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) 基因治疗为囊性纤维化(CF)的治愈带来了希望 囊性纤维化跨膜电导调节因子(CFTR)突变。 虽然还没有成为现实，但这项提案的目标是 成功靶向人类呼吸道上皮细胞的病毒载体。 目前，病毒载体在感染呼吸道上皮细胞方面效率低下， 在很大程度上，是由于根尖表面没有病毒受体。一 绕过这一限制的方法是将病毒载体定向到 从根尖表面可以接触到的受体。识别和 鉴定这些蛋白质是利用它们作为靶标的第一步。 用于病毒载体。尿激酶型纤溶酶原激活剂受体(UPAR) 顶端由人呼吸道上皮细胞表达。这项建议将进一步研究 UPAR作为基因转移靶向分子的效用。其在以下方面的表达 不同的上皮细胞类型，与潜在的辅助受体相关， 将检查吞噬能力和吞噬机制。 此外，还将设计一个七个氨基酸的uPAR结合肽(U7p)。 转入两种基因转移载体的外衣壳蛋白，腺病毒(Ad)和 腺相关病毒2型(AAV2)。U7p修饰的病毒蛋白将是 分析它们结合aR的能力。然后，病毒颗粒将在 UPAR表达细胞。此外，另一个潜在的受体系统，erbB2和 3，将被视为潜在的靶向蛋白。两种受体都是 由人的呼吸道上皮细胞表达。它们的表达模式、细胞类型 专一性和内部化能力将被分析为潜在的 靶向基因转移载体的分子。顶端识别其他 人呼吸道上皮细胞表达的蛋白质将产生更多潜力 以分子为目标。将使用表面生物素化方法来处理它们 与世隔绝。蛋白质将通过MALDI-MS进行分离和鉴定。特别的 令人感兴趣的是那些内化的顶端蛋白，因为它们允许 进入牢房。这项提议的主要目标是产生 候选靶向分子，希望提高基因的效率 把载体转移到呼吸道。