Elucidating Genotype-Phenotype Relationship of Polygenic Dilated Cardiomyopathies

阐明多基因扩张型心肌病的基因型-表型关系

• 批准号: 10532433
• 负责人: THOMAS QUERTERMOUS
• 金额: $ 12.86万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-16 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10532433
• 关键词: 3-Dimensional; Bioinformatics; Biological Assay; Blood Circulation; CRISPR screen; CRISPR/Cas technology; Cardiac Myocytes; Cardiovascular system; Cell Communication; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Congenital cardiomyopathy; Coupled; Databases; Development; Dilated Cardiomyopathy; Down-Regulation; Drug Screening; Drug Targeting; Endothelial Cells; Etiology; Family; Fibroblasts; Gene Expression; General Population; Genes; Genetic; Genetic study; Genome; Genomics; Genotype; Grant; Heart; Heritability; Human Genetics; Individual; Journals; Knowledge; Libraries; Mediating; Modeling; Molecular; Morbidity - disease rate; Paper; Pathogenesis; Pathogenicity; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Phenotype; Prevalence; Protocols documentation; Publications; Publishing; Qi; Research Personnel; Risk; Role; Signal Transduction; Techniques; Technology; Therapeutic; Tissue Engineering; Tungsten; Up-Regulation; Variant; base; cardiac tissue engineering; cell type; clinical phenotype; cohort; combinatorial; cost; cost efficient; design and construction; druggable target; gene function; gene interaction; genetic variant; genome editing; indexing; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; mortality; new therapeutic target; next generation sequencing; novel; recruit; screening; single-cell RNA sequencing; stem cells; therapeutically effective; transcriptome sequencing

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.

项目概要 扩张型心肌病 (DCM) 是全球发病率和死亡率的主要原因，据估计 普通人群的患病率为 1:250。最近的人类遗传学研究表明，复杂的 DCM 的遗传性可能是由多基因原因共同造成的。然而，它们之间复杂的相互作用 人们对各种遗传变异知之甚少，这阻碍了 DCM 有效疗法的开发。到 为了实现后者，我们将通过采用诱导多能干细胞衍生的最先进的方法 心肌细胞 (iPSC-CM) 平台与组织工程、单细胞 RNA 测序 (scRNA-seq) 相结合， 和 CRISPR/dCas9 基因组筛选技术，以探究基因变异之间的相互作用 DCM 的病因学并确定多基因 DCM 治疗的新药物靶点。在目标 1 中，我们将生成 iPSC 来自同一家庭队列的 20 名多基因 DCM 患者和 10 名对照品系，并创建了 120 个基因组 使用 CRISPR/Cas9 编辑同基因 iPSC 系。然后我们将区分多基因 DCM iPSC 和 iPSC-CM 对应的同基因 iPSC 系。使用生物信息分析，我们将生成一个基因 表达“评分指数”来评估每个遗传变异的致病性以及不同遗传变异之间的相互作用 遗传变异。在目标 2 中，我们将生成 3D 工程心脏组织 (EHT) 以阐明其功能 多基因 DCM 变异在 3D 水平上的后果。我们将制作由 iPSC-CM、iPSC- 组成的 3D EHT 来自这 20 种多基因 DCM 的衍生内皮细胞 (iPSC-EC) 和 iPSC 衍生成纤维细胞 (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/dCas9 基因组筛选 方法与 scRNA-seq 技术相结合，鉴定适合作为多基因 DCM 药物靶点的基因 iPSC-CM。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成药的药物。 与多基因 DCM 相关的基因。总的来说，拟议的目标将帮助我们全面阐明 多基因 DCM 的遗传和分子基础，并发现针对患者特异性的新药物靶点 疗法。