CELL BIOLOGY OF AIRWAY EPITHELIAL GENE TRANSFER

本项目检验的假设是， 载体和孔的顶端结构域之间的相互作用- 分化的（WD）气道上皮细胞构成了 高效的基因转移因为我们假设 载体结合和内化步骤是限速的， 项目将集中在气道上皮细胞生物学，因为它涉及 这些方面的载体细胞相互作用。具体目标1将定义 WD人气道顶端区的屏障和靶点 上皮细胞结合形态学和免疫组织化学研究 将定量与基因转移相关的糖萼组分， 潜在的靶受体的分布[七个跨膜（7- TM）与生长/营养]的顶侧与基底侧域， 最后，每个细胞膜的内部化能力， 连接，集中在不同的气道区域（支气管与 细支气管的）。具体目标2将检验假设 “修改宿主”，以增加载体对病毒的接触， WD细胞的基底外侧结构域和/或基底细胞将增加基因表达。 传输效率提出了两种广泛的方法：氧化损伤 对上皮，非特异性地增加渗透性;和细胞 选择性增加载体渗透的生物学方法 紧密连接（TJ）。第3章测试 假设载体可以被“修饰”以靶向一类受体 (the 7-TM），其在顶膜中表达并内化 在激动剂刺激后。载体将被导向P2 Y/2受体 和其他7-TM受体，使用双特异性和/或连接至修饰的 天然配体作为“同源”部分。对于两个“修改 宿主”和“修饰载体”的策略， 研究将评估每种药物的有效性和安全性， approach.对于所有的研究，我们将采用一系列的载体， 包括AAV、慢病毒载体和腺病毒载体。互补 将采用模型系统，包括WD人体气液 接口和小鼠鼻和气管模型用于体内研究。 我们的目标是开发有效的基因转移到WD气道上皮 CF肺的大小气道中的细胞。

This Project tests the hypothesis that the initial steps in the interaction between the vector and the apical domain of well- differentiated (WD) airway epithelia constitute a principal barrier to efficient to efficient gene transfer. Because we hypothesize that both the vector binding and internalization steps are rate limiting, the project will focus on the cell biology of airway epithelia as it relates to these aspects of vector-cell interactions. Specific Aim 1 will define the barriers and targets in the apical domain of WD human airway epithelia. A combination of morphologic and immunohistochemical studies will quantitate the glycocalyleal components pertinent to gene transfer, the distribution of potential target receptors [seven transmembrane (7- TM) versus growth/trophic] on apical versus basolateral domains, the capacity for internalization of each membrane, and finally, the tight junctions, focussing on different airway regions (bronchial versus bronchiolar) within the lung. Specific Aim 2 will test the hypothesis that "modification of the host" to increase access of vectors to the basolateral domain of WD cells and/or basal cells will increase gene transfer efficiency. Two broad approaches are proposed: oxidant damage to epithelium, increasing permeability non-specifically; and cell biologic approaches to selectively increase the permeation of vectors through the tight junctions (TJ). Specific Aim 3 will test the hypothesis that vectors can be "modified" to target a class of receptor (the 7-TM) that are expressed in the apical membrane and internalized after agonist stimulation. Vectors will be directed to P2Y/2 receptors and other 7-TM receptors, using bis-specific and/or linked to modified natural ligands as the "cognate" moieties. For both "modifications of the host" and "modification of the vector" strategies, the proposed studies will assess both the efficiency and the safety aspects of each approach. For all studies, we will employ a spectrum of vectors, including AAV, lentiviral vectors, and adenoviral vectors. Complementary model systems will be employed, including the WD human air-liquid interface and the mouse nasal and tracheal models for in vivo studies. Our goal is to develop efficient gene transfer to WD airway epithelial cells in both the large and small airways of the CF lung.