PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM), a disease of abnormal heart muscle thickening, is the most common form of genetic heart disease in the United States affecting upwards of 1 in 500 people. While HCM- causing mutations are found in various sarcomere protein-encoding genes, over one-third of all HCM-causing mutations occur in the Myosin Heavy Chain 7 (MYH7) gene, which encodes for beta-myosin heavy chain, a motor ATPase that incorporates into the thick filament of cardiac muscle and plays a major role in cardiac contraction. Missense mutations within MYH7 allow the incorporation of mutant myosin heads into cardiac sarcomeres, which leads to increased cardiomyocyte energy consumption, hypercontractility, and the initiation of the disease progression of HCM. For patients, complications of HCM include heart failure, arrhythmia, and sudden cardiac death. As HCM has no cure aside from transplant, there is an urgent need for novel therapeutic strategies. CRISPR-Cas9-mediated genome editing has emerged as an attractive method to correct and potentially cure genetically-based diseases. CRISPR-Cas9 and its variants, including base editing and prime editing, allow targeted genomic editing at specific loci. Base editing allows precise single nucleotide edits, which makes it an ideal therapeutic tool to correct the hundreds of documented HCM-causing missense mutations. Several studies have successfully demonstrated the use of adeno-associated virus (AAV)-mediated gene editing to modify disease-causing mutations in various tissues and rescue pathological phenotypes in small and large animal models. In particular, various studies have shown robust gene editing in the heart to treat the muscle disease Duchenne muscular dystrophy, raising the prospect of successful gene editing in the heart to treat other cardiac diseases. The Specific Aims of this study seek to demonstrate base editing as a cure for MYH7 mutations of hypertrophic cardiomyopathy in 1) human induced pluripotent stem cell-derived cardiomyocytes and 2) a mouse model of HCM. Both models of HCM contain the same pathogenic mutation commonly found in patients and the mouse model faithfully recapitulates the human phenotype of hypertrophic cardiomyopathy. The central hypothesis of this proposal is that AAV delivery of base editing components to each of these models will prevent HCM and revert established HCM. This proposal will assess gene editing on-target and off-target efficiencies and changes in cardiac function and pathology. Furthermore, these studies will develop and optimize a base editing strategy to cure hypertrophic cardiomyopathy and will lay a foundation for the development of gene editing therapies for the hundreds of other HCM-causing genetic mutations.