ABSTRACT The goal of this proposal is to complete the remaining pre-clinical proof of concept and safety studies required to file a pre-IND application with the FDA for a first-in-class bicistronic AAV gene therapy for patients with Limb Girdle Muscular Dystrophy Type R9 (LGMDR9). Gene therapy has recently shown great promise in treating some forms of muscular dystrophy as a gene replacement technology, wherein a normal functional version of the gene mutated in the disease is introduced back into the muscles of the patient. Such gene replacement technologies are designed to prevent further development of disease symptoms, but this strategy will do little to reverse disease in patients where loss of muscle mass and strength is already present. To accomplish disease reversal for muscular dystrophy patients, a bicistronic, or two gene, therapy is needed wherein disease prevention is accomplished through gene replacement while a second gene function is provided to rebuild muscle size and strength. We have developed such a bicistronic Adeno Associated Virus (AAV) gene therapy for this purpose. Such bicistronic vectors will be compared to monocistronic (one gene) vectors to demonstrate their superiority in reversing disease symptoms in a model of LGMDR9 (formerly called LGMD2i). Experiments will be done to understand the optimal formulation needed for an effective bicistronic gene therapy for LGMDR9 and also determine the safety of such a treatment. If successful, these experiments would provide a platform to design new improved therapies almost two dozen other forms of LGMD and Congenital Muscular Dystrophy (CMD).