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Engineered Stem Cells for Cardiac Repair

用于心脏修复的工程干细胞

基本信息

批准号：
10078963
负责人：
Charles E Murry
金额：
$ 66.56万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
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/10078963
关键词：
ATP Synthesis Pathway ATP phosphohydrolase Actins Acute myocardial infarction Adult Affect Aftercare Animals Binding Biochemical CRISPR/Cas technology Canis familiaris Cardiac Cardiac Myocytes Cardiotonic Agents Cell Line Cell Survival Cell Therapy Cell Transplantation Cells Cellular Metabolic Process Chronic Cicatrix Contracts Cost efficiency Coupled Data Degradation Pathway Dependovirus Depressed mood Devices Drug Delivery Systems Echocardiography Engineering Enzymes Equilibrium Family suidae Feedback Gap Junctions Gene Targeting Goals Heart Heart Transplantation Heart failure Hip region structure Human Human Engineering Immunocompromised Host In Vitro Infarction Ischemia Left Left Ventricular Function Longevity Macaca nemestrina Magnetic Resonance Imaging Measurement Mediating Mitochondria Mitotic Modeling Modification Mus Muscle Muscle Cells Muscle Contraction Mutation Myocardial Myocardial Infarction Myocardial Reperfusion Myocardium Myosin ATPase Nature Nucleotides Nude Rats Operative Surgical Procedures Pathway interactions Performance Phosphocreatine Phosphorylation Phosphotransferases Physiological Power stroke Production Proteins Pump Rattus Recovery Relaxation Reperfusion Injury Respiration Rest Ribonucleotide Reductase Rodent Rodent Model Stress Structure Tertiary Protein Structure Testing Therapeutic Time Tissues Toxic effect Transgenic Organisms Transplantation Ubiquitination Variant Ventricular Ventricular Function Viral Vector Work adeno-associated viral vector base cardiac repair cell replacement therapy coronary artery occlusion design energy balance engineered stem cells experimental study expression vector gene therapy graft function heart function human embryonic stem cell human stem cells improved improved functioning improved outcome in vivo induced pluripotent stem cell mouse model nonhuman primate novel overexpression prevent protein degradation repair function repair strategy response small molecule stem cells treatment strategy vector

项目摘要

ABSTRACT. This project is built around years of collaborative work between Drs. Murry and Regnier studying human embryonic stem cell derived cardiomyocytes (hESC-CMs) as a potential cell replacement strategy for cardiac repair following myocardial infarction (MI). Our group has shown that hESC-CMs and human inducible pluripotent SC-CMs (hiPS-CMs) can be produced at a scale and purity that permit testing in rodent models and the animal most likely to predict the human response: the non-human primate (NHP; Macaca nemestrina). We have demonstrated the ability of these cells to engraft in rodent models, covering the entire scar, and electrically integrate with host tissue to improve left ventricular performance. This project is 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 will test 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 will develop 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 will use 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.

抽象的。该项目是基于博士之间多年的合作而建立的。穆里和雷尼尔研究人类胚胎干细胞衍生的心肌细胞（hESC-CM）作为潜在的细胞替代品心肌梗死（MI）后心脏修复的策略。我们的小组已经证明 hESC-CM 和人类诱导多能 SC-CM (hiPS-CM) 的生产规模和纯度可允许在啮齿动物模型和最有可能预测人类反应的动物：非人类灵长类动物（NHP；猕猴 (Macaca nemestrina)。我们已经证明了这些细胞移植到啮齿动物模型中的能力，涵盖了整个疤痕，并与宿主组织电结合以改善左心室性能。该项目基于两个基本发现：1) 2-脱氧 ATP (dATP) 是一种有效的天然心肌收缩力的核苷酸刺激剂（通过改善肌球蛋白与肌动蛋白的结合以及在心肌收缩后更快的分离）动力冲程），以及 2） hiPSC-CM 过度表达 dATP 合成的限速酶、核糖核苷酸还原酶 (RNR) 既能增强收缩性，又能通过间隙连接将 dATP 输送到心脏的其他部分。因此，我们将测试以下假设：通过工程设计 hiPSC-CM 来提高 RNR (RNR-hiPSC-CM) 将改善 MI 细胞替代疗法的结果（与对照 hiPSC-CM 相比），改善心肌收缩力移植心肌和天然心肌。我们的方法有几个非常新颖的方面。 1）这是第一个建议使用细胞核苷酸操作来改善体内心脏功能。 2）该方法不是仅限于用功能更好的移植物替代丢失的组织（使用 hiPSC-CM），但也可能在很大程度上有益于心肌梗死后低下的天然心肌功能。 3) 首次使用工程化 hiPSC-CM 提供有效的小分子疗法 (dATP)，这是一种改善心肌的天然化合物收缩。这有效地使 hiPSC-CM 成为具有心脏特异性递送和影响。目标 1 将开发和测试 RNR 的工程突变，以提高其稳定性和活性心肌细胞及其滴定 hiPSC-CM 中产生的 dATP 水平的能力。目标2将使用从目标 1 中选择的 RNR 变体的 AAV 载体，以研究它们改善心脏功能的能力心肌梗塞和心力衰竭的小鼠模型。目标 3 将产生工程 hiPS 细胞系，分化后充当 dATP“供体细胞”，用于移植到急性 MI 中并更具挑战性慢性心肌梗死无胸腺大鼠模型，以确定其改善非非胸腺移植以外的功能的能力工程化 hiPSC-CM。我们将评估这些影响的持续性并确定长期影响这些细胞系的稳定性和活力。我们预计移植物和移植物的收缩力都会显着改善具有 RNR-hiPSC-CM 的天然心肌与 hiPSC-CM 的比较，这种效应将受到 dATP 的调节移植细胞的生产能力。这些研究的结果将阐明这种技术的潜力联合细胞和小分子疗法可改善甚至改善衰竭心脏的泵功能。