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

项目摘要/摘要 肥厚性心肌病(HCM)是一种心肌异常增厚的疾病，是世界上最常见的心肌肥厚疾病 美国常见的遗传性心脏病，每500人中就有1人受到影响。而HCM- 在各种肌节蛋白编码基因中发现了导致突变的基因，在所有引起肥厚性心肌病的基因中，有三分之一以上是导致突变的 肌球蛋白重链7(MYH7)基因发生突变，它编码β-肌球蛋白重链，a 运动ATPase结合到心肌的粗丝中，在心脏中起主要作用 收缩。MYH7内的错义突变允许突变的肌球蛋白头部进入心脏 肌节，这导致心肌细胞能量消耗增加，过度收缩，并启动 对肥厚型心肌炎的疾病进展进行研究。对于患者来说，肥厚性心肌炎的并发症包括心力衰竭、心律失常和 心源性猝死。由于HCM除了移植之外没有治愈的方法，因此迫切需要新的 治疗策略。 CRISPR-Cas9介导的基因组编辑已成为一种有吸引力的纠正和 有可能治愈基于基因的疾病。CRISPR-Cas9及其变体，包括基本编辑和主要 编辑，允许在特定位置进行有针对性的基因组编辑。碱基编辑允许精确的单核苷酸编辑， 这使得它成为纠正数百个已记录的引起肥厚型心肌病的误解的理想治疗工具 突变。一些研究已经成功地证明使用腺相关病毒(AAV)介导的 基因编辑以修改各种组织中的致病突变并挽救动物的病理表型 小动物模型和大动物模型。特别是，各种研究表明，心脏中强大的基因编辑 治疗肌肉疾病Duchenne肌营养不良症，提高了成功进行基因编辑的前景 心脏治疗其他心脏疾病。 这项研究的具体目的是试图证明碱基编辑是治愈MYH7突变的方法 肥厚性心肌病：1)人诱导的多能干细胞来源的心肌细胞和2)a 人巨噬细胞瘤小鼠模型。这两种类型的肥厚型心肌炎含有在患者中常见的相同的致病突变 小鼠模型忠实地概括了人类肥厚型心肌病的表型。这个 这一提议的中心假设是，AAV将基本编辑组件传递到每个模型 将阻止HCM并恢复已建立的HCM。这项提案将评估目标上和目标外的基因编辑 心功能和病理的效率和变化。此外，这些研究将发展和 优化基础编辑策略以治疗肥厚型心肌病，并将为 开发针对数百种其他引起HCM的基因突变的基因编辑疗法。

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.