Pathogenic hotspots illuminate mechanism and therapeutic potential in arrhythmogenic cardiomyopathy

致病热点阐明致心律失常性心肌病的机制和治疗潜力

• 批准号: 10633507
• 负责人: Victoria Parikh
• 金额: $ 77.18万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-05 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10633507
• 关键词: Adult; Arrhythmia; Arrhythmogenic Right Ventricular Dysplasia; Binding; C-terminal; Calcium; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular system; Cell membrane; Cells; Cytoplasm; Data; Desmosomes; Disease; Dominant-Negative Mutation; Engineering; Evaluation; Exons; Failure; Genes; Genetic; Genetic Transcription; Genome engineering; Guide RNA; Heart failure; In Vitro; Individual; Inherited; Ions; Left; Libraries; Maps; Measures; Membrane; Methods; Minority; Molecular Abnormality; Mus; Nonsense-Mediated Decay; Pathogenicity; Pathology; Patients; Phenotype; Preparation; Prevalence; Protein Isoforms; RNA Binding; RNA Recognition Motif; RNA Splicing; Reagent; Role; Structure; System; Techniques; Technology; Tertiary Protein Structure; Therapeutic; Tissues; Transcript; Transgenic Organisms; Validation; Variant; Ventricular; Work; arrhythmogenic cardiomyopathy; autosome; design; disease phenotype; engineering design; gene interaction; gene replacement; gene replacement therapy; genome editing; in vivo; induced pluripotent stem cell; inherited cardiomyopathy; innovation; novel; novel therapeutics; overexpression; plakophilin 2; prime editing; prospective; single-cell RNA sequencing; sudden cardiac death; theories; therapeutic genome editing; transcriptome sequencing

PROJECT SUMMARY Recent exponential advancement of genome engineering technology has revived enthusiasm for its implementation in genetic cardiomyopathies. This is especially promising for arrhythmogenic cardiomyopathy (ACM), a cause of sudden cardiac death and end stage heart failure. Most early genome engineering therapies have focused on gene replacement; however, a significant minority of ACM variants likely act via dominant negative disease mechanisms that will not respond to gene replacement therapy. RNA Binding Motif 20 (RBM20) and plakophilin 2 (PKP2) are genes associated with deadly forms of ACM in which there are both dominant negative and haploinsufficient pathogenic variants. Variants in these genes cause cardiomyopathy and arrhythmia by disrupting global cardiomyocyte transcriptional splicing and desmosomal structure, respectively. hat these variants are clustered in pathogenic hotspots that align to known and novel functional protein domains, indicating that focused study of these hotspots can illuminate differential disease mechanisms and potentially reduce the burden of therapeutic design. Our central hypothesis is that variants in pathogenic hotspots of RBM20 and PKP2 have differential downstream mechanisms that converge on ACM disease phenotypes, and that these pathogenic hotspots allow the design of a genome engineering strategy to edit many pathogenic variants with a single reagent. In Aim 1, we will identify haploinsufficient vs. dominant negative variants in RBM20. We then use high throughput genome engineering techniques to create a library of these variants in induced pluripotent stem cell cardiomyocytes. We will apply a combination of single cell library preparation and long read RNAseq to define the downstream consequences of each disease mechanism on splicing of known and novel RBM20 targets. In Aim 2, we focus on a novel dominant negative mechanism for C-terminal PKP2 truncating variants in which they lose their plasma membrane localization, sequestering critical desmosome components in the cytoplasm. We will use variant effect mapping to define downstream mechanisms of a library of pathogenic PKP2 truncating variants, and will define the role of a novel PKP2 interactor on PKP2 membrane localization. In Aim 3, we will extend our work showing the feasibility of single prime editing (PE) reagents for correction of multiple variants in a pathogenic hotspot in vitro: We will design engineered prime editing (epe)gRNAs with the newest high efficiency PEmax construct for the PKP2 C- terminus hotspot and dominant negative RBM20 RS domain hotspot in vitro. We will then use innovative methods to package PEmax in AAVMYO to correct two pathogenic murine Rbm20 RS domain variants in vivo using the same epegRNA. We will go on to measure the effect of this editing on deep ACM phenotypes. In summary, this project will capitalize on our identification of pathogenic hotspots in RBM20 and PKP2 to provide a comprehensive evaluation of variant-level disease mechanism in these genes, and demonstrate the potential of hotspot directed prime editing as a tractable genome engineering therapeutic.

项目总结 最近基因组工程技术的指数级进步重新燃起了人们对其 在遗传性心肌病中的应用。这对致心律失常的心肌病尤其有希望。 (ACM)，心源性猝死和终末期心力衰竭的原因。大多数早期的基因组工程疗法 都专注于基因替换；然而，相当少数的ACM变体可能通过显性 对基因替代疗法不起作用的阴性疾病机制。RNA结合基序20 (RBM20)和血小板亲和素2(PKP2)是与致死型ACM相关的基因，在这些基因中都有 显性阴性和单倍体不足的致病变异。这些基因的变异会导致心肌病 通过破坏整体心肌细胞转录剪接和桥粒结构而导致心律失常， 分别进行了分析。这些变异是否聚集在致病热点中，这些热点与已知和新的功能 蛋白质结构域，表明对这些热点的重点研究可以阐明不同的疾病机制 并有可能减轻治疗设计的负担。我们的中心假设是致病基因的变异 RBM20和PKP2的热点具有不同的下游机制，在ACM疾病上趋同 表型，以及这些致病热点允许基因组工程策略的设计编辑 用一种试剂产生多种致病变种。在目标1中，我们将确定单倍性不足与显性 RBM20中的负变异体。然后，我们使用高通量基因组工程技术来创建一个文库 在诱导的多能干细胞心肌细胞中的这些变体。我们将应用单细胞的组合 文库准备和长时间阅读RNAseq以确定每种疾病的下游后果 已知和新的RBM20靶的剪接机制。在目标2中，我们关注一种新的显性否定 C-末端PKP2截断变异体失去质膜定位的机制， 将关键的桥粒成分隔离在细胞质中。我们将使用变体效果映射来定义 下游机制的致病PKP2截断变异体文库，并将定义一个新的作用 PKP2相互作用蛋白对PKP2膜定位的影响。在目标3中，我们将扩展我们的工作，展示 单一基础编辑(PE)试剂用于校正体外致病热点中的多个变异：我们将 为PKP2 C设计最新的高效PEmax构建工程优质编辑(EPE)gRNAs 终末热点和显性负性RBM20RS结构域体外热点。然后我们将使用创新的 方法将PEmax包装在AAVMYO中，用于体内纠正两种致病小鼠RBM20RS结构域变异 使用相同的epegRNA。我们将继续测量这种编辑对深层ACM表型的影响。在……里面 总结，这个项目将利用我们在RBM20和PKP2中发现的致病热点来提供 对这些基因变异水平的致病机制进行了综合评价，并展示了其潜在的作用。 热点导向的优质编辑作为一种易于处理的基因组工程疗法。