Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line.

Adeno-associated virus-2 (AAV) can integrate in a site-specific manner to human chromosome 19 and is currently in phase I clinical trials for cystic fibrosis (CF) at Johns Hopkins Hospital. The goal of this study was to determine the fate of recombinant AAV containing the CFTR cDNA (AAV-CFTR) in an immortalized pseudotetraploid CF bronchial epithelial cell line (IB3-1) established from a patient with CF. Fluorescence in situ hybridization (FISH) and Southern blotting of DNA from IB3-1 cells infected with wild-type (wt) or recombinant AAV-CFTR were performed. CFRH2, an IB3-1 cell line with an estimated 15-20 integrated copies of CFTR cDNA, was used to test FISH sensitivity. All metaphase spreads had integrated copies: a single site in 36 of 56 (64.3%) and two sites within the same metaphase spread in 20 of 56 (35.7%). 3-CF-8, an IB3-1 cell line with integration of a partial CFTR cDNA (3.9 kb) was also analyzed by FISH. Integration was observed in 56 of 157 (35.7%) metaphase spreads examined. IB3-1 cells infected with wild-type AAV showed integration in 51 of 86 (59%) metaphase spreads examined. Of 51 integrations, 48 (94%) were to chromosome 19. Examination of 67 metaphase chromosome spreads of IB3-1 cells infected with AAV-CFTR vector (Azero) identified four integrations (6%) to different chromosomes. No integration was to chromosome 19 which differs significantly (P < 0.0001) from wild-type AAV. We then analyzed the A35 cell line, a clone of Azero selected for stable CFTR expression. Genomic DNA from A35 cells did not show a single site of integration; however episomal AAV-CFTR sequences were abundant in the low molecular weight DNA fraction. Examination of 68 metaphase chromosome preparations identified eight distinct integrations, none to chromosome 19. These studies show that FISH is sensitive for the detection of a partial CFTR cDNA integration. Wild-type AAV integrates in a predominantly site-specific fashion. Recombinant AAV-CFTR integrates at low frequency in a nonspecific manner and persists in episomal form in this epithelial cell line.

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.

CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR-transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl- secretory pathways in addition to itself conducting Cl-.

Alternate translation initiation codons can create functional forms of cystic fibrosis transmembrane conductance regulator.

To evaluate the function of transmembrane domain 1 (TMD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and the methionines that function in translation initiation, a series of progressive 5' truncations in TMD1 were created to coincide with residues that might serve as translation initiation codons. Expression of the mutants in Xenopus oocytes demonstrated that internal sites in TMD1 can function as initiation codons. In addition, all of the mutants that progressively removed the first four transmembrane segments (M1-M4) of TMD1 expressed functional cAMP-regulated Cl- channels with ion selectivity identical to wild-type CFTR but with reduced open probability and single channel conductance. Further removal of transmembrane segments did not produce functional Cl- channels. These data suggest that segments M1-M4 are not essential components of the conduction pore or the selectivity filter of CFTR.

Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine.

The ilv-leu operon of Bacillus subtilis is regulated in part by transcription attenuation. The cis-acting elements required for regulation by leucine lie within a 683-bp fragment of DNA from the region upstream of ilvB, the first gene of the operon. This fragment contains the ilv-leu promoter and 482 bp of the ilv-leu leader region. Spontaneous mutations that lead to increased expression of the operon were shown to lie in an imperfect inverted repeat encoding the terminator stem within the leader region. Mutations within the inverted repeat of the terminator destroyed most of the leucine-mediated repression. The remaining leucine-mediated repression probably resulted from a decrease in transcription initiation. A systematic analysis of other deletions within the ilv-leu leader region identified a 40-bp region required for the derepression that occurred during leucine limitation. This region lies within a potential RNA stem-and-loop structure that is probably required for leucine-dependent control. Deletion analysis also suggested that alternate secondary structures proximal to the terminator are involved in allowing transcription to proceed beyond the terminator. Additional experiments suggested that attenuation of the ilv-leu operon is not dependent on coupling translation to transcription of the leader region. Our data support a model proposed by Grundy and Henkin (F. J. Grundy and T. M. Henkin, Cell 74:475-482, 1993) in which uncharged tRNA acts as a positive regulatory factor to increase gene expression during amino acid limitation.