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

Recombinant adeno-associated virus (rAAV) has emerged as a promising gene t herapy vector. Its development, however, has been hampered by the lack of a readily available efficient production method. We investigated the possibi lity of establishing permanent cell lines for the production of rAAV with a new Epstein-Barr-virus (EBV)-based episomal AAV rep-cap plasmid (pCEF-rep/ cap). HeLa and 293 cells were stably transfected with plasmids that carry t he AAV2 rep/cap genes under transcriptional control of their endogenous pro moters (p5, p19 and p40) either on the pCEP-rep/cap or an integrated (plM45 ) plasmid. For the ease of monitoring transgene expression in live cells, a rAAV vector expressing gfp (the green fluorescent protein gene, rAAV-gfp/n eo) was used. Establishment of stable transfected cell lines with these pla smids proved feasible but their usefulness was limited because of their ins tability. Within 8-12 weeks after their establishment, stably transfected r ep-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 establish ment of rAAV production cell lines. These experiments should aid the furthe r development of efficient rAAV production protocols. Copyright (C) 2001 S. Karger AG, Basel.