Successful gene therapy for cystic fibrosis (CF) lung disease requires efficient in vivo gene transfer to the airway epithelium. The target tissue for gene transfer approaches for the treatment of the pulmonary manifestations of CF is the well- differentiated (WD) ciliated airway epithelium. Among the most promising vectors for CF gene therapy are the adenoviral vectors (AdV). However, AdV are inefficient gene transfer vectors for the respiratory epithelium in vivo and efforts are being directed at increasing the efficiency of AdV-mediated gene transfer. We have speculated that the gene transfer discrepancy is due to phenotypic differences for WD compared to PD airway cells and analyses of the early interactions of AdV with airway epithelial cells shows that decreased efficiency of gene transfer is due to a limited entry of AdV across the apical membrane of WD cells reflecting as many as three independent rate-limiting steps at the apical surface: (1) reduced specific AdV-attachment; (2) the absence of alphavbeta3/5 integrins; and (3) a reduced rate of AdV-internalisation. In an effort to increase gene transfer efficiency to airway epithelial this proposal will re-target AdV to 'novel' receptor-types, present on the apical surface of airway epithelium in vivo, which on activation by agonist internalise into clathrin-coated vesicles. This proposal will focus on the B2-kinin receptor (B2k) as a candidate target receptor since in addition to being present in the airway, this receptor utilises the nonamer peptide, bradykinin (BK), as an agonist. It is proposed that after validation of the B2-kinin receptor as a utile target, AdV will be engineered to express BK on the virus capsid coat in order to test this targeting strategy.
The specific aims of this proposal are: 1. To validate B2k as an effective target on the lumenal surface of airway epithelium that is capable of mediating efficient AdV-mediated gene transfer with bi-specific antibody linkers; and, 2. To generate genetically modified AdV encompassing BK, so redirecting AdV tropism to B2k.
