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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要基因组工程技术的最新指数进步恢复了对其的热情遗传性心肌病的实施。这对于心律不齐的心肌病特别有希望（ACM），这是心脏突然死亡和终结阶段心力衰竭的原因。大多数早期基因组工程疗法专注于基因替代；但是，很少有ACM变体可能通过主导作用不对基因替代疗法反应的阴性疾病机制。 RNA结合基序20 （RBM20）和plakophilin 2（PKP2）是与致命形式的ACM相关的基因致病性变异的主要阴性和单倍损害。这些基因的变体引起心肌病通过破坏全球心肌细胞的转录剪接和脱染色体结构，而心律不齐，分别。帽子这些变体聚集在与已知和新功能相符的致病热点中蛋白质结构域，表明对这些热点的重点研究可以照亮差异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性的变体 RBM20和PKP2的热点具有差异下游机制，这些机制会融合ACM疾病表型，这些病原热点允许设计基因组工程策略来编辑许多带有单个试剂的致病变体。在AIM 1中，我们将确定单倍宽松与主导地位 RBM20中的负变体。然后，我们使用高吞吐量基因组工程技术来创建库在诱导多能干细胞心肌细胞中的这些变体中。我们将使用单个单元的组合图书馆的准备和长期读取RNASEQ，以定义每种疾病的下游后果已知和新型RBM20目标剪接的机制。在AIM 2中，我们专注于新颖的负面负面 C末端PKP2截断变体的机制，其中失去了质膜定位，隔离细胞质中的临界朝向小体成分。我们将使用变体效果映射来定义致病性PKP2截断变体库的下游机制，并将定义新颖的作用 PKP2相互作用器在PKP2膜定位上。在AIM 3中，我们将扩展工作表明的可行性在体外，用于校正多种变体的单次序编（PE）试剂：我们将设计工程的Prime编辑（EPE）GRNA具有最新的PKP2 C-最新高效率Pemax结构体外末端热点和主要的负RB20 RS域热点。然后，我们将使用创新在Aavmyo中打包Pemax的方法以纠正体内的两个致病鼠RBM20 RS域变体使用相同的Epegrna。我们将继续衡量该编辑对深度ACM表型的影响。在总而言之，该项目将利用我们在RBM20和PKP2中对病原热点的识别以提供对这些基因中变异级疾病机制的全面评估，并证明了潜力 Hotspot将Prime编辑定向为一种可拖动的基因组工程治疗。