Human CRISPRa Cardiac Reprogramming: Model Characterization and Epigenetic Mechanisms

项目总结/摘要 心脏病的发病率和死亡率的优势是由于心肌的缺血性损伤。 心肌由于心肌细胞的再生能力有限，缺血性损伤导致心肌缺血。 永久性心肌死亡，随后纤维化和瘢痕形成。心脏重编程是一个有前途的新的 再生方法，具有通过将驻留的成纤维细胞转化为 心肌样细胞。在心脏重编程首次报道后的十年研究中， 该领域在提高其方法的效率和理解信令方面取得了重大进展 信号通路、转录组学变化和染色质重塑对心脏重编程很重要。我们 长期目标是通过改善我们的心脏，帮助促进心脏重编程在人类健康中的应用。 技术和加深我们对重编程过程的了解。心脏中的一个常数 重编程方法是通过外源基因的强制表达来诱导重编程。 转录因子，通常由逆转录病毒载体编码。我们开发了一种新的人类心脏模型， 使用基于CRISPR的基因激活（CRISPRa）来促进MEF 2C，GATA 4和TBX 5的重编程 表达，并通过将此模型与我们目前的方法进行比较，我们可以获得对基础生物学的新见解 重新编程的过程。目的1是表征我们的基因的分子、表型和功能特征。 CRISPRa模型。肌节结构和间隙连接模式将通过免疫荧光法测定， 将使用RNA-seq研究转录组学变化。通过荧光报告分子的钙通量以及 细胞收缩的观察结果将被测量为功能结果。目的二是研究表观遗传 CRISPRa诱导的重编程的变化和机制。MEF 2C的表观遗传动力学， GATA 4和TBX 5以及其他基因将被研究，CRISPRa独特的表观基因组重塑 将通过ChIP-seq和ATAC-seq确定重编程。潜在的机制，导致 独特的表观遗传特征将通过RNAi以及常规的CRISPR编辑来探索。总体而言，这 这项研究将为一种新的心脏重编程形式提供关键的见解，这些见解将加深我们对心脏重编程的认识。 细胞身份，细胞可塑性和细胞命运决定的观点。此外，通过开发CRISPRa- 基于该模型，我们为人类心脏重编程试验开辟了一条潜在的途径。进一步 研究表明，心脏重编程可以变得足够安全和有效，用于人类再生目的。我会 在世界著名科学家李谦博士的指导下完成这项工作， 导师制我所在的机构有充足的资源来开展我计划的工作，并为以下方面提供了许多机会： 学习和专业成长，详见培训计划。在完成这项研究后，我将准备好 在我成为一名独立资助的医生科学家的道路上，下一阶段的培训。

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.