DISEASE MODELING AND PHENOTYPIC DRUG SCREENING FOR DYSTROPHIC CARDIOMYOPATHY

项目总结 目标：我们建议利用一系列基于人IPSC的心脏微生理系统 研究营养不良心肌细胞成熟是必要的假说的复杂性增加 阐明正确的疾病表型在体外的发展。其目的是创造一个多方面的筛查 使用我们团队开发的几项核心技术来评估心肌不同方面的系统 机电功能。我们已经开发出从患者尿液中设计多能干细胞的能力， 实现了从受试者身上进行非侵入性细胞采样。此外，我们还收集了初步数据 证明组合成熟刺激在健康和抗肌萎缩蛋白缺失中的应用 心肌细胞有助于区分疾病的表型。基于这些成就，我们假设使用 一套有针对性的功能分析与适当的成熟刺激相结合将提供更多 全面了解肌营养不良症的疾病进展。焦点/目标：我们建议的 研究的重点是使用具有协同作用的技术来增强心脏 不同细胞处理对干细胞来源心肌细胞表型发育的影响 机械装置。具体地说，我们将研究纳米衬底的结构组织效应， 甲状腺激素对核受体信号的影响及let-7对代谢信号通路的影响 MicroRNA在健康心肌细胞发育过程中的过度表达及其dystrophin缺失 使用CRISPR-Cas9基因编辑技术创建的对应物。纳米形貌微电极阵列 将用于评估电生理功能(目标1)，而纳米颗粒细胞片堆叠 该技术将用于创建3D心脏贴片，用于分析收缩功能(目标2)以及 组织3D脑室结构以评估压力产生和每搏输出量(目标3)。这其中的每一个 系统将被用来评估一组药物改善营养不良表型的潜力。 为这项研究选择的化合物针对一系列已知的代谢、结构和信号通路 与肌营养不良病理的不同方面有关。多功能化分析 因此，每个化合物的端点将提供有关以下可能影响的更全面的信息 当给人类病人使用药物时。从具有更高吞吐量水平的平台向 那些仿生程度较高的人，当工作从目标1过渡到目标3时，构成了一种自然的 “漏斗”式的药物筛选过程。使用更简单的多路复用模型确定的候选人将重新 使用提供更接近自然组织的表示的系统进行评估，并提供生理 类似于在患者中监测的终点。因此，提出的研究成熟度的方法 营养不良表型发育可能为下一代筛查过程提供框架 旨在用生理上越来越有代表性的动物模型取代动物试验 心肌。

PROJECT SUMMARY Goal: We propose to utilize a series of novel human iPSC-based cardiac microphysiological systems of increasing complexity to investigate the hypothesis that maturation of dystrophic cardiomyocytes is necessary to elucidate correct disease phenotype development in vitro. The aim is to create a multifaceted screening system using several core technologies developed by our group to evaluate different aspects of myocardial electromechanical function. We have developed the ability to engineer pluripotent stem cells from patient urine, enabling non-invasive cell sampling from human subjects. Furthermore, we have collected preliminary data demonstrating that application of combinatorial maturation stimuli to healthy and dystrophin-null cardiomyocytes helps stratify the disease phenotype. Based on these achievements, we posit that the use of a targeted set of functional assays in combination with appropriate maturation stimuli will provide a more comprehensive understanding of disease progression in muscular dystrophy. Focus/Aim: Our proposed research focuses on the use of techniques with the potential to act synergistically to enhance cardiac phenotype development in stem cell-derived cardiomyocytes through manipulation of different cellular mechanisms. Specifically, we will investigate the effect of structural organization by nanopatterned substrates, nuclear receptor signaling by thyroid hormone, and alterations in metabolic signaling pathways by Let-7 microRNA over-expression on the development of healthy cardiomyocytes and their dystrophin-null counterparts created using CRISPR-Cas9 gene editing technology. Nanotopographic microelectrode arrays will be used to evaluate electrophysiological function (Aim 1), while nanopatterned cell sheet stacking technology will be used to create 3D cardiac patches for analyzing contractile function (Aim 2) as well as organized 3D ventricle structures for assessing pressure generation and stroke volume (Aim 3). Each of these systems will be used to evaluate a panel of drugs for their potential to ameliorate the dystrophic phenotype. The compounds chosen for this study target a range of metabolic, structural, and signaling pathways known to be associated with different aspects of muscular dystrophy pathology. The analysis of multiple functional endpoints for each compound will therefore provide more comprehensive information on the likely effect of drugs when administered to human patients. The movement from platforms with higher levels of throughput to those with higher degrees of biomimicry, as the work transitions from Aim 1 to Aim 3, constitutes a natural “funneling” of the drug screening process. Candidates identified using simpler multiplexed models will be re- evaluated using systems that offer closer representations of the native tissue, and provide physiological endpoints analogous to those monitored in patients. As such, the proposed method for studying maturation and dystrophic phenotype development could provide the framework for a next generation screening process geared towards replacing animal testing with increasingly physiologically representative models of the myocardium.