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个死于心力衰竭。我们最近发现了四种细胞的组合- 周期调节剂可诱导成年心肌细胞稳定的胞质分裂，以取代心肌梗死后的胞质分裂损失 梗塞具体地说，腺病毒过表达细胞周期蛋白依赖性激酶1（CDK 1）、CDK 4、细胞周期蛋白B1和 细胞周期蛋白D1（统称为4F，即四个因子）在有丝分裂后的小鼠，大鼠， 和人心肌细胞。细胞周期调节因子的过度表达是通过蛋白酶体自限性的， 介导的蛋白质产物在心肌细胞中的降解。在体内，使用Mosaic 双标记（MADM）小鼠模型的分析显示，15-20%的成年心肌细胞表达MADM。 4F细胞分裂稳定，急性或亚急性心肌梗死后心功能明显改善， 心肌梗死目前，我们的方法是诱导心肌细胞增殖的最稳健的方法; 然而，由于担心在其它器官中的致瘤潜力，在人体中的临床应用受到限制。我们 体外初步数据显示，在其他细胞类型（如神经元）中，4F连续诱导细胞增殖， 5-6在7天内连续几轮。因此，在本提案中，我们将重点关注使细胞周期 在心肌细胞中的诱导更临床适用且避免任何致癌潜力。 我们假设，一个短暂的和心肌细胞特异性的表达是诱导一个周期的心肌细胞凋亡所必需的。 以避免对其他组织的任何潜在的不利影响。我们的初步数据显示 TNNT 2衍生的4F在NIL中的表达在体外和体内强烈诱导增殖，并且改善了 心肌梗死后的心功能在这里，我们将严格证明这种药物的有效性和安全性。 由TNNT 2的心脏特异性启动子驱动的编码4F表达的多顺反子NIL（TNNT-4F-NIL）在 诱导心肌细胞分裂改善大鼠和猪心肌梗死后心功能的实验研究 模型以及来自健康和梗塞心脏的人心脏切片中的原位。在这里，我们的目标首先是充分 证明了TNNT-4F-NIL仅在体内心肌细胞中诱导增殖的功效和特异性。 其次，我们将在大鼠和猪中测试TNNT-4F-NIL的功能功效并进行初步安全性研究 心力衰竭的模型。最后，我们将证明TNNT-4F-NIL在诱导成年人成纤维细胞中的功效。 心肌细胞在人心肌片上的原位增殖及对衰竭心脏收缩功能的改善作用 切片这项研究将解决所有临床前疗效测试，并进行初步的安全性研究，其中一个最 有希望的方法来再生心脏。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.