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

说明(申请人提供)：心血管疾病(CVD)在美国仍然是主要的死亡原因。在心血管疾病的各种疾病中，心肌梗死(MI)，通常被称为心脏病发作，是导致大量CVD死亡的直接原因，也是其他形式CVD死亡的一个促成因素。随着每年报告的120万新发或复发的心肌梗塞病例，其中超过三分之一导致死亡，改进现有的治疗方法和开发新的、更有效的MI疗法是当务之急。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，由于我们无法在没有转基因的情况下改变蛋白质及其在体内的表达水平，这一知识在临床环境中的转换受到阻碍。Alpha B-Crystallin(1BC)是一种蛋白质，在模拟心肌梗死的各种模型中已被证实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蛋白输送到细胞和组织中，而不需要使用转基因、病毒转移或转基因。这一独特的系统利用了细胞穿透肽(CPP)的特性，它具有移动自身和货物穿过细胞膜的能力。我们设计了一种系统，可以在1CB蛋白和CPP之间建立可逆联系，从而允许1BC进入细胞。一旦进入细胞，两个分子之间的连接就被切断，释放出细胞内的1BC蛋白。我们已经证明，该系统在体外有效地工作，可以将1BC输送到培养中的细胞。此外，将与CPP多肽相连的1BC注入小鼠左室壁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方面进行进一步的研究，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中的细胞免受渗透、活性氧和低氧应激源的能力。我们还将在体外模型中检测该系统以提供I/R保护的方式将1BC输送到心脏的能力。最后，我们将检测输送系统将1BC输送到梗死区内的心肌细胞的能力，并在体内的MI小鼠模型中保护组织免受损伤和细胞死亡。 与公共健康相关：在美国，心脏病发作是导致大量死亡的原因，也是导致其他形式的心脏病相关死亡的主要因素。这项拟议的研究探索了一种将保护性分子直接引入心脏细胞的新技术，目的是保护心脏免受心脏病发作造成的损害。

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.