Human CRISPRa Cardiac Reprogramming: Model Characterization and Epigenetic Mechanisms

项目摘要/摘要 心脏疾病的发病率和死亡率主要是由于心脏的缺血性损害。 心肌。由于心肌细胞再生能力有限，缺血性损伤会导致 永久性心肌死亡，随后是纤维化和疤痕形成。心脏重编程是一种很有前途的新技术 一种再生方法，通过将驻留的成纤维细胞转化为 心肌细胞样细胞。在第一份心脏重编程报告之后的十年研究中， 该领域在提高其方法的效率和理解信号方面取得了重大进展 对心脏重编程很重要的途径、转录改变和染色质重构体。我们的 长期目标是帮助催化心脏重编程对人类健康的应用，通过改善我们的 技术，并通过加深我们对重新编程过程的知识。心脏中的一个常量 重编程方法已经通过强制表达外源基因来诱导重编程 转录因子，通常由逆转录病毒载体编码。我们已经开发出一种新的人体心脏模型 使用基于CRISPR的基因激活(CRISPRa)来促进MEF2C、GATA4和TBX5的重新编程 表达，通过将这个模型与我们目前的方法进行比较，我们可以对基础生物学有新的见解 重新编程的过程。目标1是表征我们的分子、表型和功能特征 CRISPRA模型。肌节结构和缝隙连接模式将通过免疫荧光进行分析，以及 转录的变化将使用RNA-seq进行研究。通过荧光报告的钙离子通量以及 对细胞收缩的观察将作为功能结果进行测量。目标2是研究表观遗传学 CRISPRA诱导的重编程变化及其机制。MEF2C的表观遗传动力学， 将研究GATA4、TBX5和其他基因，以及CRISPRA特有的表观基因组重塑 重新编程将通过CHIP-SEQ和ATAC-SEQ确定。潜在的机制导致 独特的表观遗传学特征将通过RNAi和传统的CRISPR编辑进行探索。总体而言，这 研究将提供对一种新形式的心脏重新编程的关键见解，这些见解将深化我们的 关于细胞特性、细胞可塑性和细胞命运决定的观点。此外，通过开发CRISPRA- 基于模型，我们已经为在人类身上进行心脏重编程试验开辟了一条潜在的途径。使用进一步的 研究表明，心脏重编程可以变得足够安全和有效，以达到人类再生的目的。这就做 在世界著名科学家李倩博士的指导下完成了这项工作 师徒关系。我的机构为我计划的工作提供了充足的资源，并提供了许多机会 在我的培训计划中详细说明了学习和职业成长。在完成这一团契后，我将准备好 为我成为一名独立资助的内科科学家的下一阶段培训做准备。

PROJECT SUMMARY / ABSTRACT The preponderance of morbidity and mortality from cardiac disease results from ischemic damage to the myocardium. Because of the limited regenerative capacity of cardiomyocytes, ischemic damage results in permanent myocardial death followed by fibrosis and scar formation. Cardiac reprogramming is a promising new regenerative approach that has the potential to restore myocardium by converting resident fibroblasts into cardiomyocyte-like cells. In the decade of research that has followed the first report of cardiac reprogramming, the field has made significant strides in improving the efficiency of its methods and in understanding the signaling pathways, transcriptomic changes, and chromatin remodelers that are important for cardiac reprogramming. Our long-term goal is to help catalyze the application of cardiac reprogramming to human health by improving our techniques and by deepening our knowledge of the reprogramming process. A constant in cardiac reprogramming methods has been induction of reprogramming through forced expression of exogenous transcription factors, usually encoded by a retroviral vector. We have developed a new model of human cardiac reprogramming that uses CRISPR-based gene activation (CRISPRa) to promote MEF2C, GATA4, and TBX5 expression, and by comparing this model to our current methods, we can gain new insights into the basic biology of the reprogramming process. Aim 1 is to characterize the molecular, phenotypic, and functional features of our CRISPRa model. Sarcomeric structure and gap junction patterns will be assayed by immunofluorescence, and transcriptomic changes will be studied using RNA-seq. Calcium flux through a fluorescent reporter as well as observations of cell contraction will be measured as functional outcomes. Aim 2 is to study the epigenetic changes and mechanisms of reprogramming induced by CRISPRa. The epigenetic dynamics at MEF2C, GATA4, and TBX5 and other genes will be studied, and the epigenomic remodeling unique to CRISPRa reprogramming will be determined through ChIP-seq and ATAC-seq. The potential mechanisms that lead to unique epigenetic features will be explored through RNAi as well as conventional CRISPR editing. Overall, this study will provide key insights into a novel form of cardiac reprogramming, insights that will deepen our perspectives on cell identity, cell plasticity, and cell fate determination. Furthermore, by developing a CRISPRa- based model, we have opened a potential avenue for trial of cardiac reprogramming in humans. With further study, cardiac reprogramming can become safe and effective enough for human regenerative purposes. I will complete this work under the advisement of Dr. Li Qian, a world-renowned scientist with a passion for mentorship. My institution has ample resources for the work I have planned, and it offers many opportunities for learning and professional growth as detailed in my training plan. Upon completing this fellowship, I will be ready for the next phase of training in my path to becoming an independently funded physician scientist.