice patterns may affect skeletal muscle function is not known. Since mdx4cv mice are known to exhibit a fragmented synaptic phenotype, we also examined neuromuscular junctions (NMJs) in treated and untreated mice and found that µUtrn prevented synapse fragmentation and also restored the depth of synaptic folds. Interestingly, the NMJs in µUtrn-treated muscles exhibited a more highly branched architecture of the synaptic folds, and this may have compensated for the reduced number of fold openings observed in untreated mice. µUtrn treatment also provided partial restoration of the reduced Achilles myotendinous junction folds seen in mdx4cv mice, and did not lead to either the myotendinous strain injury or the ringed fiber formation associated with micro-dystrophinΔR4-R23/ΔCT mediated therapy. Importantly, physiological studies indicated that IM treated tibialis anterior muscles maintained peak force production and exhibited partially improved specific force production. Overall, rAAV6-CK8-µUtrn treatment of mdx4cv mice provided major improvements in many muscle parameters. However, it did not overcome all dystrophic deficits, and it modified the normal costameric lattice structure. Further improvements might be achieved by expressing µUtrn via regulatory cassettes with higher relative activity in 1, 2a and 2d/x fibers, and by further modifications of µUtrn (ΔR4-R21/ΔCT)'s design.