Characterization of permanent cell lines that contain the AAV2 rep-cap genes on an Epstein-Barr-virus-based episomal...

Recombinant adeno-associated virus (rAAV) has emerged as a promising gene therapy vector. Its development, however, has been hampered by the lack of a readily available efficient production method. We investigated the possibility of establishing permanent cell lines for the production of rAAV with a new Epstein-Barr-virus (EBV)-based episomal AAV rep-cap plasmid (pCEP-rep/cap). HeLa and 293 cells were stably transfected with plasmids that carry the AAV2 rep/cap genes under transcriptional control of their endogenous promoters (p5, p19 and p40) either on the pCEP-rep/cap or an integrated (pIM45) plasmid. For the ease of monitoring transgene expression in live cells, a rAAV vector expressing gfp (the green fluorescent protein gene, rAAV-gfp/neo) was used. Establishment of stable transfected cell lines with these plasmids proved feasible but their usefulness was limited because of their instability. Within 8-12 weeks after their establishment, stably transfected rep-cap cell lines invariably lost their function. In addition, the rAAV-gfp/neo vector we used was susceptible to mutation in stably transfected HeLa cells. Our observations demonstrate specific problems both at the level of rep/cap gene function and the rAAV genome that can occur with the establishment of rAAV production cell lines. These experiments should aid the further development of efficient rAAV production protocols.