Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV–microdystrophin gene therapy in DMD patients.
Duchenne muscular dystrophy (DMD) is an X-linked inherited disease affecting ∼1:5,000 male births, leading to a highly debilitating and ultimately life-limiting muscle-wasting condition. DMD is caused by mutations in the gene coding for dystrophin, a cytoskeletal protein that is critical to the stability and function of myofibres in skeletal and cardiac muscle1,2. Dystrophin establishes a mechanical link between cytoskeletal actin and the extracellular matrix in muscle fibres through the dystrophin-associated protein complex, and when dystrophin is absent the mechanical and signalling functions of the costamer are compromised3. DMD-affected boys develop muscle weakness during the first years of life, and although palliative treatments are available (essentially physiotherapy, assisted ventilation and glucocorticoids) they become wheelchair-bound generally before the age of 15 years. Serious, life-threatening muscle wasting and respiratory and cardiac complications arise in late teens, and patients rarely survive into their fourth decade.
This is the first time a gene therapy based on rAAV-MD gene delivery by LR isolated limb perfusion and without immunosuppression has been performed in young adult GRMD dogs, showing clear prevention of degeneration/regeneration, fibrosis, magnetic resonance imaging and NMR changes and loss in muscle strength.
Unlike rodents, dog immunity shares many common features with its human counterpart with a full development before birth, although the maturity of the immune system completes after birth. An important observation of our study, in view of clinical translation, was the lack of systemic adverse effects and anti-cMD1 T-cell immune responses at the highest, efficacious dose of cMD1 vector, associated with long-term persisting transgene expression despite the occasional and transient detection of anti-cMD1 IgGs in most of the dogs. Importantly, the transient humoral immune response was apparently without serious consequences in the transduced muscles or in peripheral organs.