GENE-TRANSFER TO FRESHLY ISOLATED HUMAN RESPIRATORY EPITHELIAL-CELLS IN-VITRO USING A REPLICATION-DEFICIENT ADENOVIRUS CONTAINING THE HUMANCYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CDNA

Cystic fibrosis (CF) results from mutations of the CF transmembrane co nductance regulator (CFTR) gene and subsequent defective regulation of cAMP-stimulated chloride (CI-) permeability across the apical membran e of epithelial cells. In vitro transfer of normal CFTR cDNA corrects this defect, and studies in experimental animals have shown successful gene transfer to airway epithelium in vivo using a recombinant adenov iral vector containing the human CFTR cDNA (AdCFTR), supporting the fe asibility of in vivo AdCFTR-mediated gene therapy for the respiratory manifestations of CF. One step in applying this therapy to CF patients is to evaluate the safety and efficacy of AdCFTR-mediated gene transf er in the actual target for human gene therapy, human airway epitheliu m. The present study demonstrates that AdCFTR restores cAMP-stimulated Cl- permeability in human CF bronchial epithelial cells. In addition, the study utilizes freshly isolated human airway epithelial cells fro m the nose and/or bronchi of normal individuals and/or individuals wit h CF to demonstrate that after in vitro AdCFTR-mediated gene transfer: (i) AdCFTR DNA does not replicate as a function of dose and time; (ii ) CF epithelial cells express AdCFTR-mediated normal human CFTR mRNA; and (iii) CF epithelial cells, including terminally differentiated cil iated cells (the most common airway epithelial cell type), express the normal human CFTR protein. Together, these data support the use of Ad CFTR in human gene therapy trials and suggest that biologic efficacy s hould be achievable in vivo.