Elucidating Genotype-Phenotype Relationship of Polygenic Dilated Cardiomyopathies

项目总结 扩张型心肌病(DCM)是全世界发病率和死亡率的主要原因，据估计 在普通人群中的患病率为1：250。最近的人类遗传学研究表明，这种复合体 扩张型心肌病的遗传性可能是多基因原因的组合。然而，它们之间的复杂相互作用 对各种遗传变异的了解较少，阻碍了扩张型心肌炎有效治疗方法的发展。至 为了实现后者，我们将使用最先进的方法，通过使用诱导多能干细胞来源的 心肌细胞(IPSC-CM)平台与组织工程、单细胞RNA测序(scRNA-seq)、 和CRISPR/dCas9基因组筛选技术，以询问遗传变异之间的相互作用 扩张型心肌病的病因学和寻找治疗多基因扩张型心肌病的新药物靶点。在目标1中，我们将生成IPSC 来自20个多基因扩张型心肌病患者和10个来自同一家庭队列的对照，创建了120个基因组。 用CRISPR/Cas9编辑等基因IPSC品系。然后我们将区分多基因DCM IPSCs和 与IPSC-CMS等基因系相对应。利用生物信息学分析，我们将产生一个基因 表达“得分指数”来评估每个基因变体的致病性以及不同基因变体之间的相互作用 基因变异。在目标2中，我们将生成3D工程心脏组织(EHTS)来阐明功能 3D水平上的多基因DCM变异的后果。我们将制作由IPSC-CMS、IPSC-CMS和IPSC-CMS组成的3D EHTS 这20个多基因DCM来源的内皮细胞(IPSC-ECs)和IPSC来源的成纤维细胞(IPSC-FBS) 患者及其基因组编辑的等基因IPSC系，使我们能够模拟复杂的细胞-细胞相互作用 并了解不同小区类型之间的串扰。在目标3中，我们将使用CRISPR/dCas9基因组筛选 结合scRNA-seq技术筛选适合作为多基因扩张型心肌病药物靶点的基因 IPSC-CMS。这项新技术提供了一种独特且经济高效的方法来系统地筛选可用药物 与多基因扩张型心肌病相关的基因。总而言之，拟议的目标将有助于我们全面阐明 多基因扩张型心肌病的遗传学和分子基础及针对患者的新药物靶点的发现 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.