Duchenne muscular dystrophy gene therapy in the canine model

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by loss of function mutations in the dystrophin gene, resulting in the absence of dystrophin, a structural protein in muscle. DMD is the most common form of inherited muscle disease in childhood, with an incidence of 1 in 5000 live male births worldwide. The dystrophin-null mdx mouse has been the most widely used animal model for DMD research over the last 30 years. Dystrophin-deficient DMD dogs have also gained prominence as a highly relevant preclinical animal model due to their high phenotypic homology to human DMD patients. Preclinical treatment studies in these dogs are expected to better inform and guide clinical trials in human patients. However, there are still significant gaps in our understanding of the disease pathogenesis and gene therapy in the canine model. The goals of my dissertation work were to establish reagents and methodologies to study preclinical treatment in the canine model, and subsequently characterize the disease pathogenesis and gene therapy in DMD dogs. To this end, I first characterized 65 epitope-specific human dystrophin monoclonal antibodies for their reactivity in canine skeletal and cardiac muscle by both immunofluorescence (IF) staining and western blot. I found species-specific, tissue-specific and assay-specific patterns of reactivity in these antibodies. Importantly, out of the 65 antibodies that I characterized, I recognized 15 antibodies that worked well for canine tissue on both IF staining and western blot, which are recommended for DMD research in the canine model. ... Dystrophin-independent gene therapy for DMD takes advantage of disease-modifying genes that are either structural and/or functional homologues of dystrophin, or alternative targets that are involved in disease pathogenesis. One such alternative target gene is the sarcoplasmic reticulum calcium ATPase 2a (SERCA2a), a pump that transports calcium ions from the cytoplasm into the sarcoplasmic reticulum. I show that SERCA2a expression and activity are impaired, and that calcium homeostasis is dysregulated in DMD dog skeletal muscle. Furthermore, gene therapy with human SERCA2a restored expression and activity of the pump, and improved several aspects of muscle function and histopathology in DMD dog skeletal muscle. In summary, this dissertation work advances our knowledge of the disease pathogenesis and gene therapy prospects in the canine model of DMD, a highly relevant and valuable preclinical DMD model.