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.

项目摘要 最近基因组工程技术的指数级进步重新唤起了人们对它的热情。 在遗传性心肌病中的应用。这对于致心肌病尤其有希望 (ACM)是心脏性猝死和终末期心力衰竭的原因。大多数早期的基因工程疗法 已经专注于基因替换;然而，ACM变异的一个显着的少数可能通过显性作用， 对基因替代疗法没有反应的负面疾病机制。RNA结合基序20 （RBM 20）和plakophilin 2（PKP 2）是与致命形式的ACM相关的基因，其中既有 显性阴性和单倍不足致病性变体。这些基因的变异导致心肌病 和心律失常，通过破坏整体心肌细胞转录剪接和桥粒结构， 分别这些变异聚集在致病热点，与已知的和新的功能性 蛋白质结构域，这表明对这些热点的集中研究可以阐明不同的疾病机制 并潜在地减少治疗设计的负担。我们的中心假设是致病基因的变异 RBM 20和PKP 2的热点具有不同的下游机制，其会聚于ACM疾病 表型，并且这些致病热点允许设计基因组工程策略来编辑 用一种试剂就能检测出多种致病性变异。在目标1中，我们将确定单倍不足与显性 RBM 20中的阴性变体。然后，我们使用高通量基因组工程技术来创建文库， 在诱导多能干细胞心肌细胞中的这些变体。我们将应用单个细胞的组合 文库制备和长读段RNAseq，以确定每种疾病的下游后果 已知的和新的RBM 20靶标的剪接机制。在目标2中，我们关注一种新的显性否定 C-末端PKP 2截短变体的机制，其中它们失去质膜定位， 将关键的桥粒组分隔离在细胞质中。我们将使用变体效果映射来定义 下游机制的致病性PKP 2截短变异体库，并将定义一个新的作用， PKP 2相互作用子对PKP 2膜定位的影响。在目标3中，我们将扩展我们的工作，展示以下可行性： 单引物编辑（PE）试剂，用于校正体外致病热点中的多个变体：我们将 设计工程化的引物编辑（epe）gRNA，具有PKP 2 C的最新高效PEmax构建体， 末端热点和显性阴性RBM 20 RS结构域热点。我们将利用创新 在AAVMYO中包装PEmax以在体内校正两种致病性鼠Rbm 20 RS结构域变体的方法 使用相同的epegRNA。我们将继续测量这种编辑对深度ACM表型的影响。在 总之，本项目将利用我们对RBM 20和PKP 2中致病热点的鉴定， 全面评价这些基因在变异水平上的致病机制，并展示其潜在的 热点导向的引物编辑作为一种易处理的基因组工程治疗。