Engineered Stem Cells for Cardiac Repair

用于心脏修复的工程干细胞

• 批准号: 10293039
• 负责人: Charles E Murry
• 金额: $ 5.4万
• 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 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10293039
• 关键词: ATP Synthesis Pathway; Actins; Acute; Award; Binding; Cardiac; Cardiac Myocytes; Cardiotonic Agents; Cell Line; Cell Survival; Cell Therapy; Cell Transplantation; Cells; Chronic; Depressed mood; Devices; Dilated Cardiomyopathy; Drug Delivery Systems; Engineering; Environment; Enzymes; Gap Junctions; Heart; Heart Contractilities; Heart failure; Hip region structure; Ischemia; Modeling; Muscle Contraction; Muscular Dystrophies; Mutation; Myocardial Infarction; Myocardial Ischemia; Myocardium; Myosin ATPase; Nucleotides; Nude Rats; Parents; Power stroke; Pump; Rattus; Research Project Grants; Rest; Ribonucleotide Reductase; Testing; Tissues; Transgenic Model; Translational Research; Transplantation; Variant; adeno-associated viral vector; base; cardiac repair; cell replacement therapy; engineered stem cells; experimental study; gene therapy; graft function; heart function; improved; improved functioning; improved outcome; in vivo; mouse model; novel; overexpression; parent project; small molecule

ABSTRACT. The parent project is built around 20 years of mechanistic and translational research based on two fundamental discoveries: 1) 2-deoxy ATP (dATP) is a potent natural nucleotide stimulant of cardiac contractility (via improved myosin binding to actin & faster detachment after the power stroke), and 2) hiPSC- CMs that overexpress the rate-limiting enzyme for dATP synthesis, ribonucleotide reductase (RNR), have both increased contractility and deliver dATP to the rest of the heart via gap junctions. Thus we are testing the hypothesis that engineering hiPSC-CMs to elevate RNR (RNR-hiPSC-CMs) will improve outcomes in cell replacement therapy for MI (compared with control hiPSC-CMs), improving contractility of both graft and native myocardium. There are several highly novel aspects to our approach. 1) It is the first proposed use of cellular nucleotide manipulation to improve in vivo cardiac function. 2) The approach is not limited to replacement of lost tissue (with hiPSC-CMs) with a better functioning graft, but may also substantially benefit the post-MI depressed function of native myocardium. 3) The first use of engineered hiPSC-CMs to deliver what is effectively a small molecule therapy (dATP), a natural compound that improves heart muscle contraction. This effectively makes hiPSC-CMs a drug delivery device with cardiac specific delivery and effects. Aim 1 develops and test engineered mutations in RNR that increase it’s stability and activity in cardiomyocytes and their ability to titrate increasing levels of dATP produced in hiPSC-CMs. Aim 2 uses AAV vectors for RNR variants, selected from Aim 1, to investigate their capacity to improve cardiac function in a mouse model of myocardial infarct and heart failure. Aim 3 will produce engineered hiPS cell lines that will act as dATP ‘donor cells’ following differentiation, for transplantation into acute MI and more challenging chronic MI athymic rat models to determine their capacity to improve function beyond transplantation of non-engineered hiPSC-CMs. We will evaluate the persistence of these effects and determine the long-term stability and viability of these cell lines. We expect significant contractile improvement of both the graft and native myocardium with RNR-hiPSC-CMs vs. hiPSC-CMs and this effect will be modulated by the dATP producing capacity of the transplanted cells. Results from these studies will elucidate the potential of this combination cell- and small molecule therapy to ameliorate or even improve pump function in failing hearts. This supplement, as the candidates research project will extend the project with 2 aims. Aim 1 will investigate the mechanism by which cardiac muscle using dATP is less susceptible to reductions in contractile strength when pH is reduced, such as occurs in ischemia. Aim 2 will determine whether elevation (rescue) of cardiac function can occur in a different model of dilated cardiomyopathy (than MI), that occurring in Deuchenne’s Muscular Dystrophy (DMD) using a rat transgenic model.

摘要。母项目是围绕20年的机械和转化研究而建立的 基于两个基本发现：1）2-脱氧ATP（dATP）是一种有效的天然核苷类心脏兴奋剂 收缩性（通过改善的肌球蛋白与肌动蛋白的结合和动力性中风后更快的分离），和2）hiPSC- 过表达dATP合成限速酶核糖核苷酸还原酶（RNR）的CM， 增加收缩力，并通过间隙连接将dATP输送到心脏的其余部分。我们正在测试。 假设工程化hiPSC-CM以提高RNR（RNR-hiPSC-CM）将改善细胞中结果 MI的替代治疗（与对照hiPSC-CM相比），改善移植物和天然细胞的收缩性 心肌我们的方法有几个非常新颖的方面。1)这是第一次提出使用蜂窝 核苷酸操作以改善体内心脏功能。2)该方法不仅限于更换丢失的 组织（具有hiPSC-CM）具有更好的功能移植物，但也可能大大有益于MI后抑郁 天然心肌的功能。3)第一次使用工程hiPSC-CM来提供有效的小型 分子疗法（dATP），一种改善心肌收缩的天然化合物。这有效地使 hiPSC-CM是一种具有心脏特异性递送和作用的药物递送装置。 目的1开发和测试RNR中的工程突变，以增加其在体内的稳定性和活性。 心肌细胞和它们滴定hiPSC-CM中产生的增加水平的dATP的能力。Aim 2使用AAV RNR变体的载体，选自目标1，以研究它们改善心脏功能的能力， 小鼠心肌梗死和心力衰竭模型。目的3将产生工程hiPS细胞系， 作为分化后的dATP“供体细胞”，用于移植到急性MI和更具挑战性的慢性MI中 无胸腺大鼠模型，以确定它们在移植非工程化的 hiPSC-CM。我们将评估这些影响的持续性，并确定长期稳定性和可行性 这些细胞系。我们预期移植心肌和自体心肌的收缩都有显著改善， RNR-hiPSC-CM相对于hiPSC-CM的dATP产生能力，并且这种作用将通过RNR-hiPSC-CM的dATP产生能力来调节。 移植细胞这些研究的结果将阐明这种细胞和小细胞结合的潜力。 分子治疗，以改善或甚至改善衰竭心脏的泵功能。 这个补充，作为候选人的研究项目将扩展该项目有2个目标。目标1将调查 使用dATP的心肌对收缩力降低不敏感的机制 当pH值降低时，例如在局部缺血中发生时。目标2将确定是否升高（抢救）心脏 功能可以发生在不同的扩张型心肌病模型中（而不是MI），发生在Deuchenne’s 使用大鼠转基因模型的肌营养不良症（DMD）。