Cell Plasticity-Based Reprogramming Strategies to Enhance Human Myocardial Regeneration

充血性心力衰竭(CHF)通常是由心肌梗死(MI)引起的，仍然是主要的 在西方，心脏死亡的原因。近期充血性心力衰竭的治疗策略主要集中在外源性干细胞 细胞管理在很大程度上令人失望-可能是由于植入物存活率和与 宿主心肌。细胞重新编程，允许心脏成纤维细胞原位转分化为 诱导心肌细胞样细胞(ICMs)代表了一种新的心肌再生策略 干细胞移植的许多挑战。然而，最近的研究发现，重新编程的因素一直是 诱导啮齿动物细胞转分化不能重新编程人类细胞表明人类细胞对 与啮齿动物细胞相比，重新编程--这是该领域面临的一个重要新挑战。我们已经开发了两个 典型的“前可塑性”细胞重编程策略来测试我们的中心假设，即这些策略可以 被用来关键地促进人类细胞重新编程，作为改善心肌梗塞后心脏功能的一种手段。这些 促进可塑性的策略是：1)重新编程途径的转录激活(通过p63下调 和河马途径覆盖)，以及2)使用内皮细胞分化来诱导一种“跨细胞”状态 因子(ETV2或血管内皮生长因子)将成纤维细胞转分化为内皮细胞媒介。我们的推论 假说是，这些前塑性策略的有效性可以归因于它们抑制KEY 高级物种中表观遗传介体对基因激活的重新编程。这项工作的新颖性在于 反映在我们就这些发现提交的三项美国专利申请中。我们的具体目标是 相应地，旨在测试这两种促可塑性策略都可以增强人类 并检验第三个假设，即这些策略可用于增强细胞 重新编程，从而改善在体心肌梗死后的心功能。为了实现这些目标，我们将利用 单细胞RNA-Seq和ATAC-Seq用于鉴定心脏分化基因和表观遗传因子 在人和啮齿动物细胞中被差异抑制，并由促可塑性策略诱导，从而发生逆转 工程师“从这些因素中衍生出一种优化的精密重新编程鸡尾酒。我们还将使用CRISPR VS基于载体的转基因过表达策略，以优化这些重新编程因子的传递。我们 将在大鼠冠状动脉结扎中测试这些策略，然后在临床前的猪MI模型中测试(AAV- 中介)全身给药策略与直接给药策略。实现这些目标可能会改变方向 在心脏细胞重编程的新领域的努力，并有助于阐明治疗的新临床策略 瑞士法郎。 1

Congestive heart failure (CHF) typically occurring as a result of myocardial infarction (MI) remains the leading cause of cardiac mortality in the West. Recent CHF treatment research strategies focused on exogenous stem cell administration have been largely disappointing - likely due to poor implant survival and integration into the host myocardium. Cellular reprogramming, allowing the in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs), represents a novel myocardial regenerative strategy that may abrogate many challenges of stem cell delivery. However, recent findings that reprogramming factors which consistently induce rodent cell transdifferentiation fail to reprogram human cells suggest that human cells are resistant to reprogramming compared to rodent cells - an important new challenge to this field. We have developed two prototypical “pro-plasticity” cell reprogramming strategies to test our central hypothesis that these strategies can be used to critically facilitate human cell reprogramming as a means to improve post-MI cardiac function. These pro-plasticity strategies are: 1) transcriptional activation of reprogramming pathways (via p63 downregulation and Hippo pathway override), and 2) induction of a “trans-cellular” state using an endothelial cell differentiation factor (ETV2 or VEGF) to transdifferentiate fibroblasts into an endothelial cell intermediary. Our corollary hypothesis is that the efficacy of these pro-plasticity strategies can be ascribed to their de-repression of key reprogramming gene activation by epigenetic mediators in higher-order species. The novelty of this work is reflected in the three US patent applications we have filed regarding these discoveries. Our specific aims are accordingly designed to test the hypothesis that each of these two pro-plasticity strategies can enhance human cardio-differentiation and to test a third hypothesis that these strategies can be used to enhance cellular reprogramming and thereby improve post-infarct cardiac function in vivo. In pursuit of these aims, we will use single-cell RNA-seq and ATAC-Seq to identify cardio-differentiating genes and epigenetic factors that are differentially repressed in human vs rodent cells and induced by pro-plasticity strategies and thereby “reverse engineer” an optimized precision reprogramming cocktail derived from these factors. We will also use CRISPR vs vector-based transgene overexpression strategies to optimize deliver of these reprogramming factors. We will test these strategies in a rat coronary ligation and then a pre-clinical, porcine MI model, testing (AAV- mediated) systemic vs direct myocardial administration strategies. Accomplishment of these aims could redirect efforts in the novel field of cardiac cellular reprogramming and help elucidate a new clinical strategy for treating CHF. 1