Genome Editing of Human iPSCs to Study Inherited Hypertrophic Cardiomyopathy

人类 iPSC 的基因组编辑研究遗传性肥厚型心肌病

基本信息

批准号：
10213111
负责人：
Lei Stanley Qi
金额：
$ 66.97万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-10 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10213111
关键词：
3-Dimensional Adult Affect Animal Model Basic Science Binding Proteins Biological Biology Biomedical Engineering Blood Circulation CRISPR interference Calcium Cardiac Cardiac Myocytes Cardiomyopathies Cardiovascular Diseases Cardiovascular system Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Coculture Techniques Communication Complex Data Developmental Biology Disease Disease Progression Endothelial Cells Engineering Evaluation Familial Hypertrophic Cardiomyopathy Fibroblasts Functional disorder Funding G-Protein-Coupled Receptors General Population Genes Genomics Heart Heart failure Human Hypertrophic Cardiomyopathy In Vitro Individual Inherited Journals Lead Mediator of activation protein Medicine Modeling Molecular Mutation Myosin ATPase Nonsense-Mediated Decay Onset of illness Paper Pathogenesis Pathology Pathway interactions Patients Pattern Phenotype Physiological Proteomics Publishing Qi Technology Therapeutic Tissue-Specific Gene Expression Translational Research Tungsten Wing cardiac tissue engineering cell type data management disease-in-a-dish fetal genome editing in vitro Assay in vitro Model in vivo induced pluripotent stem cell interest molecular phenotype new technology new therapeutic target novel therapeutic intervention recruit screening single cell sequencing single-cell RNA sequencing statistics stem cells sudden cardiac death therapeutic target tool transcriptome sequencing

项目摘要

PROJECT SUMMARY Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people in the general population and is a leading cause of heart failure and sudden cardiac death. Despite the significant clinical impact and ongoing basic and translational research, the molecular mechanisms leading to disease onset and progression are poorly understood. Until now, no specific therapeutic approach has been established. In vitro modeling of cardiovascular diseases is of high interest as human adult cardiomyocytes are difficult to isolate and propagate long-term in culture, and animal models have often proven non-predictive of human pathophysiology. Patient- specific human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and genome editing represent novel technologies for modeling cardiomyopathies. In the past funding period, we were able to recruit ~150 HCM patients. Preliminary data shows diastolic dysfunction and aberrant calcium handling in iPSC-CMs with mutations in myosin binding protein C3 (MYBPC3). Furthermore, preliminary data shows a molecular phenotype in MYBPC3 iPSC-CMs characterized by an activation of non-sense mediated decay (NMD) pathway. However, a major limitation of iPSC-CMs is their immature state as well as the non- physiological conditions of in vitro assays used so far. Here we propose to increase the significance of modeling HCM by i) incorporating 2D micropatterned iPSC-CMs and 3D engineered heart tissues (EHTs) to enhance cellular maturation; by ii) co-culturing iPSC-CMs with iPSC-derived endothelial cells (iPSC-ECs) and cardiac fibroblasts (iPSC-CFs) in 3D EHTs, and by (iii) using single cell RNA-seq and proteomics approaches to elucidate molecular mechanisms of HCM cellular crosstalk. Finally, we will evaluate the significance of the NMD pathway in HCM pathogenesis by using the CRISPR-interference (CRISPRi) and CRISPR-activation (CRISPa) technology, which allows us to screen expeditiously the functional relevance of inhibition or activation of a set of genes followed by downstream functional evaluation to localize the target genes with therapeutic potentials.

项目概要肥厚型心肌病 (HCM) 影响普通人群中五百分之一的人，是主要原因心力衰竭和心源性猝死。尽管具有重大的临床影响和持续的基础和转化研究中，导致疾病发生和进展的分子机制尚不清楚明白了。到目前为止，尚未建立具体的治疗方法。体外建模由于人类成年心肌细胞难以分离和繁殖，心血管疾病受到高度关注长期培养中，动物模型常常被证明不能预测人类病理生理学。病人- 特定的人类诱导多能干细胞衍生心肌细胞 (iPSC-CM) 和基因组编辑代表了心肌病建模的新技术。在过去的资助期间，我们能够招募约 150 名 HCM 患者。初步数据显示舒张功能障碍和钙处理异常肌球蛋白结合蛋白 C3 (MYBPC3) 发生突变的 iPSC-CM。此外，初步数据显示 MYBPC3 iPSC-CM 中的分子表型以无义介导的衰变激活为特征（NMD）途径。然而，iPSC-CM 的一个主要限制是其不成熟状态以及非迄今为止使用的体外测定的生理条件。在这里我们建议增加重要性通过 i) 结合 2D 微图案 iPSC-CM 和 3D 工程心脏组织 (EHT) 来建模 HCM 促进细胞成熟； ii) 将 iPSC-CM 与 iPSC 衍生的内皮细胞 (iPSC-EC) 共培养，以及 3D EHT 中的心脏成纤维细胞 (iPSC-CF)，以及 (iii) 使用单细胞 RNA 测序和蛋白质组学方法阐明 HCM 细胞串扰的分子机制。最后，我们将评估该项目的意义利用 CRISPR 干扰 (CRISPRi) 和 CRISPR 激活研究 HCM 发病机制中的 NMD 通路（CRISPa）技术，使我们能够快速筛选抑制或激活一组基因，然后进行下游功能评估，以定位目标基因治疗潜力。