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.

项目概要 最近基因组工程技术的指数级进步重新燃起了人们对其的热情 遗传性心肌病的实施。这对于致心律失常性心肌病尤其有希望 （ACM），导致心源性猝死和终末期心力衰竭的原因。最早期的基因组工程疗法 专注于基因替换；然而，极少数 ACM 变体可能通过显性作用发挥作用 对基因替代疗法没有反应的阴性疾病机制。 RNA 结合基序 20 (RBM20) 和 plakophilin 2 (PKP2) 是与致死性 ACM 相关的基因，其中同时存在 显性失活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来引起心律失常， 分别。这些变异聚集在与已知和新功能相一致的致病热点中 蛋白质结构域，表明对这些热点的集中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变异 RBM20 和 PKP2 的热点具有不同的下游机制，共同导致 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 使用单一试剂即可检测多种致病变异。在目标 1 中，我们将识别单倍体不足与显性 RBM20 中的负变体。然后，我们使用高通量基因组工程技术来创建文库 诱导多能干细胞心肌细胞中的这些变异。我们将应用单细胞的组合 文库制备和长读 RNAseq 来定义每种疾病的下游后果 已知和新型 RBM20 靶标的剪接机制。在目标 2 中，我们关注一种新颖的显性阴性 C 端 PKP2 截短变体的机制，其中它们失去质膜定位， 将关键的桥粒成分隔离在细胞质中。我们将使用变量效应映射来定义 致病性 PKP2 截短变体库的下游机制，并将定义新的作用 PKP2 膜定位上的 PKP2 相互作用子。在目标 3 中，我们将扩展我们的工作，展示以下项目的可行性： 用于在体外校正致病热点中的多个变异的单引物编辑（PE）试剂：我们将 使用最新的高效 PEmax 构建体为 PKP2 C 设计工程引物编辑 (epe)gRNA 体外末端热点和显性失活 RBM20 RS 结构域热点。然后我们将使用创新的 将 PEmax 包装到 AAVMYO 中以纠正体内两种致病性鼠 Rbm20 RS 结构域变异的方法 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 总之，该项目将利用我们对 RBM20 和 PKP2 致病热点的鉴定来提供 对这些基因的变异水平疾病机制进行全面评估，并展示其潜力 热点定向初等编辑作为一种易于处理的基因组工程疗法。