Induction of Cardiomyocyte Proliferation via Transient Expression of Cell Cycle Factors as a Promising Therapy for Heart Failure

在美国，每10个65岁以上的人中就有1人死于心力衰竭。我们最近发现了四个细胞的组合- 在成年心肌细胞中诱导稳定的胞质分裂以弥补心肌细胞在心肌梗死后丢失的周期调节剂 脑梗塞。具体地说，腺病毒过表达细胞周期蛋白依赖性激酶1(CDK1)、CDK4、细胞周期蛋白B1和 Cyclin D1(统称为4F，即四种因子)能有效地诱导有丝分裂后的小鼠、大鼠、 和人类心肌细胞。细胞周期调节因子的过度表达是通过蛋白酶体自我限制的- 介导的蛋白产物在心肌细胞中的降解。在体内，使用马赛克进行血统追踪 对双标记(MADM)小鼠模型的分析表明，15%-20%的成年心肌细胞表达 4F细胞分裂稳定，急性或亚急性后心功能明显改善 心肌梗死。目前，我们的方法是诱导心肌细胞增殖的最有力的方法； 然而，由于对其他器官致癌潜力的担忧，临床应用受到了限制。我们的 体外初步数据显示，在其他类型的细胞(如神经元)中，4F持续诱导细胞增殖 7天内连续5-6轮。因此，在这项提案中，我们将重点介绍使细胞周期 在心肌细胞中诱导更适合临床应用，避免任何致癌潜力。 我们假设，需要一种短暂的、心肌细胞特异性的表达来诱导一个周期的 心肌细胞增殖，避免对其他组织产生任何潜在的不利影响。我们的初步数据显示 TNNT2衍生的4F在NIL细胞中的表达在体内外均有较强的促增殖作用，并得到改善。 心肌梗塞后的心功能。在这里，我们将严格证明这种方法的有效性和安全性。 TNNT2心脏特异性启动子(TNNT-4F-NIL)调控的4F编码基因多顺反子NIL 诱导大鼠和猪心肌梗死后心肌细胞分裂及改善心功能的实验研究 模型以及来自健康和梗死心脏的人心脏切片的原位研究。在这里，我们的目标首先是全面 证明TNNT-4F-NIL仅在体内诱导心肌细胞增殖的有效性和特异性。 其次，我们将测试TNNT-4F-NIL的功能有效性，并在大鼠和猪身上进行初步的安全性研究 心力衰竭的模型。最后，我们将展示TNNT-4F-NIL诱导成人人类 人心脏切片中心肌细胞的原位增殖和改善衰竭心脏的收缩功能 切片。这项研究将解决所有临床前疗效测试，并执行初步的安全性研究之一 有希望再生心脏的方法。这项研究的成功完成将使第一个 在人体临床试验中。

Heart failure kills 1 in 10 people over the age of 65 in the USA. We recently identified a combination of four cell- cycle regulators that induces stable cytokinesis in adult cardiomyocytes to replace their loss following myocardial infarction. Specifically, adenoviral overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 (collectively known as 4F, i.e. four factors), efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome- mediated degradation of the protein products in cardiomyocytes. In vivo, lineage tracing using the Mosaic Analysis of Double Marker (MADM) mouse model revealed that 15–20% of adult cardiomyocytes expressing the 4F underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Currently, our approach is the most robust method to induce cardiomyocyte proliferation; however, clinical applicability in humans is limited by concerns for tumorigenic potential in other organs. Our preliminary data in vitro shows that in other cell types (e.g. neurons), 4F induce cell proliferation continuously for 5-6 successive rounds over 7 days. Therefore, in this proposal we will focus on approaches to make cell cycle induction in cardiomyocytes more clinically applicable and avoid any oncogenic potential. We hypothesize that a transient and cardiomyocyte-specific expression is needed to induce one cycle of cardiomyocyte proliferation to avoid any potential adverse effects on other tissues. Our preliminary data shows that TNNT2 derived 4F expression in NIL is robustly inducing proliferation in vitro and in vivo and improved cardiac function after myocardial infarction. Here, we will rigorously demonstrate the efficacy and safety of this polycistronic NIL encoding 4F expression driven by the cardiac specific promoter of TNNT2 (TNNT-4F-NIL) in inducing cardiomyocyte division and improving cardiac function after myocardial infarction in vivo in rat and pig models as well as in situ in human heart slices from healthy and infarcted hearts. Here, we aim first to fully demonstrate the efficacy and specificity of TNNT-4F-NIL to induce proliferation only in cardiomyocytes in vivo. Secondly, we will test the functional efficacy and perform initial safety studies for TNNT-4F-NIL in rat and pig models of heart failure. Lastly, we will demonstrate the efficacy of TNNT-4F-NIL in inducing adult human cardiomyocyte proliferation in situ in human heart slices and improving contractile function of failing human heart slices. This study will address all preclinical efficacy testing and perform initial safety studies for one of the most promising approaches to regenerate the heart. The successful completion of this study will allow the start of first in human clinical trial.