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， 随后对内皮细胞进行遗传修饰，用于各种治疗目的，包括诱导血管生成、预防再狭窄、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，腺病毒在介导内皮基因转移方面是最有效的，但高载体浓度是必要的，伴随着与载体相关的不良事件的风险。在这种情况下，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提高的基因转移到内皮细胞的效率，这将导致递送给患者的载体剂量的相应减少。为了实现增加基因转移到内皮细胞的潜力，必须首先表征基因转移到内皮细胞的生物学。在这种情况下，该提议的中心假设是，评估腺病毒在内皮细胞中的运输将产生提高基因转移到内皮细胞的效率的策略。为了实现这一假设，将在内皮细胞的不同生理状态（包括静息或活化）的背景下评价腺病毒与内皮细胞的相互作用。 内皮细胞，如亚汇合培养物、汇合极化单层或受伤极化单层。这些策略将为了解腺病毒嗜性改变对基因转移到内皮细胞效率的影响奠定基础。该项目集中于两个目标：（1）表征腺病毒在内皮细胞的不同生理状态下的结合和细胞内运输的效率;和（2）表征腺病毒通过替代细胞表面受体感染后进入内皮细胞和运输。

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.