Engineered Stem Cells for Cardiac Repair

摘要：母项目建立在两项基本发现的基础上，跨越了20年的机械和 翻译研究：1)2-脱氧三磷酸腺苷(DATP)是一种强大的天然核苷酸刺激剂，可通过 增加肌球蛋白与肌动蛋白的结合，并在强力中风后更快地脱离)，以及2)hiPSC-CMS 过表达dATP合成限速酶，即核糖核苷酸还原酶(RNR)，既表现出 通过缝隙连接增加心脏的收缩能力和dATP输送到心脏的其余部分。因此，我们是 研究改变HiPSC-CMS以增加RNR(RNR-hiPSC-CMS)可改善预后的假说 在心肌梗死的细胞替代治疗中(与对照HiPSC-CMS相比)，通过增加两者的收缩 移植物和自体心肌。我们的技术包含了几个高度创新的元素。1)这是 首次提出将细胞核苷酸修饰作为改善体内心脏功能的一种手段。2) 这项技术并不局限于用功能更强的移植物取代丢失的组织(用HiPSC-CMS)，但可能 也明显有利于心肌梗死后自主心肌的抑制功能。3)修改后的第一次应用 HiPSC-CMS提供一种增强心肌收缩的小分子治疗(DATP)。这 本质上将hiPSC-CMS转化为心脏特异的药物输送系统。 目标1是产生和表征RNR的工程突变，以增强其在 心肌细胞，以及它们滴定hiPSC-CMS产生的不断增加的dATP的能力。目标2 研究目标1中确定的RNR变异在小鼠心脏功能改善中的能力。 使用AAV载体检测心肌梗死和心力衰竭。AIM 3将产生经过工程改造的髋关节细胞系， 分化后，将作为dATP‘供体细胞’移植到急性心肌梗死和更有问题的慢性心肌梗死 建立心律失常大鼠模型，测试其增加功能的能力，超越非设计的HiPSC-CMS。我们 将评估这些影响的持久性以及细胞系的长期存活和稳定性。我们预计会有一个 RNR-hiPSC-CMS与hiPSC-CMS相比，移植心肌和天然心肌的收缩性能均有显著改善。 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疗法改善或恢复泵功能的潜力 心力衰竭时的表现。 作为候选人的研究项目，这一补充将通过追求两个目标来扩大研究范围 目标。目标1将研究允许心肌利用的机械和结构机制 当pH降低时，如在缺血时，dATP对收缩强度的损失不那么敏感。 目的2将研究在不同的心肌病模型(扩张型)中是否可以改善心功能 而不是MI)，利用Duchenne肌营养不良(DMD)扩张型心肌病模型 转基因大鼠。

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.