Engineered Stem Cells for Cardiac Repair

摘要。这个项目是建立在默里博士和雷尼尔之间多年的合作工作 研究人类胚胎干细胞衍生的心肌细胞（hESC-CMs）作为潜在的细胞替代品 心肌梗死（MI）后心脏修复策略。我们的研究小组已经表明，hESC-CM和 人诱导型多能SC-CM（hiPS-CM）可以以允许在以下条件下测试的规模和纯度生产： 啮齿类动物模型和最有可能预测人类反应的动物：非人灵长类动物（NHP; Macaca nemestrina）。我们已经证明了这些细胞在啮齿类动物模型中移植的能力，包括 整个疤痕，并与宿主组织电整合，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）是一种有效的天然药物， 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），和2）hiPSC-CM，其过表达dATP合成的限速酶，核糖核苷酸 还原酶（RNR）具有增加的收缩性并通过间隙连接将dATP递送至心脏的其余部分。 因此，我们将检验以下假设：工程化hiPSC-CM以提高RNR（RNR-hiPSC-CM）将改善HiPSC-CM的表达。 MI的细胞替代疗法的结果（与对照hiPSC-CM相比），改善了 移植心肌和自体心肌。我们的方法有几个非常新颖的方面。1)是第一 提出使用细胞核苷酸操作来改善体内心脏功能。2)该方法不 仅限于用功能更好的移植物替换丢失的组织（用hiPSC-CM），但也可以基本上 有利于心肌梗死后抑制自体心肌的功能。3)首次使用工程化的hiPSC-CM， 提供有效的小分子疗法（dATP），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的药物递送装置， 方面的影响.目标1将开发和测试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。目标2将使用 RNR变体的AAV载体，选自目标1，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。目的3将产生工程hiPS细胞系， 在分化后作为dATP“供体细胞”，用于移植到急性MI中， 慢性心肌梗死无胸腺大鼠模型，以确定其改善功能的能力，超过移植非心肌梗死的能力。 工程化的hiPSC-CM。我们将评估这些影响的持续性，并确定长期 这些细胞系的稳定性和活力。我们预期移植物和移植物的收缩性都有显著改善。 RNR-hiPSC-CM与hiPSC-CM的天然心肌，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一点的潜力。 联合细胞和小分子治疗以改善或甚至改善衰竭心脏中的泵功能。

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.