Cell Plasticity-Based Reprogramming Strategies to Enhance Human Myocardial Regeneration

基于细胞可塑性的重编程策略增强人类心肌再生

• 批准号: 10391463
• 负责人: Todd K Rosengart
• 金额: $ 62.86万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391463
• 关键词: ATAC-seq; Acute; Applications Grants; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Differentiation process; Cells; ChIP-seq; Chronic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Congestive Heart Failure; Coronary; DIF factor; Data; Differentiated Gene; Down-Regulation; EFRAC; Endothelial Cells; Epigenetic Process; Family member; Family suidae; Fibroblasts; GATA4 gene; Gene Activation; Gene Expression; Generations; Genes; Genetic Transcription; Histone Deacetylase; Human; Implant; In Situ; In Vitro; Injections; Legal patent; Ligation; Mediating; Mediator of activation protein; Modeling; Myocardial; Myocardial Infarction; Myocardium; Natural regeneration; Outcome; Pathway interactions; Phenotype; Predisposition; Property; Rattus; Regimen; Repression; Research; Resistance; Reverse engineering; Rodent; Role; TP53 gene; Testing; Transcriptional Activation; Transgenes; Treatment Failure; Vascular Endothelial Growth Factors; Work; adeno-associated viral vector; base; cell type; clinically relevant; design; heart function; high throughput screening; improved; in vivo; morphogens; mortality; novel; overexpression; pre-clinical; preconditioning; regenerative approach; restraint; screening; single-cell RNA sequencing; small hairpin RNA; stem cell delivery; stem cell therapy; stem cells; transcription factor; transcriptome sequencing; transdifferentiation; treatment research; vector; vector control

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

充血性心力衰竭（CHF）通常是由于心肌梗死（MI）引起的， 导致西方心脏病死亡的原因近年来CHF治疗研究策略的重点是外源性干细胞 细胞给药在很大程度上是令人失望的-可能是由于植入物存活和整合到 宿主心肌细胞重编程，允许心脏成纤维细胞原位转分化为 诱导的心肌细胞样细胞（iCM），代表了一种新的心肌再生策略，可以废除 干细胞移植的许多挑战。然而，最近的研究发现，重编程因素， 诱导啮齿动物细胞转分化不能重编程人细胞，这表明人细胞对 与啮齿动物细胞相比，这是该领域的一个重要的新挑战。我们开发了两个 原型“前可塑性”细胞重编程策略，以测试我们的中心假设，这些策略可以 用于关键性地促进人类细胞重编程，作为改善MI后心脏功能的手段。这些 前可塑性策略是：1）重编程途径的转录激活（通过p63下调 和Hippo途径覆盖），和2）使用内皮细胞分化诱导“跨细胞”状态， 在一些实施方案中，细胞因子（ETV 2或VEGF）可用于使成纤维细胞转分化成内皮细胞中介。我们的推论 一个假说是，这些促可塑性策略的功效可以归因于它们对关键的去抑制。 重编程基因激活的表观遗传介质在高阶物种。这项工作的新奇在于 这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 因此，设计来测试这两种前可塑性策略中的每一种都可以增强人类的可塑性的假设。 并测试第三个假设，即这些策略可用于增强细胞分化， 重编程，从而改善体内梗死后心脏功能。为了实现这些目标，我们将使用 单细胞RNA-seq和ATAC-Seq鉴定心脏分化基因和表观遗传因子， 在人类与啮齿类动物细胞中差异抑制，并由促可塑性策略诱导，从而“逆转” “工程师”从这些因素中获得优化的精确重编程鸡尾酒。我们还将使用CRISPR 相对于基于载体的转基因过表达策略，以优化这些重编程因子的递送。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-1）。 介导的）全身与直接心肌给药策略。这些目标的实现可以改变 在心脏细胞重编程的新领域的努力，并帮助阐明一个新的临床策略， 瑞士法郎。 1