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：该母项目建立在两个基本发现的基础上，跨越了20年的机械和 翻译研究：1）2-脱氧ATP（dATP）是一种有效的天然核苷酸刺激剂的心脏收缩力（通过 增加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-CM 过表达dATP合成的限速酶核糖核苷酸还原酶（RNR）， 增加收缩力和dATP通过间隙连接递送到心脏的其余部分。因此，我们 研究改变hiPSC-CM以增加RNR（RNR-hiPSC-CM）改善结局的假设 在MI的细胞替代治疗中（与对照hiPSC-CM相比），通过增加两者的收缩性， 移植物和天然心肌。我们的技术结合了几个高度创新的元素。1)这是 这是第一次提供细胞核苷酸修饰作为改善体内心脏功能的手段。（二） 该技术并不局限于用功能更强的移植物替换丢失的组织（用hiPSC-CM）， 对心肌梗死后抑制的自体心肌功能也有明显的保护作用。3)第一次应用修改 hiPSC-CM用于递送增强心肌收缩的小分子治疗（dATP）。这 基本上将hiPSC-CM转化为心脏特异性药物递送系统。 目的1是产生和表征RNR中的工程化突变，所述工程化突变增强RNR的稳定性和活性， 心肌细胞，以及它们滴定hiPSC-CM中产生的增加量的dATP的能力。目的2 研究了目标1中鉴定的RNR变化改善小鼠模型心脏功能的能力， 心肌梗塞和心力衰竭的研究。目标3将产生工程化的hiPS细胞系， 在分化后，将作为dATP“供体细胞”移植到急性MI和更有问题的慢性MI中， MI大鼠模型，以测试其增加功能超过非设计的hiPSC-CM的能力。我们 将评估这些效应的持续性以及细胞系的长期存活和稳定性。殷切地盼望 使用RNR-hiPSC-CM与hiPSC-CM相比，在移植物和天然心肌中的显著收缩改善。 CMs，这将由移植细胞的dATP生产能力控制。这些调查将使 这种细胞和小分子联合治疗改善或恢复泵的潜力 心脏衰竭的表现 这一增加，作为候选人的研究项目，将扩大研究的范围，通过追求两个 目标.目的1将研究机械和结构机制，使心肌利用 当pH降低时，dATP对收缩强度损失不太敏感，如在缺血期间发生的那样。 目的2将研究在不同的心肌病模型（扩张型）中是否可以改善心脏功能 代替MI），利用以表达的Duchenne肌营养不良（DMD）扩张型心肌病模型， 转基因大鼠