Development of a new system for the delivery of protective molecules to the heart

描述（由申请人提供）：在美国，心血管疾病（CVD）仍然是导致死亡的主要原因。在心血管疾病的各种情况下，心肌梗死（MI），更常见的称为心脏病发作，是导致大量CVD死亡的直接原因，也是导致其他形式CVD死亡的一个因素。每年报告120万例心肌梗死新发或复发病例，其中三分之一以上导致死亡，因此改进目前的治疗方法和开发新的、更有效的心肌梗死治疗方法是当务之急。心脏研究人员已经确定了许多保护心脏免受I/R损伤的蛋白质。然而，将这些知识转化到临床环境受到阻碍，因为我们无法在没有转基因的情况下修改蛋白质及其在体内的表达水平。α B-晶体蛋白（1BC）是一种蛋白质，已被确定在各种模拟心肌梗死的模型中具有强大的心脏保护作用。此外，已经确定了增强蛋白质保护作用的修饰。我们已经在开发一种系统方面迈出了第一步，该系统可以将1BC蛋白传递到细胞和组织中，而无需使用转染、病毒转移或转基因。这种独特的系统利用细胞穿透肽（CPP）的特性，CPP具有移动自身和货物穿过细胞膜的能力。我们设计了一个系统，在1CB蛋白和CPP之间建立可逆的链接，允许1BC进入细胞。一旦进入细胞，两个分子之间的连接就会断裂，释放出细胞内的1BC蛋白。我们已经证明该系统在体外有效工作，可以将1BC传递到培养细胞中。此外，将与CPP肽相关的1BC注射到小鼠左心室壁上，可导致常驻心肌细胞摄取1BC蛋白。我们建议在细胞培养和动物模型中进行进一步的研究，以继续开发该系统并在已建立的心脏模型中测试其性能。这些研究包括评估输送系统保护培养细胞免受渗透、活性氧和缺氧胁迫的能力。我们还将在离体模型中研究该系统以提供I/R保护的方式将1BC输送到心脏的能力。最后，我们将在心肌梗死小鼠体内模型中检测递送系统将1BC递送到梗死区心肌细胞并保护组织免受损伤和细胞死亡的能力。

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) continues to be the leading cause of death in the U.S. Of the various conditions that fall under the umbrella of cardiovascular disease, myocardial infarction (MI), known more commonly as a heart attack, is directly responsible for a significant number of the deaths attributed to CVD, and is a contributing factor in mortality attributed to other forms of CVD. With 1.2 million new or recurrent cases of MI reported every year, more than a third of which result in death, improving on current treatments and developing new, more effective treatments for MI are top priorities. Cardiac researchers have identified many proteins that that protect the heart against I/R injury. However, translation of this knowledge to the clinical setting is hampered by our inability to modify proteins and their expression levels in vivo without transgenesis. Alpha B- Crystallin (1BC) is a protein that has been identified as having robust cardioprotective effects in various models which mimic myocardial infarction. Furthermore, modifications have been identified which enhance the protective effects of the protein. We have taken the first steps in developing a system to deliver 1BC proteins to cells and tissue without using transfection, viral transfer or trangenesis. This unique system utilizes the properties of cell penetrating peptides (CPP) which have the ability to move themselves and cargo across cell membranes. We have devised a system that creates a reversible linkage between the 1CB protein and the CPP allowing for the delivery of 1BC into cells. Once inside the cell, the linkage between the two molecules is cleaved, freeing the 1BC protein within the cell. We have demonstrated that this system works effectively in vitro and can deliver 1BC to cells in culture. Furthermore, injecting 1BC linked to the CPP peptide into the left ventricle wall of mouse hearts results in uptake of the 1BC protein by resident cardiomyocytes. We are proposing further studies in both cell culture and animal models, to continue the development of this system and to test its performance in established cardiac models. The studies include assessing the ability of the delivery system to protect cells in culture from osmotic, reactive oxygen species and hypoxic stressors. We will also examine the ability of the system to deliver 1BC to hearts, in an ex vivo model, in a manner that provides protection from I/R. Lastly, we will examine the ability of the delivery system to deliver 1BC to cardiomyocytes within the infarct zone and protect the tissue from damage and cell death in an in vivo mouse model of MI. PUBLIC HEALTH RELEVANCE: Heart attacks are responsible for a significant number of deaths in the U.S. and are a major contributing factor to other forms of heart disease related death. The proposed research explores a new technique to introduce protective molecules directly in to the cells of the heart, with the goal of protecting the heart against damage caused by a heart attack.