RECOMBINANT ADENOASSOCIATED VIRUS (AAV-CFTR) VECTORS DO NOT INTEGRATEIN 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 fo r cystic fibrosis (CF) at Johns Hopkins Hospital. The goal of this stu dy was to determine the fate of recombinant AAV containing the CFTR cD NA (AAV-CFTR) in an immortalized pseudotetraploid CF bronchial epithel ial 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 perfor med, CFRH2, an IB3-1 cell line with an estimated 15-20 integrated copi es of CFTR cDNA, was used to test FISH sensitivity. All metaphase spre ads had integrated copies; a single site in 36 of 56 (64.3%) and two s ites 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 a lso analyzed by FISH. Integration was observed in 56 of 157 (35.7%) me taphase spreads examined. IB3-1 cells infected with wild-type AAV show ed integration in 51 of 86 (59%) metaphase spreads examined. Of 51 int egrations, 48 (94%) were to chromosome 19. Examination of 67 metaphase chromosome spreads of IB3-1 cells infected with AAV-CFTR vector (A0) identified four integrations (6%) to different chromosomes. No integra tion was to chromosome 19 which differs significantly (P < 0.0001) fro m wild-type AAV. We then analyzed the A35 cell line a clone of A0 sele cted for stable CFTR expression. Genomic DNA from A35 cells did not sh ow a single site of integration however episomal AAV-CFTR sequences we re abundant in the low molecular weight DNA fraction Examination of 68 metaphase chromosome preparations identified eight distinct integrati ons, none to chromosome 19. These studies show that FISH is sensitive for the detection of a partial CFTR cDNA integration. Wild-type AAV in tegrates in a predominantly site-specific fashion. Recombinant AAV-CFT R integrates at low frequency in a nonspecific manner and persists in episomal form in this epithelial cell line.