DISEASE MODELING AND PHENOTYPIC DRUG SCREENING FOR DYSTROPHIC CARDIOMYOPATHY

营养不良性心肌病的疾病建模和表型药物筛选

基本信息

批准号：
10116566
负责人：
Deok-Ho Kim
金额：
$ 52.09万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-15 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10116566
关键词：
3-Dimensional Achievement Address Adult Animal Testing Animals Architecture Biological Assay Biomedical Engineering CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiomyopathies Cell Culture Techniques Cell Density Cell Survival Cells Characteristics Clinical Trials Complex Cues Cytoskeleton Data Development Disease Progression Disease model Drug Screening Drug Targeting Duchenne muscular dystrophy Dystrophin EFRAC Electrophysiology (science)Engineering Ensure Environment Exhibits Exposure to Extracellular Matrix Failure Functional disorder Generations Genes Goals Heart Heart Rate Human In Vitro Individual Investigation Kinetics L-Type Calcium Channels Left ventricular structure Measures Mediating Membrane Metabolic Methods MicroRNAs Microelectrodes Modeling Monitor Movement Muscular Dystrophies Myocardial Myocardial dysfunction Myocardium Nature Nitric Oxide Nuclear Receptors Organoids Oxidative Stress Pathway Pathology Pathway interactions Patients Pharmaceutical Preparations Phenotype Physiological Pluripotent Stem Cells Process Property Pump Receptor Signaling Relaxation Research Role Sampling Series Signal Pathway Signal Transduction Stimulus Stroke Volume Structure Symptoms System Techniques Technology Testing Thyroid Hormones Time Tissue Engineering Tissues Urine Validation Ventricular Work base combinatorial disease phenotype drug discovery drug efficacy dystrophic cardiomyopathy fetal heart function heart rate variability hormonal signals human model human stem cells human subject human tissue immunocytochemistry improved induced pluripotent stem cell microphysiology system nanopattern next generation novel organizational structure overexpression pre-clinical pressure response screening stem cells success therapy design three-dimensional modeling

项目摘要

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.

项目概要目标：我们建议利用一系列新型的基于人类 iPSC 的心脏微生理系统增加营养不良心肌细胞成熟必要性这一假设的复杂性阐明体外正确的疾病表型发展，目的是建立多方面的筛查。系统采用我组开发的多项核心技术来评估心肌的不同方面我们已经开发出从患者尿液中改造多能干细胞的能力，此外，我们还收集了初步数据。证明组合成熟刺激对健康和肌营养不良蛋白无效的应用基于这些成就，心肌细胞有助于对疾病表型进行分层。一系列有针对性的功能测定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：我们提出的建议。研究重点是使用具有协同作用潜力的技术来增强心脏功能通过操纵不同细胞来实现干细胞衍生心肌细胞的表型发育具体来说，我们将研究纳米图案基底对结构组织的影响，甲状腺激素的核受体信号转导以及 Let-7 的代谢信号转导途径的改变 microRNA 过度表达对健康心肌细胞及其抗肌营养不良蛋白缺失的发育的影响使用 CRISPR-Cas9 基因编辑技术创建的手臂。将用于评估电生理功能（目标 1），而纳米图案细胞片堆叠技术将用于创建 3D 心脏贴片，用于分析收缩功能（目标 2）以及组织 3D 心室结构，用于评估压力产生和每搏输出量（目标 3）。系统将用于评估一组药物改善营养不良表型的潜力。本研究选择的化合物针对一系列已知的代谢、结构和信号传导途径与肌营养不良症病理学的不同方面进行相关的多功能分析。因此，每种化合物的终点将提供有关其可能影响的更全面的信息将药物从具有更高吞吐量的平台转移到人类患者身上。那些仿生程度较高的人，随着作品从目标 1 过渡到目标 3，构成了自然的使用更简单的多重模型确定的药物筛选过程的“漏斗”将被重新定义。使用能够更接近地表示天然组织并提供生理学信息的系统进行评估终点类似于在患者中监测的终点，因此，提出了研究成熟的方法。营养不良表型的发展可以为下一代筛选过程提供框架旨在用越来越具有生理代表性的模型取代动物测试心肌。