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

• 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
• 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

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、有功能的心肌细胞修复和再生受损的心脏仍然是一个艰巨的挑战 用于心血管药物。心脏损伤后，成纤维细胞进入损伤区，通过各种途径 这些过程会主动损害收缩功能。将瘢痕组织内的心脏成纤维细胞转化为功能性 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。 我们首先确定了miRNAs、miR组合(miR-1、miR-133a、miR-208a和miR-499- 5P)直接将心脏成纤维细胞重新编程为心肌细胞，而其他人则使用 转录因子产生同样的效果。重要的是，使用慢病毒或逆转录病毒传递miR组合导致了 心脏功能略有改善，但意义重大。此应用程序的重点是改进miR组合 通过寻找提高效率、细胞特异性和有效性的方法来治疗。初步 研究已经确定了一种腺相关病毒(AAV)血清型，它专门针对成纤维细胞， 此外，在体内表现出更高的转导效率。此外，TLR3的激活，强烈地 增强miR组合在体外定向重编程，可能是一种提高有效性的新方法。最后， 几种转录抑制物已被确定为miR组合靶点。这些转录抑制物 抑制关键的心脏转录因子的表达，提示miR组合导向的机制 重新编程。本项目将对三个重叠区域进行调查。目标1将专注于优化 MiR组合的体外效率、有效性和细胞特异性。为此，我们将测试自我是否- 互补(Sc)AAV2/1表达多顺反子miR组合导致成纤维细胞特异性递送 和平号套餐。药理学药物将定义TLR3激活的适当水平和持续时间 MiR组合介导的重新编程的优化增强。Aim 2将在体内解决同样的问题。 斑点追踪超声心动图将确定活体局部室壁运动的变化。组织学将会 量化纤维化水平和谱系追踪将评估成纤维细胞向心肌细胞的重新编程。 将使用药理学药物来确定TLR3激活是否增强了功能改善 这是在miR como对成纤维细胞重新编程之后。目标3将定义miR组合的机制 促进重新编程。功能获得和功能丧失的方法将决定转录的作用 MiR组合中的抑制子指示了重新编程。荧光素酶分析将被用来描述物理 MiR组合内的miRNAs与转录抑制物之间的相互作用。诱饵抑制 方法将确定核转录因子B，TLR3途径中的关键转录因子，在miR组合中的作用 定向重新编程。 这些研究的结果将为提高效率、有效性和 心脏修复和再生的直接重编程作为治疗的细胞特异性。