Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.
We developed a drug-free regional intravenous (r.i.) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and 8 to an entire limb in the nonhuman primate (NHP), and compared the results with those produced by intramuscular (i.m.) delivery of the same dose of vector. We show that r.i. delivery of both serotypes was remarkably well tolerated with no adverse side-effects. After i.m., muscle transduction was restricted to the site of injection with a high number of vector copies per cell for rAAV1. In contrast, although r.i. delivery resulted in a lower vector copy per cell, it was detectable in the vast majority of muscles of the injected limb. The amounts of circulating infectious rAAV were similar for both serotypes and modes of delivery. At autopsy at up to 34 months after vector administration, similar biodistribution patterns were found for both vectors and for both modes of delivery, with numerous organs found to be positive for vector sequence when assayed using PCR and Southern blot. Altogether, we demonstrated that r.i. is a simple and efficient transduction protocol in NHPs, resulting in higher expression of the transgene with a lower number of vector genomes per cell. However, regardless of the mode of delivery, concerns continue to be raised by the presence of vector sequences detected at distant sites.
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