Trafficking of adenovirus vectors in endothelial cells

描述(由申请人提供)： 内皮细胞的解剖位置使其成为体内基因转移的理想靶点 血管内皮细胞随后的基因修饰用于各种治疗目的，包括诱导血管生成、防止再狭窄、血管成形术后、抑制肿瘤中的血管生长，以及作为治疗遗传性和获得性疾病的治疗性蛋白质的来源。在所有可用的基因转移载体中，腺病毒在介导血管内皮细胞基因转移方面是最有效的，但高载体浓度是必要的，与载体相关的不良事件的风险随之而来。在此背景下，使用腺病毒载体将基因转移到内皮细胞的策略可以受益于向内皮细胞转移基因的效率的提高，这将导致输送给患者的载体剂量相应减少。为了实现增加向内皮细胞的基因转移的潜力，必须首先对向内皮细胞转移基因的生物学进行表征。在此背景下，这项建议的中心假设是，对腺病毒在内皮细胞中的运输进行评估将产生提高向内皮细胞转移基因效率的策略。为了实现这一假设，我们将在内皮细胞处于不同的生理状态(包括静息或激活)的情况下评估腺病毒与内皮细胞的相互作用。 内皮细胞作为亚融合培养物，融合极化单层或损伤极化单层。这些策略将为了解改变的腺病毒趋向性对血管内皮细胞基因转移效率的影响奠定基础。本项目主要致力于两个目标：(1)研究不同生理状态下内皮细胞中腺病毒结合和细胞内转运的效率；(2)确定腺病毒感染后通过交替的细胞表面受体进入和转运内皮细胞的特征。

DESCRIPTION (provided by applicant): The anatomic location of the endothelial cell makes it an ideal target for in vivo gene transfer with consequent genetic modification of the endothelial cell for a variety of therapeutic purposes, including induction of angiogenesis, prevention of restenosis, following angioplasty, suppression of vessel growth in tumors, and as a source of therapeutic proteins for treatment of hereditary and acquired disorders. Of all gene transfer vectors available, adenovirus has been most effective at mediating endothelial gene transfer, but high vector concentrations are necessary, with the attendant risks for adverse events related to the vector. In this context, strategies using adenovirus vectors to transfer genes to endothelial cells could benefit from improved efficiency of gene transfer to endothelial cells which would lead to a corresponding decrease in dose of vector delivered to the patient. To realize the potential for increasing gene transfer to endothelial cells, the biology of gene transfer to endothelial cells must first by characterized. In this context, the central hypothesis of this proposal is that the evaluation of adenovirus trafficking in endothelial cells will yield strategies for improved efficiency of gene transfer to endothelial cells. To pursue this hypothesis, adenovirus interaction with endothelial cells will be evaluated in the context of varying physiological states of endothelial cells including resting or activated endothelial cells, as sub-confluent cultures, confluent polarized monolayers, or wounded polarized monolayers. These strategies will lay a foundation for understanding the impact of altered adenovirus tropism on efficiency of gene transfer to endothelial cells. This project is focused on two aims: (1) To characterize efficiency of adenovirus binding and intracellular trafficking in varying physiological states of endothelial cells; and (2) To characterize adenovirus entry and trafficking in endothelial cells subsequent to infection via alternate cell surface receptors.