CONSTRUCTION OF RECOMBINANT ADENOASSOCIATED VIRUS VECTOR CONTAINING THE RAT PREPROINSULIN-II GENE

We have investigated a possible delivery system for the rat preproinsu lin II gene (rl(2)) utilizing a recombinant adeno-associated virus (rA AV) vector system, with the long-term goal of engineering stably infec ted insulin-producing cell lines. The rAAV vector was chosen because i t is a safe and nonpathogenic method for gene transfer. The plasmid pB C1SBI (ATCC) was purified and digested with restriction enzymes SspI a nd StuI to release a fragment containing the Rous sarcoma virus long t erminal repeat (RSV-LTR) promoter-driven rat preproinsulin II gene (rl (2)). Subsequently, the RSV-rI(2) gene fragment was cloned into the Ba mHI site of rAAV vector plasmid pWP-19 to produce the rI(2) recombinan t plasmid designated pLP-1. The pWP-19 also encodes the AAV inverted t erminal repeats for integration and replication and the herpes virus t hymidine kinase promoter-driven gene for neomycin resistance (neo(R)). The cell line 293 (ATCC) was then cotransfected with pLP-1 and helper plasmid pAAVW), which is required for viral replication. The rAAV gen ome, now containing rI(2), was rescued using adenovirus and packaged i nto mature AAV virions termed vLP-1. Finally, human pancreatic adenoca rcinoma cells (HPAC; ATCC) were exposed to vLP-1, selected for G418 re sistance, and screened for insulin production. Successful rescue was c onfirmed by Southern blot analysis using the rl(2) gene probe derived from the original plasmid, The final titer of 1.25 x 10(9) particles! mi was determined by DNA slot blots using pLP-1 as the standard. HPAC cells were infected with vLP-1 (termed HPAC/rI(2)). Integration of the rI(2) genome in G418-resistant clones was confirmed by Southern blot analysis and again after 6 months in culture by amplification of the r I(2) gene by PCR, Insulin gene transcription was confirmed by RT-PCR. We have developed a rAAV-mediated gene transfer system for the rat pre proinsulin II gene. Successful transduction and stable integration of rI(2) into HPAC was achieved. Production of insulin by HPAC/rI(2) was confirmed by RIA and RT-PCR, validating this system as an effective ap proach to experimental gene therapy. (C) 1997 Academic Press.