CRISPR-induced cardiovascular progenitor cells to repair myocardial infarction

项目摘要/摘要 与心肌梗死(MI)相关的心血管疾病仍然是死亡的主要原因 全世界。细胞重新编程为心血管祖细胞(CPC)，绕过 多能性诱导，为心脏修复提供了一种有前景的方法，通过同时 新生血管和心肌生成。当前研究中的一个重大差距是不清楚 了解直接重编程的机制。内源性遗传位点的激活 (被抑制性染色质标记遮挡的)在起始细胞中被认为是一个重要的 高质量完全可编程电池的标准。因此，我们试图获得一种新的CPC来源 通过直接操纵内源基因。为此，我们建议组合使用 重新编程技术和基于CRISPR的工具。CRISPR诱导的基因激活优于 由于其有效性、设计简单性而采用cDNA超表达的传统技术 以及避免需要额外转导转录因子复合体。我们的预赛 数据表明，CRISPR诱导的细胞具有CPC的特性，包括克隆性、自体和 更新，和心脏三重潜能。重要的是，CRISPR诱导的CP(ICPC)可以钝化 心肌梗死小鼠移植后心功能恶化，心肌梗死面积缩小。这些发现是 为在临床前环境中进一步系统研究CRISPR-ICPC奠定了基础。因此， 提出了三个具体目标，以开发一个技术平台来生成新的CRISPR-ICPC，研究 细胞重编程机制，并评价ICPC在心肌梗死治疗中的应用。在AIM-1中，一个新的 非病毒CRISPR系统将被开发用于使用临床可接受的基因来产生ICPC 将对载体系统以及新的ICPC的生物学和功能特性进行表征。在AIM-2中， 介导iCPC生成的表观遗传因素将是实验的重点。长的非编码RNA 并将研究有针对性的表观遗传介体，以剖析细胞的机制 重新编程。在AIM-3中，ICPC将使用细胞贴片技术进行植入，并了解它们的细胞命运 决定将在梗塞的心脏中被追踪。差异化的ICPC的范围和作用将是 由活体成像确定，并与使用细胞消融方法的其他后果区分开来。 综上所述，这一建议将为心肌梗死治疗提供一种新的融合再生策略 CRISPR技术和组织工程在CPC开发中的应用。

Project Summary/Abstract Cardiovascular disease associated with myocardial infarction (MI) remains a major cause of death worldwide. Cell reprogramming into cardiovascular progenitor cells (CPCs), bypassing the process of pluripotency induction, has provided a promising approach for cardiac repair through simultaneous neovascularization and cardiomyogenesis. One significant gap in current research is an unclear understanding of the mechanisms of direct reprogramming. The activation of endogenous genetic loci (that are occluded by repressive chromatin marks) in starting cells has been considered an important criterion of high-quality fully reprogrammed cells. Therefore, we attempt to obtain a new source of CPCs through direct manipulation of endogenous genes. To this end, we propose a combined use of reprogramming technologies and CRISPR-based tools. CRISPR-induced gene activation is superior to conventional techniques employing cDNA overexpression due to its effectiveness, simplicity of design and avoidance of the need for additional transduction of transcription factor complexes. Our preliminary data demonstrates that CRISPR-induced cells possess the properties of CPCs including clonality, self- renewal, and cardiac tri-potentiality. Importantly, the CRISPR-induced CPCs (iCPCs) can blunt the worsening of heart function and reduce infarct size after transplantation in MI mice. These findings are the basis for a further systematic investigation of CRISPR-iCPCs in a preclinical setting. Accordingly, three Specific Aims are proposed to develop a technical platform to generate new CRISPR-iCPCs, study cell-reprogramming mechanisms, and evaluate the application of iCPCs for MI therapy. In Aim-1, a new non-viral CRISPR system will be developed for generation of iCPCs using a clinically acceptable gene vector system and the biological and functional features of new iCPCs will be characterized. In Aim-2, epigenetic factors mediating iCPC generation will be the focus of experiments. Long non-coding RNAs and targeted epigenetic mediators will be investigated to dissect the mechanisms of cellular reprogramming. In Aim-3, iCPCs will be implanted using cell patch techniques, and their cell fate decisions will be tracked in the infarcted heart. The extent and role of differentiated iCPCs will be determined by in vivo imaging and distinguished from other consequences using a cell ablation approach. In conclusion, this proposal will provide a new regeneration strategy for MI therapy through convergence of CRISPR technology and tissue engineering in CPC development.