Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is the most common, disabling and lethal muscle disease, afflicting one of every 3500 males. Recently, we have generated a series of highly truncated mini-dystrophin genes that had large deletions in the """"""""non-essential"""""""" regions including part of the central rod domain and the very C-terminus domain. These minigenes were small enough to be packaged into adeno-associated virus (AAV) vectors and large enough to preserve high functionality, when tested in mdx mice after local intramuscular gene delivery. However, the mdx mice are far from an ideal DMD animal model although it is a commonly used one. While manifesting many similar symptoms of the human patients such as the muscle pathology, the mdx mice do not suffer shortened lifespan and do not show overall muscle weakness and skeletal contractures as do the human patients. This phenomenon is due to the up-regulation of utrophin gene (a dystrophin analogue) that partially compensates the defects of dystrophin in the mdx mice. By knocking out both dystrophin and utrophin genes (double-KO), two teams have recently developed a severe DMD mouse model that closely reflects every major deficiency seen in the human patients including much shortened life-span, severe muscle weakness and skeletal contractures, offering a more truthful small animal model for more stringent tests of new therapeutics. In this grant proposal, we will use the newly available double-KO mice to vigorously test the hypothesis whether the novel mini-dystrophin genes are able to rescue the muscle functions locally and systemically, and more importantly, to improve the overall health and prolong the life-span of the severe DMD animal, which is key to the development of a clinically efficacious gene therapy strategy. In this proposal, we will investigate 1) biological/therapeutic functions of mini-dystrophin genes in the double-KO mice using the transgenic mouse technology; 2) therapeutic effects of mini-dystrophin genes in both young and adult double-KO mice after local intramuscular injection of AAV vectors; 3) systemic gene delivery and its therapeutic effects in large groups of muscles and the entire body; 4) alternative therapeutic genes that may offer synergistic effects along with the minigenes to benefit the dystrophic muscles.
