Novel strategy for Enhancing miRNA as a Therapeutic for Cardiac Regeneration

用新的功能性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，并通过各种途径， 过程主动损害收缩功能。将瘢痕组织内的心脏成纤维细胞转化为功能性的 心肌细胞是一种具有恢复受损心脏功能的巨大潜力的治疗方法。 我们是第一个鉴定了miRNAs的组合，miR组合（miR-1，miR-133 a，miR-208 a和miR-499- 2010）。 5 p）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 转录因子达到同样的效果。重要的是，使用慢病毒或逆转录病毒的miR组合的递送导致了在细胞内的表达。 适度但显著的心脏功能改善。该申请专注于改善miR组合 作为一种治疗方法，通过确定提高效率，细胞特异性和有效性的方法。初步 研究已经鉴定了特异性靶向成纤维细胞的腺相关病毒（AAV）血清型， 而且，在体内显示出增强的转导效率。此外，TLR 3激活，这强烈 在体外增强miR组合指导的重编程，可能是一种新的方式来增加有效性。最后， 几种转录抑制剂已被鉴定为miR组合靶。这些转录抑制剂 抑制了关键心脏转录因子的表达，提示miR组合指导的机制 重新编程本项目将调查三个相互重叠的领域。目标1将侧重于优化 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- 表达多顺反子miR组合的互补（sc）AAV 2/1导致miR的成纤维细胞特异性递送。 miR组合。药理学试剂将定义用于治疗的TLR 3活化的适当水平和持续时间。 miR组合介导的重编程的最佳增强。目标2将在体内解决相同的问题。 斑点追踪超声心动图将确定在体内局部室壁运动的变化。组织学将 定量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编程。 药理学试剂将用于确定TLR 3活化是否增强功能改善 在成纤维细胞通过miR组合重编程之后。目的3将定义miR组合 促进基因重组。获得和丧失功能的方法将决定转录因子的作用。 miR组合中的阻遏物指导重编程。荧光素酶测定将用于描绘物理 miR组合内的miRNA与转录阻遏物之间的相互作用。诱饵抑制剂 方法将确定NF κ B B，TLR 3途径中的关键转录因子，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。 直接重编程的细胞特异性用于心脏修复和再生作为治疗。

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.