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Targeting the Apical Surface of Human Airway Epithelia

靶向人类气道上皮细胞的顶端表面

基本信息

批准号：
6635396
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
项目类别：
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/6635396
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

项目摘要

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）基因治疗通过纠正囊性纤维化（CF）的发生，囊性纤维化跨膜传导调节因子（CFTR）突变。虽然尚未成为现实，但该提案的目标是成功靶向人气道上皮细胞的病毒载体。目前，病毒载体在感染气道上皮细胞方面是低效的，在很大程度上是由于顶端表面没有病毒受体。一规避这一限制的方法是将病毒载体靶向于受体，可从顶端表面。识别和鉴定这些蛋白质是利用它们作为靶点的第一步 for viral病毒vectors载体.尿激酶纤溶酶原激活物受体（uPAR）是由人气道上皮细胞顶端表达。该建议将进一步研究 uPAR作为基因转移靶向分子的效用。原核表达不同的上皮细胞类型，与潜在的共受体的关联，检查内吞能力和内吞机制。此外，将工程化七个氨基酸的uPAR结合肽（u 7 p），两种基因转移载体腺病毒（Ad）和腺相关病毒血清型2（AAV 2）。U 7 p修饰的病毒蛋白将被分析它们结合uPAR的能力。病毒颗粒将在 uPAR表达细胞。此外，另一个潜在的受体系统，erbB 2和erbB 3， 3，将作为潜在的靶向蛋白进行研究。两种受体都由人气道上皮细胞表达。它们的表达模式、细胞类型特异性和内化能力将被分析为潜在的，用于基因转移载体的靶向分子。识别其他顶点从人气道上皮细胞中表达蛋白，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。感兴趣的是那些顶端蛋白质，它们内化，因为它们允许进入细胞。该提案的主要目标是候选人靶向分子，希望提高基因治疗的效率把病毒传播到呼吸道