Adeno-associated virus (AAV) vectors expressing the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA complement the cystic fibrosis (CF) defect in vitro. Unlike other DNA virus vectors, AAV is a stably integrating virus, which could make possible long-term in vivo complementation of the CF defect in the airway epithelium. We report AAV-CFTR gene transfer and expression after infection of primary CF nasal polyp cells and after in vivo delivery of AAV-CFTR vector to one lobe of the rabbit lung through a fiberoptic bronchoscope. In the rabbit, vector DNA could be detected in the infected lobe up to 6 months after administration. A 26-amino acid polypeptide sequence unique to the recombinant AAV-CFTR protein was used to generate both oligonucleotide probes and a polyclonal antibody which allowed the unambiguous identification of vector RNA and CFTR protein expression. With these reagents, CFTR RNA and protein were detected in the airway epithelium of the infected lobe for up to 6 months after vector administration. AAV vectors do, therefore, efficiently promote in vivo gene transfer to the airway epithelium which is stable over several months. These findings indicate that AAV-CFTR vectors could potentially be very useful for gene therapy.

Cystic fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability, increased mucous sulphation, increased Na+ absorption and defective acidification of lysosomal vesicles. The CF gene encodes a protein (the cystic fibrosis transmembrane conductance regulator, CFTR) that can function as a low-conductance Cl- channel with a linear current-voltage relationship whose regulation is defective in CF patients. Larger conductance, outwardly rectifying Cl- channels are also defective in CF and fail to activate when exposed either to cyclic AMP-dependent protein kinase A or to protein kinase C. The role of the outwardly rectifying Cl- channel in CF has been questioned. We report here that expression of recombinant CF genes using adeno-associated virus vectors in CF bronchial epithelial cells corrects defective Cl- secretion, that it induces the appearance of small, linear conductance Cl- channels, and restores protein kinase A activation of outwardly rectifying Cl- channels. These results re-establish an involvement of outwardly rectifying Cl- channels in CF and suggest that CFTR regulates more than one conductance pathway in airway tissues.

Adeno-associated virus type 2 (AAV2)-based vectors are capable of stable expression in the airway epithelium and may be useful for gene therapy for human diseases, such as cystic fibrosis. Certain virus vectors, such as retroviruses, require active cell division for integration and expression, but this has not been formally evaluated in the case of AAV2. The cystic fibrosis bronchial epithelial cell line, IB3-1, which can be transduced by AAV2 vectors, was shown to undergo a decrease in DNA synthesis to undetectable levels when grown to confluence. Cultures in which < 0.1% of cells were dividing could still be efficiently transduced with AAV-lacZ or AAV-neo vectors, with a linear dose response, up to 91% with a multiplicity of 3,000 vector particles per cell. The fate of vector DNA in nondividing target cells was investigated by Southern blotting of both low molecular weight, nonintegrated DNA and high molecular weight, genomic DNA fractions. Detectable levels of vector DNA were only seen in the nonintegrated state. These results indicate that AAV2-based vectors, unlike retrovirus vectors, do not require active cell division or integration for expression to occur and thus possess a unique profile of biologic properties.