Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene

We have investigated a possible delivery system for the rat preproinsulin II gene (rl(2)) utilizing a recombinant adeno-associated virus (rAAV) vector system, with the long-term goal of engineering stably infected insulin-producing cell lines. The rAAV vector was chosen because it is a safe and nonpathogenic method for gene transfer. The plasmid pBC1SBI (ATCC) was purified and digested with restriction enzymes SspI and StuI to release a fragment containing the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter-driven rat preproinsulin II gene (rl(2)). Subsequently, the RSV-rI(2) gene fragment was cloned into the BamHI site of rAAV vector plasmid pWP-19 to produce the rI(2) recombinant plasmid designated pLP-1. The pWP-19 also encodes the AAV inverted terminal repeats for integration and replication and the herpes virus thymidine 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 genome, now containing rI(2), was rescued using adenovirus and packaged into mature AAV virions termed vLP-1. Finally, human pancreatic adenocarcinoma cells (HPAC; ATCC) were exposed to vLP-1, selected for G418 resistance, and screened for insulin production. Successful rescue was confirmed 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 rI(2) gene by PCR, Insulin gene transcription was confirmed by RT-PCR. We have developed a rAAV-mediated gene transfer system for the rat preproinsulin 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 approach to experimental gene therapy. (C) 1997 Academic Press.