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One major class of muscular dystrophy is limb-girdle muscular dystrophy (LGMD), encompassing many different subtypes. LGMD2E is one of the most severe forms of LGMD, resulting from recessive mutations in the ß-sarcoglycan (SGCB) gene, causing loss of functional protein. SGCB is a structural protein component of the dystrophin-associated protein complex (DAPC), which as a whole provides structural and mechanical stability to the sarcolemma. Due to the loss of protein, a devastating disorder ensues with widespread progressive muscle wasting, leading to loss of function. Disease progression is directly correlated with age, where the most severe cases have symptom onset in early childhood and many patients are rendered non-ambulant by their teens. In addition to skeletal muscle weakness, significant respiratory failure and fatal cardiomyopathy are common features in more severe LGMD2E patients. To date, no effective therapy exists to treat this debilitating disease. Thus, with an urgent need for a viable treatment option for LGMD2E patients, this work attempted to develop the first viral-mediated approach to restore wild-type (WT) SGCB. Providing a relevant model to test therapeutic efficacy, the Sgcb-null (sgcb-/-) mouse recapitulates the clinical phenotype and shares many of the pathologic features of LGMD2E patient biopsies including myofiber necrosis, central nucleation, inflammation, and fibrosis. We have engineered a viral mediated gene replacement therapy using adeno-associated virus (AAV) carrying a codon optimized human SGCB gene (hSGCB) driven by one of two different muscle specific promoters. We first established proof-of-principle efficacy of scAAVrh.74.tMCK.hSGCB by intramuscular (IM) injection and isolated-limb perfusion (ILP) in young and aged mice, which resulted in long-term widespread transgene expression accompanied by histological and function benefits. Notably, we saw a considerable reduction in fibrotic tissue, a significant component of the disease and a potential major obstacle of gene transfer. After switching promoters to the MHCK7 promoter which provides robust expression in cardiac tissue, we then demonstrated efficacy of systemic delivery of scAAVrh.74.MHCK7.hSGCB in treating skeletal and cardiac muscle deficits in sgcb-/- mice to provide a potential rationale for meaningful results in a clinical trial. This led to nearly 100% transgene expression in numerous muscles throughout the limbs, torso, and the heart, which was again accompanied by improvements in muscle histopathology and function, as well as increased overall activity. Importantly, a formal GLP toxicology study of AAV.hSGCB gene transfer in WT mice showed no adverse effects. In this well-defined model of LGMD2E, we have established a clinically relevant path for AAV mediated SGCB gene replacement therapy that has great promise for LGMD2E patients.After demonstrating the importance of functional deficits in dystrophic muscle to serve as outcome measures for functional recovery from SGCB gene therapy, we applied these concepts to the investigations of several other forms of LGMD, Types 2B and 2L, involving dysferlin (DYSF) and anoctomin5 (ANO5), respectively. In these studies, we optimized and validated muscle fiber isolation with a laser induced membrane repair assay to aid in thoroughly characterizing these diseases and test therapeutic interventions like viral-mediated gene transfer. Using this technique, we are able to directly measure a functional parameter of both DYSF and ANO5 and quantify the membrane repair ability in different physiological settings. Taken together, we report here an experimental method that can be a powerful tool for pre-clinical studies of muscular dystrophy. We also emphasize the importance of the pre-clinical efficacy study with SGCB gene replacement therapy and the impact it has in the translational pathway in providing a clinically meaningful therapy for LGMD2E patients.