Engineered Stem Cells for Cardiac Repair

用于心脏修复的工程干细胞

• 批准号: 10544645
• 负责人: Charles E Murry
• 金额: $ 10.7万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10544645
• 关键词: ATP Synthesis Pathway; Actins; Acute; Award; Binding; Cardiac; Cardiac Muscle Contraction; Cardiac Myocytes; Cardiomyopathies; Cardiotonic Agents; Cell Line; Cell Therapy; Cell Transplantation; Cells; Chronic; Depressed mood; Dilated Cardiomyopathy; Duchenne muscular dystrophy; Elements; Engineering; Environment; Enzymes; Exhibits; Gap Junctions; Heart; Heart Contractilities; Heart failure; Hip region structure; Investigation; Ischemia; Light; Mechanics; Modeling; Modification; Mutation; Myocardial Infarction; Myocardial Ischemia; Myocardium; Myosin ATPase; Nucleotides; Parents; Performance; Pharmaceutical Preparations; Power stroke; Production; Pump; Rattus; Research Project Grants; Rest; Ribonucleotide Reductase; Span 20; System; Techniques; Testing; Time; Tissues; Transgenic Organisms; Translational Research; Variant; adeno-associated viral vector; cardiac repair; cell replacement therapy; engineered stem cells; experimental study; gene therapy; heart function; improved; improved outcome; in vivo; innovation; mouse model; novel; overexpression; parent project; small molecule

ABSTRACT: The parent project is built on two fundamental discoveries, spanning 20 years of mechanistic and translational research: 1) 2-deoxy ATP (dATP) is a potent natural nucleotide stimulant of cardiac contractility (via increased myosin binding to actin and faster detachment following the power stroke), and 2) hiPSC-CMs overexpressing the rate-limiting enzyme for dATP synthesis, ribonucleotide reductase (RNR), exhibit both increased contractility and dATP delivery to the rest of the heart via gap junctions. Consequently, we are investigating the hypothesis that altering hiPSC-CMs to increase RNR (RNR-hiPSC-CMs) improves outcomes in cell replacement treatment for MI (as compared to control hiPSC-CMs), by increasing the contractility of both the graft and native myocardium. Our technique incorporates several highly innovative elements. 1) This is the first time that cellular nucleotide modification has been offered as a means of improving in vivo heart function. 2) The technique is not restricted to replacing lost tissue (with hiPSC-CMs) with a more functional graft but may also significantly benefit the native myocardium's post-MI depressed function. 3) The first application of modified hiPSC-CMs to deliver a small molecule treatment (dATP) that enhances cardiac muscle contraction. This essentially transforms hiPSC-CMs into a cardiac-specific medication delivery system. Aim 1 is to generate and characterize engineered mutations in RNR that enhance its stability and activity in cardiomyocytes, as well as their ability to titrate increasing quantities of dATP produced in hiPSC-CMs. Aim 2 investigates the ability of RNR variations identified in Aim 1 to improve cardiac function in a mouse model of myocardial infarction and heart failure using AAV vectors. Aim 3 will generate engineered hiPS cell lines that, upon differentiation, will act as dATP 'donor cells' for transplantation into acute MI and more problematic chronic MI arrhythmic rat models to test their capacity to increase function beyond that of non-designed hiPSC-CMs. We will assess the persistence of these effects and the cell lines' long-term survival and stability. We anticipate a significant contractile improvement in both the graft and native myocardium using RNR-hiPSC-CMs vs. hiPSC- CMs, which will be controlled by the transplanted cells' dATP producing capacity. These investigations will shed light on the potential for this combination cell- and small-molecule therapy to improve or perhaps restore pump performance in failing hearts. This addition, as the candidate's research project, will expand the scope of the study by pursuing two objectives. Aim 1 will examine the mechanical and structural mechanisms that allow cardiac muscle utilizing dATP to be less sensitive to contractile strength losses when the pH is decreased, as occurs during ischemia. Aim 2 will investigate whether cardiac function can be improved in a different cardiomyopathy model (dilated instead of MI), utilizing a Duchenne’s Muscular Dystrophy (DMD) dilated cardiomyopathy model expressed in a transgenic rat.

摘要：父项目建立在两个基本发现的基础上，涵盖了20年的机械和 翻译研究：1）2-脱氧ATP（DATP）是一种潜在的自然核肽刺激剂（通过 肌球蛋白与肌动蛋白的结合增加，并在动力冲程之后更快地脱离）和2）HIPSC-CMS 过表达DatP合成的限速酶，核糖核苷酸还原酶（RNR），两者都暴露 通过间隙连接增加了收缩性和DATP向其他心脏的交付。因此，我们是 调查了改变HIPSC-CM提高RNR（RNR-IHIPSC-CMS）的假设可改善预后 在MI的细胞替代处理中（与对照HIPSC-CMS相比），通过增加两者的收缩力 移植物和本地心肌。我们的技术结合了几个高度创新的元素。 1）这是 首次提供了细胞核核苷酸修饰，以改善体内心脏功能。 2） 该技术不仅限于用功能更大的移植物代替丢失的组织（用HIPSC-CMS），但可以 还显着受益于本地心肌的MI后抑郁功能。 3）修改后的第一个应用 HIPSC-CMS提供小分子处理（DATP），可增强心肌收缩。这 基本上，将HIPSC-CMS转换为心脏特异性的药物输送系统。 目标1是在RNR中生成和表征工程突变，以增强其稳定性和活动 心肌细胞及其在HIPSC-CMS中滴定增加数量的DATP的能力。目标2 研究在AIM 1中鉴定出的RNR变化的能力，以改善小鼠模型中的心脏功能 使用AAV向量的心肌梗塞和心力衰竭。 AIM 3将生成工程的臀部细胞系， 分化后，将充当Datp“供体细胞”，以移植到急性MI和更多有问题的慢性 MI心律不齐的大鼠模型测试其增加功能超出未设计的HIPSC-CM的功能的能力。我们 将评估这些作用的持久性以及细胞系的长期生存和稳定性。我们期待一个 使用RNR-HIPSC-CMS与HIPSC- CMS，将由移植的细胞的DATP生产能力控制。这些调查将丢弃 阐明这种组合细胞和小分子疗法的潜力，以改善或恢复泵 失败的心脏表现。 作为候选人的研究项目，这一增加将通过追求两个来扩大研究范围 目标。 AIM 1将检查允许使用心脏肌肉的机械和结构机制 当pH降低时，DATP对收缩强度损失的敏感性降低了，如缺血期间所发生的那样。 AIM 2将研究在不同的心肌病模型中是否可以改善心脏功能（扩张） 而不是MI），使用Duchenne的肌肉营养不良（DMD）扩张的心肌病模型 转基因大鼠。