Elucidating Genotype-Phenotype Relationship of Polygenic Dilated Cardiomyopathies

项目摘要 扩张型心肌病（DCM）是全球发病率和死亡率的主要原因， 一般人群中的患病率为1：250。最近的人类遗传学研究表明， 扩张型心肌病的遗传性可能是多基因原因的组合。然而， 对各种遗传变异的了解较少，阻碍了DCM有效治疗方法的开发。到 实现后者，我们将使用最先进的方法，通过采用诱导多能干细胞衍生的 心肌细胞（iPSC-CM）平台与组织工程结合，单细胞RNA测序（scRNA-seq）， 和CRISPR/dCas 9基因组筛选技术，以询问基因组中遗传变异之间的相互作用。 DCM病因学和鉴定用于多基因DCM治疗的新药物靶点。在目标1中，我们将生成iPSC 来自同一家族队列的20名多基因DCM患者和10名对照，并创建120个基因组- 使用CRISPR/Cas9编辑的等基因iPSC系。然后，我们将分化多基因DCM iPSC， 将等基因iPSC系对应于iPSC-CM。利用生物信息学分析，我们将生成一个基因 表达“得分指数”来评估每个遗传变异体的致病性和不同基因之间的相互作用。 基因变异在目标2中，我们将生成3D工程化心脏组织（EHTs），以阐明功能 多基因DCM变体在3D水平的后果。我们将制作由iPSC-CM、iPSC- 来自这20个多基因DCM的iPSC-EC和iPSC-衍生的成纤维细胞（iPSC-FB 患者及其基因组编辑的同基因iPSC系，这使我们能够模拟复杂的细胞间相互作用， 并了解不同细胞类型之间的串扰。在目标3中，我们将使用CRISPR/dCas 9基因组筛选 结合scRNA-seq技术鉴定适合作为多基因DCM药物靶点的基因的方法 iPSC-CM。这项新技术提供了一种独特的和具有成本效益的方法来系统地筛选可药用的药物， 与多基因DCM相关的基因。总的来说，拟议的目标将有助于我们全面阐明 多基因DCM的遗传和分子基础，并发现新的药物靶点， 治疗学

PROJECT SUMMARY Dilated cardiomyopathy (DCM) is a leading cause of morbidity and mortality worldwide, with an estimated prevalence of 1:250 in the general population. Recent human genetic studies suggest that the complex heritability of DCM may be due to a combination of polygenic causes. However, the complex interactions among various genetic variants are less understood, hampering the development of effective therapeutics for DCM. To achieve the latter, we will use state-of-the-art approaches by employing the induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) platform coupled with tissue engineering, single cell RNA sequencing (scRNA-seq), and CRISPR/dCas9 genome screening technologies to interrogate the interplay between genetic variants in the etiology of DCM and identify novel drug target for polygenic DCM treatment. In Aim 1, we will generate iPSC lines from 20 polygenic DCM patients and 10 controls from the same family cohort, and create 120 genome- edited isogenic iPSC lines using CRISPR/Cas9. We will then differentiate the polygenic DCM iPSCs and corresponding isogenic iPSC lines to iPSC-CMs. Using bioinformatic analysis, we will generate a gene expression “score index” to assess the pathogenicity of each genetic variant and interactions between different genetic variants. In Aim 2, we will generate 3D engineered heart tissues (EHTs) to elucidate functional consequences of polygenic DCM variants at the 3D level. We will make 3D EHTs consisting of iPSC-CMs, iPSC- derived endothelial cells (iPSC-ECs), and iPSC-derived fibroblasts (iPSC-FBs) from these 20 polygenic DCM patients and their genome-edited isogenic iPSC lines, which allow us to simulate complex cell-cell interactions and understand the crosstalk among different cell types. In Aim 3, we will use CRISPR/dCas9 genome screening approach in combination with scRNA-seq technology to identify genes suitable as drug targets in polygenic DCM iPSC-CMs. This novel technique provides a unique and cost-efficient way to systemically screen for druggable genes associated with polygenic DCM. Collectively, the proposed aims will help us comprehensively elucidate the genetic and molecular basis of polygenic DCM and discover novel drug targets for patient-specific therapeutics.