Genome Editing of Human iPSCs to Study Inherited Hypertrophic Cardiomyopathy

项目总结 肥厚性心肌病(HCM)在普通人群中每500人中就有1人受到影响，是主要原因 心力衰竭和心源性猝死。尽管有重大的临床影响和正在进行的基本和 翻译研究，导致疾病发生和发展的分子机制很差 明白了。到目前为止，还没有建立起具体的治疗方法。人血清白蛋白体外模拟 心血管疾病引起高度关注，因为成人心肌细胞很难分离和繁殖 长期的培养和动物模型往往被证明不能预测人类的病理生理学。病人- 特异性人诱导多能干细胞来源的心肌细胞及其基因组编辑 代表了建模心肌病的新技术。在过去的资助期，我们能够 招募~150名肥厚型心肌炎患者。初步数据显示，高血压患者的舒张期功能障碍和异常的钙处理 肌球蛋白结合蛋白C3(MYBPC3)突变的IPSC-CMS。此外，初步数据显示， MYBPC3 IPSC-CMS无义介导衰变激活的分子表型 (NMD)途径。然而，IPSC-CMS的一个主要局限性是其状态不成熟以及不成熟。 目前所使用的体外检测的生理条件。在这里，我们建议增加 通过i)结合2D微图案化IPSC-CMS和3D工程心脏组织(EHTS)来建模HCM以 促进细胞成熟；通过II)与IPSC来源的内皮细胞(IPSC-ECs)共培养IPSC-CMS和 3DEHTS中的心脏成纤维细胞(IPSC-CFs)，以及(III)使用单细胞RNA-SEQ和蛋白质组学方法 目的：阐明HCM细胞串扰的分子机制。最后，我们将评估 CRISPR干扰(CRISPRi)和CRISPR激活在肥厚性心肌病发病中的NMD途径 (CRISPA)技术，它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。

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.