Long-term functional adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog

Background: Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle-wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno-associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of microdystrophin in dystrophic dogs and a lack of long-term microdystrophin expression associated with a CD8 ⁺T-cell response in DMD patients suggests that the development of improved microdystrophin genes and delivery strategies is essential for successful clinical trials in DMD patients. Methods: We have previously shown the efficiency of mRNA sequence optimization of mouse microdystrophin in ameliorating the pathology of dystrophic mdx mice. In the present study, we generated adeno-associated virus (AAV)2/8 vectors expressing an mRNA sequence-optimized canine microdystrophin under the control of a muscle-specific promoter and injected intramuscularly into a single canine X-linked muscular dystrophy (CXMDj) dog. Results: Expression of stable and high levels of microdystrophin was observed along with an association of the dystrophin-associated protein complex in intramuscularly injected muscles of a CXMDj dog for at least 8weeks without immune responses. Treated muscles were highly protected from dystrophic damage, with reduced levels of myofiber permeability and central nucleation. Conclusions: The data obtained in the present study suggest that the use of canine-specific and mRNA sequence-optimized microdystrophin genes in conjunction with a muscle-specific promoter results in high and stable levels of microdystrophin expression in a canine model of DMD. This approach will potentially allow the reduction of dosage and contribute towards the development of a safe and effective AAV gene therapy clinical trial protocol for DMD.