Novel strategy for Enhancing miRNA as a Therapeutic for Cardiac Regeneration

增强 miRNA 作为心脏再生治疗的新策略

基本信息

批准号：
9237608
负责人：
Victor J Dzau
金额：
$ 39.75万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-15 至 2020-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9237608
关键词：
Address Adult Agonist Area Bioinformatics Biological Assay Cardiac Cardiac Myocytes Cardiovascular system Cell Fate Control Cells Cicatrix Clinical Clinical effectiveness Data Dependovirus Echocardiography Effectiveness Fibroblasts Fibrosis Future Gene Expression Genes Genetic Transcription Heart Histology Impairment In Vitro Infection Injury Invaded Knockout Mice Luciferases Mediating Medicine Messenger RNA Methods MicroRNAs Motion Myocardial Myocardial Infarction Natural Immunity Natural regeneration Pathway interactions Pharmacology Process Recovery of Function Reporting Retroviridae Role SOX6 gene Serotyping Signal Pathway Signal Transduction Specificity Subfamily lentivirinae TLR3 gene Testing Therapeutic Tissues Transcription Repressor/Corepressor Viral Virus base cardiac regeneration cardiac repair functional improvement functional restoration gain of function heart function improved in vivo induced pluripotent stem cell inhibitor/antagonist injured insight loss of function novel novel strategies repaired response transcription factor transdifferentiation transduction efficiency vector

项目摘要

Repair and regeneration of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiovascular medicine. Following cardiac injury, fibroblasts enter injury zone and through various processes actively impair contractile function. Converting cardiac fibroblasts within scar tissue into functional cardiomyocytes is a therapeutic approach that has great potential to restore the function of an injured heart. We were the first to identify a combination of miRNAs, miR combo (miR-1, miR-133a, miR-208a, and miR-499- 5p) that directly reprogrammed cardiac fibroblasts into cardiomyocytes, whereas others have used transcription factors to the same effect. Importantly, delivery of miR combo using lentivirus or retrovirus led to a modest, but significant, improvement in cardiac function. This application is focused on improving miR combo as a therapeutic by identifying ways to enhance efficiency, cell specificity, and effectiveness. Preliminary studies have identified an Adeno-associated virus (AAV) serotype that specifically targets fibroblasts and, moreover, displays enhanced transduction efficiency in vivo. Furthermore, TLR3 activation, which strongly augments miR combo directed reprogramming in vitro, may be a novel way to increase effectiveness. Finally, several transcriptional inhibitors have been identified as miR combo targets. These transcriptional inhibitors repressed the expression of key cardiac transcription factors, suggesting a mechanism for miR combo directed reprogramming. Three overlapping areas will be investigated in this project. Aim 1 will focus on optimizing the efficiency, effectiveness, and cell specificity of miR combo in vitro. To that end we will test if the self- complementary (sc)AAV 2/1 expressing a polycistronic miR combo results in the fibroblast-specific delivery of miR combo. Pharmacological agents will define the appropriate level and duration of TLR3 activation for optimal enhancement of miR combo mediated reprogramming. Aim 2 will address the same questions in vivo. Speckle tracking echocardiography will determine changes in regional wall motion in vivo. Histology will quantify fibrosis levels and lineage tracing will evaluate reprogramming of fibroblasts into cardiomyocytes. Pharmacological agents will be used to determine if TLR3 activation enhances the functional improvements that follow fibroblast reprogramming by miR combo. Aim 3 will define the mechanism by which miR combo promotes reprogramming. Gain- and loss-of-function approaches will determine the role of the transcriptional repressors in miR combo directed reprogramming. Luciferase assays will be used to delineate the physical interaction between the miRNAs within miR combo and the transcriptional repressors. Decoy inhibitor approaches will define the role of NFB, the key transcription factor in the TLR3 pathway, in miR combo directed reprogramming. Findings from these studies will provide important new insights into improving the efficiency, effectiveness, and cell specificity of direct reprogramming for cardiac repair and regeneration as a therapy.

用新的功能性心肌细胞修复和再生受伤的心脏仍然是一项艰巨的挑战用于心血管医学。心脏损伤后，成纤维细胞进入损伤区域并通过各种途径。过程主动损害疤痕组织内的心脏成纤维细胞的收缩功能。心肌细胞是一种治疗方法，具有恢复受损心脏功能的巨大潜力。我们是第一个鉴定出 miRNA 组合、miR 组合（miR-1、miR-133a、miR-208a 和 miR-499- 5p）直接将心脏成纤维细胞重编程为心肌细胞，而其他人则使用重要的是，使用慢病毒或逆转录病毒传递 miR 组合会产生相同的效果。心脏功能得到适度但显着的改善该应用的重点是改善 miR 组合。通过确定提高效率、细胞特异性和有效性的方法来作为治疗方法。研究已经确定了一种专门针对成纤维细胞的腺相关病毒 (AAV) 血清型，此外，在体内表现出增强的转导效率。此外，TLR3 激活，这强烈。在体外增强 miR 组合定向重编程，可能是提高有效性的新方法。几种转录抑制剂已被确定为 miR 组合靶标。抑制关键心脏转录因子的表达，提示 miR 组合定向的机制该项目将研究三个重叠领域，重点是优化。为此，我们将测试 miR 组合的体外效率、有效性和细胞特异性。互补的 (sc)AAV 2/1 表达多顺反子 miR 组合，导致成纤维细胞特异性递送 miR组合将确定TLR3激活的适当水平和持续时间。 miR组合介导的重编程的最佳增强将在体内解决相同的问题。斑点追踪超声心动图将确定体内局部室壁运动的变化。量化纤维化水平，谱系追踪将评估成纤维细胞重编程为心肌细胞的情况。将使用药物来确定 TLR3 激活是否会增强功能改善 miR 组合对成纤维细胞重编程后的结果将定义 miR 组合的机制。促进重编程的方法将决定转录的作用。 miR组合定向重编程中的阻遏物将用于描述物理。 miR 组合内的 miRNA 与转录抑制因子之间的相互作用。方法将定义 NFκB（TLR3 通路中的关键转录因子）在 miR 组合中的作用定向重编程。这些研究的结果将为提高效率、有效性和效率提供重要的新见解。直接重编程用于心脏修复和再生的细胞特异性作为一种疗法。