Targeting the Apical Surface of Human Airway Epithelia

靶向人类气道上皮细胞的顶端表面

• 批准号: 6517963
• 负责人: Paola Drapkin Vermeer
• 金额: $ 9.82万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517963
• 关键词: apical membrane; binding proteins; endocytosis; gene expression; gene therapy; mass spectrometry; matrix assisted laser desorption ionization; membrane proteins; protein engineering; protein structure function; respiratory epithelium; technology /technique development; transfection /expression vector; urokinase

DESCRIPTION (provided by applicant) Gene therapy offers hope of a cure for cystic fibrosis (CF) by correcting mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). Though not yet a reality, the goals of this proposal are aimed at generating viral vectors that successfully target epithelial cells of the human airway. Presently, viral vectors are inefficient at infecting the airway epithelia due, in large part, to the absence of viral receptors at the apical surface. One approach to circumventing this limitation is to target viral vectors to receptors that are accessible from the apical surface. Identifying and characterizing these proteins is a first step towards utilizing them as targets for viral vectors. The urokinase plasminogen activator receptor (uPAR) is apically expressed by human airway epithelia. This proposal will further study uPAR's utility as a gene transfer-targeting molecule. Its expression in different epithelial cell types, association with potential co-receptors, ability to endocytose and mechanism of endocytosis will be examined. Additionally, a seven amino acid uPAR binding peptide (u7p) will be engineered into outer capsid proteins of two gene transfer vectors, adenovirus (Ad) and adeno-associated virus serotype 2 (AAV2). U7p-modified viral proteins will be analyzed for their ability to bind uPAR. Viral particles will then be tested on uPAR expressing cells. Moreover, another potential receptor system, erbB2 and 3, will be examined as potential targeting proteins. Both receptors are expressed by human airway epithelia. Their expression pattern, cell type specificity and ability to internalize will be analyzed for potential as targeting molecules for gene transfer vectors. Identifying other apically expressed proteins from human airway epithelia will generate more potential targeting molecules. A surface biotinylation approach will be used for their isolation. Proteins will be separated and identified by MALDI-MS. Of particular interest are those apical proteins that internalize because they allow for entry into the cell. The major goals of this proposal are to generate candidate-targeting molecules with the hope of increasing efficiency of gene transfer vectors to the airways.

描述（由申请人提供） 基因疗法通过校正提供了治愈囊性纤维化（CF）的希望 囊性纤维化跨膜电导调节剂（CFTR）的突变。 尽管还没有现实，但该提案的目标旨在产生 成功靶向人气道上皮细胞的病毒载体。 目前，病毒载体在感染应得的气道上皮时效率低下， 在很大程度上，在顶部表面没有病毒受体。一 规避此限制的方法是将病毒向量靶向 可以从顶部表面进入的受体。识别和 表征这些蛋白是利用它们作为目标的第一步 对于病毒载体。尿激酶纤溶酶原激活剂受体（UPAR）为 由人类气道上皮的顶端表达。该建议将进一步研究 UPAR作为基因转移靶向分子的效用。它的表达 不同的上皮细胞类型，与潜在的共受体相关， 将检查内吞作用和内吞作用机理的能力。 此外，将设计一个七个氨基酸UPAR结合肽（U7P） 进入两个基因转移载体腺病毒（AD）和 腺相关病毒血清型2（AAV2）。 U7P修饰的病毒蛋白将是 分析了它们绑定UPAR的能力。然后将在病毒颗粒上测试 UPAR表达细胞。此外，另一个潜在的受体系统ERBB2和 3将被检查为潜在靶向蛋白。两个受体都是 由人类气道上皮表达。它们的表达模式，细胞类型 特异性和内在化能力将被分析为潜力 靶向基因转移载体的分子。顶端识别其他 来自人类气道上皮的蛋白质将产生更多的潜力 靶向分子。表面生物素化方法将用于其 隔离。蛋白质将通过MALDI-MS分离和鉴定。特别 兴趣是那些内在化的根尖蛋白，因为它们允许 进入单元格。该提案的主要目标是产生 候选靶向分子，希望提高基因效率 将向量转移到气道。