Limb-girdle muscular dystrophy (LGMD) type 2E results from mutations in the beta-sarcoglycan gene causing loss of functional protein. It is characterized by muscle weakness and progressive muscle wasting. The sarcoglycans (alpha, beta, gamma, and delta-SG) are structural proteins localized at the cell membrane of muscle fibers that together with dystrophin and other proteins make up the dystrophin-associated protein complex (DAPC). Loss of functional protein in this complex results in an unstable sarcolemma leading to eventual myofiber death. To date, no effective therapy exists to treat this debilitating disease. The goal of this study is to demonstrate efficacy of AAV-mediated beta-sarcoglycan gene transfer in beta-SG knock-out mice to provide proof-of-principle for gene replacement therapy for LGMD2E. A previous study by our group investigating AAV gene replacement therapy to treat LGMD2D led to a successful clinical trial in LGMD2D patients (alpha-sarcoglycan deficiency), which will provide a solid platform for this study. We generated a self-complementary AAVrh74 vector containing a codon optimized human beta-Sarcoglycan gene (hSGCB) driven by the muscle specific tMCK promoter. Following direct intramuscular injection to demonstrate potency of our vector, we treated beta-SG KO mice via a clinically relevant vascular delivery model to deliver AAV.tMCK.hSGCB in two doses (1x1011 and 5x1011 vg) to the lower limb muscles. We had clear demonstration of dose dependent beta-SG expression and improvement in dystrophic pathology with greater than 90% beta-SG expression in lower limb muscles following vascular delivery at high dose following 3months of treatment. Functional outcome measures performed on the extensor digitorum longus (EDL) muscle from these mice showed an improvement in specific force generation and protection from eccentric contraction induced injury. This pre-clinical study is pivotal for establishing proof-of-principle for translation of AAV.hSGCB gene transfer in LGMD2E patients.