Engineered Stem Cells for Cardiac Repair

抽象的。母项目是建立在大约20年的机械和翻译研究的基础上的 基于两个基本发现：1)2-脱氧三磷酸腺苷(DATP)是一种有效的心脏天然核苷酸刺激剂 收缩能力(通过改善肌球蛋白与肌动蛋白的结合，以及在强力中风后更快地脱离)，以及2)HiPSC- 过度表达dATP合成限速酶的细胞质雄性不育系，核糖核苷酸还原酶(RNR)，两者都有 增加收缩能力，并通过缝隙连接将dATP输送到心脏的其余部分。因此，我们正在测试 假设工程hiPSC-CMS提高RNR(RNR-hiPSC-CMS)将改善细胞结局 心肌梗死的替代疗法(与对照HIPSC-CMS相比)，改善移植物和天然心肌的收缩性能 心肌。我们的方法有几个非常新奇的方面。1)这是第一次提出使用蜂窝 改善体内心脏功能的核苷酸操作。2)这种方法并不局限于替换丢失的 组织(带有hiPSC-CMS)功能更好的移植物，但也可能实质上受益于心肌梗塞后抑郁 天然心肌的功能。3)首次使用工程HIPSC-CMS提供有效的小 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-CMS是一种具有心脏特异性递送和效应的药物递送装置。 目标1开发和测试RNR的工程突变，以增加其稳定性和活性 心肌细胞及其滴定在HiPSC-CMS中产生的dATP水平增加的能力。AIM 2使用AAV RNR变异体的载体，选自Aim 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。Aim 3将生产出将发挥作用的工程HIPS细胞系 作为分化后的dATP供体细胞，用于移植到急性心肌梗死和更具挑战性的慢性心肌梗死 无菌大鼠模型确定其改善非工程化移植后功能的能力 HiPSC-CMS。我们将评估这些影响的持续性，并确定其长期稳定性和可行性。 这些细胞系中。我们希望移植心肌和天然心肌的收缩性能都能得到显著改善。 RnR-hiPSC-CMS与hiPSC-CMS相比，这种效应将受到 移植的细胞。这些研究的结果将阐明这种细胞和小细胞组合的潜力 改善甚至改善衰竭心脏泵功能的分子疗法。 本副刊作为考生研究项目，将延续该项目的2个目标。AIM 1将调查 使用dATP的心肌不太容易受到收缩强度降低的影响的机制 当pH值降低时，如在缺血时发生。目标2将确定心脏抬高(抢救)是否 功能可以发生在不同的扩张型心肌病(不同于心肌梗死)中，发生在杜兴氏病中 使用大鼠转基因模型进行肌肉营养不良症(DMD)的研究。

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.