Base Editing Gene Correction of Pathogenic MYH7 Mutations in Models of Hypertrophic Cardiomyopathy

项目总结/摘要 肥厚型心肌病（HCM）是一种心肌异常增厚的疾病，是最常见的心肌病。 一种常见的遗传性心脏病，在美国每500人中就有1人患病。虽然HCM- 在各种肌节蛋白编码基因中发现了导致HCM的突变， 突变发生在肌球蛋白重链7（MYH 7）基因中，该基因编码β-肌球蛋白重链， 一种运动ATP酶，结合到心肌的粗丝中，并在心脏运动中起主要作用。 收缩。MYH 7内的错义突变允许突变的肌球蛋白头掺入心脏 肌节，这导致心肌细胞能量消耗增加，过度收缩， HCM的疾病进展。对于患者，HCM的并发症包括心力衰竭、心律失常和 心源性猝死由于HCM除了移植外没有治愈方法，因此迫切需要新的 治疗策略 CRISPR-Cas9介导的基因组编辑已经成为一种有吸引力的方法来纠正和 有可能治愈遗传疾病CRISPR-Cas9及其变体，包括碱基编辑和引物 编辑，允许在特定基因座进行靶向基因组编辑。碱基编辑允许精确的单核苷酸编辑， 这使得它成为一个理想的治疗工具，以纠正数百个记录在案的HCM引起的误解， 突变。几项研究已经成功地证明了使用腺相关病毒（AAV）介导的 基因编辑，以修改各种组织中的致病突变，并挽救 小型和大型动物模型。特别是，各种研究表明，心脏中的强大基因编辑， 治疗肌肉疾病杜氏肌营养不良症，提高了基因编辑成功的前景， 治疗其他心脏疾病。 本研究的具体目的是试图证明碱基编辑作为治疗MYH 7突变的方法， 在1）人诱导多能干细胞衍生的心肌细胞和2） HCM小鼠模型。两种HCM模型都含有常见于患者中的相同致病性突变 并且小鼠模型忠实地再现了肥厚型心肌病的人类表型。的 该提议的中心假设是，AAV将基础编辑组分递送到这些模型中的每一个 将防止HCM并恢复已建立的HCM。该提案将评估基因编辑的靶向和脱靶 心脏功能和病理学的效率和变化。此外，这些研究将继续发展， 优化碱基编辑策略治疗肥厚型心肌病，将为 为数百种其他导致HCM的基因突变开发基因编辑疗法。

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.