CRISPR-induced cardiovascular progenitor cells to repair myocardial infarction

CRISPR诱导心血管祖细胞修复心肌梗塞

• 批准号: 10372161
• 负责人: Jialiang Liang
• 金额: $ 54.78万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10372161
• 关键词: Ablation; Address; Biological; Bypass; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Cardiac; Cardiovascular Diseases; Cardiovascular system; Cause of Death; Cell Therapy; Cells; Chromatin; Cicatrix; Clinical; Clonality; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complementary DNA; Complex; Data; Development; Effectiveness; Embryo; Embryonic Heart; Engraftment; Ensure; Enzymes; Epigenetic Process; Feedback; Fibroblasts; Fostering; GATA4 gene; Gene Activation; Generations; Genes; Genetic; Genotype; Heart; Heterogeneity; Histones; Implant; Infarction; Insertional Mutagenesis; Investigation; MLL gene; Mediating; Mediator of activation protein; Modeling; Molecular; Mus; Myocardial Infarction; Myocardial Ischemia; Myocardium; Natural regeneration; Oncogenic; Outcome; Phenotype; Process; Property; RNA-Protein Interaction; Regulation; Research; Ribonucleoproteins; Role; Safety; Selection for Treatments; Somatic Cell; Source; Stem Cell Development; System; Techniques; Technology; Therapeutic; Tissue Engineering; Transcript; Translations; Transplantation; Treatment Efficacy; Tumorigenicity; Undifferentiated; Untranslated RNA; adverse outcome; base; cardiac regeneration; cardiac repair; cell regeneration; design; dosage; experimental study; functional restoration; gene network; genome editing; genomic locus; heart function; histone methyltransferase; imaging approach; implantation; improved; in vivo imaging; induced pluripotent stem cell; neovascularization; next generation sequencing; overexpression; pluripotency; pre-clinical; regenerative; regenerative therapy; repaired; screening; self-renewal; stem cells; tool; trait; transcription factor; translational study; vector

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.

项目总结/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 国际吧细胞重编程为心血管祖细胞（CPC），绕过了 多能性诱导为心脏修复提供了一种有前途的方法， 新血管形成和心肌形成。目前研究的一个重大差距是不清楚 了解直接重编程的机制。内源性基因位点的激活 (that被抑制性染色质标记所遮挡）被认为是重要的 高质量完全重编程细胞的标准。因此，我们试图获得新的CPC来源 通过直接操纵内源基因。为此，我们建议结合使用 重编程技术和基于CRISPR的工具CRISPR诱导的基因激活上级于 采用cDNA过表达的常规技术由于其有效性、设计简单 并且避免了对转录因子复合物的额外转导的需要。我们的初步 数据表明，CRISPR诱导的细胞具有CPC的特性，包括克隆性、自噬性、 更新和心脏三潜能。重要的是，CRISPR诱导的CPC（iCPC）可以钝化 心肌梗死小鼠移植后心脏功能恶化和梗死面积减少。这些发现 为在临床前环境中进一步系统研究CRISPR-iCPC奠定了基础。因此，委员会认为， 提出了三个具体目标，以开发一个技术平台来产生新的CRISPR-iCPC，研究 细胞重编程机制，并评估iCPC在MI治疗中的应用。在Aim-1中， 将开发非病毒CRISPR系统，用于使用临床上可接受的基因生成iCPC 载体系统和新的iCPC的生物学和功能特征将被表征。在Aim-2中， 介导iCPC产生的表观遗传因素将是实验的重点。长非编码rna 和针对性的表观遗传介质将被调查，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植入，并且它们的细胞命运 将在梗塞的心脏中跟踪决策。分化的iCPC的程度和作用将是 通过体内成像确定，并与使用细胞消融方法的其他结果区分开。 总之，该建议将通过融合为MI治疗提供新的再生策略 CRISPR技术和组织工程在CPC开发中的应用。