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Duchenne muscular dystrophy (DMD) is a severe congenital disease associated with mutation of the dystrophin gene. Supplementation of dystrophin using recombinant adeno-associated virus (rAAV) has promise as a treatment for DMD, although vector-related general toxicities, such as liver injury, neurotoxicity, and germline transmission, have been suggested in association with the systemic delivery of high doses of rAAV. Here, we treated normal or dystrophic dogs with rAAV9 transduction in conjunction with multipotent mesenchymal stromal cell (MSC) injection to investigate the therapeutic effects of an rAAV expressing microdystrophin (μDys) under conditions of immune modulation. Bone-marrow-derived MSCs, rAAV-CMV-μDys, and a rAAV-CAG-luciferase (Luc) were injected into the jugular vein of a young dystrophic dog to induce systemic expression of μDys. One week after the first injection, the dog received a second intravenous injection of MSCs, and on the following day, rAAV was intravenously injected into the same dog. Systemic injection of rAAV9 with MSCs pretreatment improves gene transfer into normal and dystrophic dogs. Dystrophic phenotypes significantly improved in the rAAV-μDys-injected dystrophic dog, suggesting that an improved rAAV-μDys treatment including immune modulation induces successful long-term transgene expression to improve dystrophic phenotypes.

To establish an efficient, safe immunosuppressive regimen of adeno-associated vector (AAV)-mediated gene therapy for Duchenne muscular dystrophy (DMD), we evaluated the effect of tacrolimus (FK506) on skeletal muscle transduction with AAV8 and AAV9 vectors expressing the LacZ and microdystrophin (M3) genes labeled by FLAG. We utilized 3- to 4-year-old Macaca fascicularis, screened for neutralizing antibodies against AAV. 3 days before AAV injection and throughout the experiment, 0.06 mg/kg tacrolimus was intravenously administered. A viral suspension of 1 × 1013 viral genomes/muscle was intramuscularly injected bilaterally at the tibialis anterior and biceps brachii muscles, which were biopsied at 8, 16, 24, and 42 weeks after injection. Without tacrolimus, AAV8- and AAV9-mediated LacZ expression disappeared 8 and 16 weeks after transduction, respectively. With tacrolimus, AAV8/9-mediated LacZ expression persisted for at least 42 weeks after injection. At 42 weeks after AAV8CMVLacZ and AAV9CMVLacZ injection, nearly 50% and 17% of muscle fibers were positive for β-galactosidase, respectively. AAV8/9-mediated M3-FLAG expression lasted for up to 42 weeks using tacrolimus. No significant generalized toxicity was observed in any monkey. These results indicate that tacrolimus administration regulated the immune response to transgenes and truncated microdystrophin in normal primates and may enhance the benefits of AAV-mediated gene therapy for DMD.

Micro-dystrophin (μDys) gene therapeutics can improve striated muscle structure and function in different animal models of Duchenne muscular dystrophy. Most studies, however, used young mdx mice that lack a pronounced dystrophic phenotype, short treatment periods, and limited muscle function tests. We, therefore, determined the relative efficacy of two previously described μDys gene therapeutics (rAAV6:μDysH3 and rAAV6:μDys5) in 6-month-old mdx mice using a 6-month treatment regimen and forced exercise. Forelimb and hindlimb grip strength, metabolic rate (VO2 max), running efficiency (energy expenditure), and serum creatine kinase levels similarly improved in mdx mice treated with either vector. Both vectors produced nearly identical dose-responses in all assays. They also partially prevented the degenerative effects of repeated high-intensity exercise on muscle histology, although none of the metrics examined was restored to normal wild-type levels. Moreover, neither vector had any consistent effect on respiration while exercising. These data together suggest that, although μDys gene therapy can improve isolated and systemic muscle function, it may be only partially effective when dystrophinopathies are advanced or when muscle structure is significantly challenged, as with high-intensity exercise. This further suggests that restoring muscle function to near-normal levels will likely require ancillary or combinatorial treatments capable of enhancing muscle strength.