CELL BIOLOGY OF AIRWAY EPITHELIAL GENE TRANSFER

这个项目测试了一种假设，即 载体与Well的顶端区域之间的相互作用 分化的(WD)呼吸道上皮构成了主要的屏障 高效到高效的基因转移。因为我们假设两者 载体绑定和内部化步骤是速率限制， 该项目将专注于呼吸道上皮的细胞生物学，因为它与 载体-细胞相互作用的这些方面。具体目标1将定义 WD人呼吸道顶区的屏障和靶点 上皮细胞。形态学和免疫组织化学相结合的研究 将对与基因转移相关的糖基化成分进行量化， 潜在靶向受体的分布[七个跨膜(7- TM)与生长/营养]在顶端区域和基侧区域上， 每层膜的内化能力，最后是紧密性 连接，集中在不同的呼吸道区域(支气管区与 细支气管炎)在肺内。《特定目标2》将检验这一假设 “修改宿主”，以增加载体对 WD细胞和/或基底细胞的基侧结构域将增加基因 转移效率。提出了两种主要的方法：氧化剂损伤 上皮细胞，非特异性增加通透性；细胞 选择性增加载体渗透性的生物学方法 通过紧密连接(TJ)。《特定目标3》将测试 载体可以被“修饰”以针对一类受体的假设 (7-TM)，在顶膜上表达并内化 在激动剂刺激后。载体将被定向到P2Y/2受体 和其他7-TM受体，使用双特异性和/或连接到修饰的 作为“同源”部分的天然配体。对于这两个“修改 “东道主”和“修改载体”战略，建议 研究将评估每种药物的效率和安全性。 接近。对于所有的研究，我们将使用一系列的载体， 包括AAV、慢病毒载体和腺病毒载体。互补性 将采用模型系统，包括WD人体气液系统 界面和小鼠鼻腔和气管模型用于活体研究。 我们的目标是开发高效的基因转移到WD呼吸道上皮细胞 CF型肺的大、小气道内均有细胞。

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.