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

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

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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.

项目摘要目标：我们建议利用一系列新型的基于IPSC的新型心脏微生物生理系统提高复杂性，以研究营养不良心肌细胞成熟的假设阐明体外正确疾病表型发育。目的是创建一个多面筛选系统使用我们小组开发的几种核心技术来评估心肌的不同方面机电功能。我们已经发展了从患者尿液中设计多能干细胞的能力，从人类受试者中实现非侵入性细胞采样。此外，我们收集了初步数据证明组合成熟刺激在健康和肌营养不良蛋白无效心肌细胞有助于分层疾病表型。基于这些成就，我们指出使用有针对性的功能测定与适当的成熟刺激结合使用将提供更多对肌肉营养不良的疾病进展的全面了解。重点/目标：我们的建议研究重点是使用技术，以协同行动以增强心脏通过操纵不同细胞的干细胞衍生心肌细胞中的表型发育特定的机制，我们将研究纳米图案底物的结构组织的效果，甲状腺激素的核接收器信号传导，以及Let-7的代谢信号通路的改变 MicroRNA过表达健康的心肌细胞及其肌营养不良蛋白无效使用CRISPR-CAS9基因编辑技术创建的对应物。纳米型微电极阵列将用于评估电生理功能（AIM 1），而纳米图案的堆叠技术将用于创建用于分析收缩功能（AIM 2）的3D心脏补丁以及有组织的3D通风结构用于评估压力产生和中风量（AIM 3）。每个系统将用于评估一组药物，以减轻营养不良的表型。为这项研究选择的化合物针对一系列已知的代谢，结构和信号传导途径与肌肉营养不良病理学的不同方面有关。多个功能的分析因此，每种化合物的端点将提供有关可能影响的更全面的信息药物送给人类患者。从具有较高吞吐量的平台到那些具有更高程度的仿生程度的人，作为从AIM 1到AIM 3的工作过渡，构成了自然药物筛查过程的“漏斗”。使用更简单的多路复用模型确定的候选人将重新验证使用提供本机组织更紧密表示的系统进行评估，并提供生理终点类似于患者监测的终点。因此，提出的研究成熟方法营养不良的表型开发可以为下一代筛查提供框架旨在用越来越多的物理代表性模型代替动物测试的模型心肌。