Human CRISPRa Cardiac Reprogramming: Model Characterization and Epigenetic Mechanisms

人类 CRISPRa 心脏重编程：模型表征和表观遗传机制

• 批准号: 10467989
• 负责人: Benjamin Keepers
• 金额: $ 3.75万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10467989
• 关键词: ATAC-seq; Address; Biological; Biological Assay; Biology; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cell Cycle; Cells; Cessation of life; ChIP-seq; Chromatin Remodeling Factor; Cicatrix; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; DNA; Elements; Epigenetic Process; Fellowship; Fibroblasts; Fibrosis; Funding; Future; GATA4 gene; Gap Junctions; Gene Activation; Gene Expression; Gene Transduction Agent; Genes; Goals; Growth; Guide RNA; Health; Heart; Heart Diseases; Histones; Human; Immunofluorescence Immunologic; Institution; Knowledge; Lead; Learning; Measures; Mediating; Mediator of activation protein; Mentorship; Methods; Modeling; Molecular; Morbidity - disease rate; Muscle; Muscle Cells; Myocardial; Myocardial Ischemia; Myocardium; Natural regeneration; Patients; Pattern; Phase; Phenotype; Physicians; Process; Proteins; RNA Interference; RNA Splicing; Reagent; Regenerative capacity; Reporter; Reporting; Research; Resources; Retroviral Vector; Role; Scientist; Signal Pathway; Structure; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Uses; Training; Transactivation; Transcriptional Activation; Transgenes; Translating; Untranslated RNA; Work; base; cell type; chromatin immunoprecipitation; chromatin remodeling; design; epigenome; epigenomics; functional outcomes; gene therapy; genetic manipulation; human model; improved; insight; molecular phenotype; mortality; new technology; novel; overexpression; regenerative; regenerative approach; regenerative therapy; release of sequestered calcium ion into cytoplasm; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; vector

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.

项目摘要 /摘要 心脏病的发病率和死亡率的优势是缺血性损害 心肌。由于心肌细胞的再生能力有限，缺血性损害导致 永久性心肌死亡，然后是纤维化和疤痕形成。心脏重编程是一个有希望的新的 再生方法，有可能通过将居民成纤维细胞转换为恢复心肌 心肌细胞样细胞。在第一个心脏重编程报告遵循的研究十年中， 该领域在提高其方法的效率和理解信号传导方面取得了重大进步 对于心脏重编程很重要的途径，转录组变化和染色质重塑剂。我们的 长期目标是通过改善我们 技术并通过加深我们对重编程过程的了解。心脏常数 重编程方法已通过强迫表达外源性诱导重编程 转录因子，通常由逆转录病毒载体编码。我们已经开发了一种新的人类心脏模型 重新编程使用基于CRISPR的基因激活（CRISPRA）来促进MEF2C，GATA4和TBX5 表达，并通过将该模型与我们当前的方法进行比较，我们可以获得对基本生物学的新见解 重编程过程。目的1是表征我们的分子，表型和功能特征 CRISPRA模型。免疫荧光将测定肉类结构和间隙连接模式，并测定 将使用RNA-Seq研究转录组变化。钙通量通过荧光记者以及 细胞收缩的观察结果将被测量为功能结果。目标2是研究表观遗传学 CRISPRA引起的重编程的变化和机制。 MEF2C的表观遗传动力学， 将研究GATA4，TBX5和其他基因，以及CRISPRA独有的表观基因组重塑 重新编程将通过chip-seq和atac-seq确定。导致的潜在机制 将通过RNAi以及常规的CRISPR编辑探索独特的表观遗传特征。总体而言，这 研究将提供对一种新型心脏重编程形式的关键见解，这将使我们加深我们 细胞身份，细胞可塑性和细胞命运确定的观点。此外，通过开发一个crispra- 基于模型，我们为人类心脏重编程试验开辟了潜在的途径。进一步 研究，心脏重编程可以安全有效地用于人类再生目的。我会 在世界知名的科学家李·齐安（Li Qian）的建议下完成这项工作，对此充满热情 指导。我的机构为我计划的工作提供了充足的资源，它为许多机会提供了很多机会 我的培训计划中详细介绍的学习和专业成长。完成此研究金后，我会准备好 对于我成为一名独立资助的医师科学家的下一阶段培训。