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 (rI₂) 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 pBC12BI (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 (rI₂). Subsequently, the RSV-rI₂ gene fragment was cloned into the BamHI site of rAAV vector plasmid pWP-19 to produce the rI₂ 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 pAAV/AD, which is required for viral replication. The rAAV genome, now containing rI₂, 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 rI₂ gene probe derived from the original plasmid. The final titer of 1.25 x 10⁹ particles/ml was determined by DNA slot blots using pLP-1 as the standard. HPAC cells were infected with vLP-1 (termed HPAC/rI₂). Integration of the rI₂ genome in G418-resistant clones was confirmed by Southern blot analysis and again after 6 months in culture by amplification of the rI₂ 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 rI₂ into HPAC was achieved. Production of insulin by HPAC/rI₂ was confirmed by RIA and RT-PCR, validating this system as an effective approach to experimental gene therapy.