Cardiomyocyte-specific modified mRNA of Pkm2 for induction of cardiac regeneration in ischemic heart failure

项目总结 慢性心肌梗死(MI)后继发的缺血性心力衰竭(HF)是主要的死亡原因 以及全世界的残疾问题。缺血性心力衰竭的高发病率和死亡率在很大程度上是由 哺乳动物成年心脏的再生能力低(因此也就是修复能力)。几种组织工程，细胞- 基于基因的治疗已被尝试用于诱导心肌梗死后的心脏再生。自.以来 心肌细胞(CM)增殖和心脏再生在斑马鱼和蝾螈中是非常健壮的过程， 在成人身上模仿这些较低系统发育生物的促增殖机制的努力 哺乳动物的心脏已经尝试使用标准的基因治疗载体。然而，这些方法， 遇到与短期、低水平或不受控制的目标交付有关的重大挑战 吉恩。在最好的情况下，这导致了糟糕的CM增殖。在其他情况下，这些战略导致了高度的 不良后果，包括病理性肥大和炎症加重。我们最近做了 研究表明，修饰的信使核糖核酸(ModRNA)是一种安全、有效、瞬时、非免疫原性、局部和 剂量控制型心脏基因传递平台。我们发现糖酵解酶丙酮酸激酶 肌肉同工酶M2(PKM2)在再生性胎儿和新生儿CMS中高表达，但在成人中不表达 心肌梗死时给予CMS是重新唤醒心脏再生的有效载体。有选择性的 将PKM2 modRNA传递到不同的细胞群体导致它们各自的比率显著增加 扩散。肌成纤维细胞等非CMS的增殖可能通过以下方式加剧病理重塑 舒张期功能受损，心肌传导中断，并引发心律失常。为了规避这一点， 我们设计了一种独特的CM特异性modRNA方法来改善心肌梗死后的心脏修复和 再生。这一建议将为modRNA介导的PKM2的传递确定最佳的治疗窗口 主动愈合期后的CMS有效逆转机电功能障碍，将 确定局部和瞬时传递PKM2促进CM增殖的分子机制 通过β-连环蛋白和下游信号转导，最终确定改变的传导特性和风险 与新形成的依赖PKM2的CMS与宿主有效整合相关的心律失常 心肌梗死后的心肌。我们建议的modRNA平台的高度可翻译特性和我们的集成式、 多学科实验计划最终面向缺血性心脏的临床适用性 失败了。

PROJECT SUMMARY Ischemic heart failure (HF) secondary to chronic post myocardial infraction (MI), is a leading cause of death and disability worldwide. The high morbidity and mortality rates in ischemic HF are caused, in large part, by the low regenerative capacity (and therefore repair) of the mammalian adult heart. Several tissue-engineering, cell- and gene-based therapies have been attempted for induction of cardiac regeneration following MI. Since cardiomyocyte (CM) proliferation and cardiac regeneration are highly robust processes in Zebrafish and Newts, efforts to mimic the pro-proliferative mechanisms of these lower phylogenetic organisms in the adult mammalian heart have been attempted using standard gene therapy vectors. Those approaches however, have encountered major challenges that are related to short-term, low, or uncontrolled delivery of the target gene. At best, this has resulted in poor CM proliferation. In other cases, these strategies have led to highly undesirable outcomes, including exacerbation of pathological hypertrophy and inflammation. We have recently shown that modified mRNA (modRNA) delivery is a safe, efficient, transient, non-immunogenic, local, and dose-controlled cardiac gene delivery platform. We have found that the glycolytic enzyme Pyruvate Kinase Muscle Isozyme M2 (Pkm2), which is highly expressed in regenerative fetal and neonatal CMs but not in adult CMs is an effective vehicle for re-awakening cardiac regeneration when delivered at the time of MI. Selective Pkm2 modRNA delivery to distinct cell populations led to a significant increase in their respective rates of proliferation. Proliferation of non-CMs such as myofibroblasts may exacerbate pathological remodeling by impairing diastolic function, disrupting myocardial conduction, and promoting arrhythmias. To circumvent this, we have designed a unique CM-specific modRNA-based approach for improving post-MI cardiac repair and regeneration. This proposal will identify the optimal therapeutic widow for modRNA-mediated delivery of Pkm2 to CMs following the phase of active healing for effective reversal of electro-mechanical dysfunction, will determine the molecular mechanisms by which local and transient delivery of Pkm2 promotes CM proliferation via β-catenin and downstream signaling, and finally determine the altered conduction properties and risk of arrhythmias associated with efficient integration of newly-formed Pkm2-dependent CMs with the host myocardium post MI. The highly translatable nature of our proposed modRNA platform and our integrative, multi-disciplinary experimental plan are ultimately geared towards clinical applicability for ischemic heart failure.