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 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.
Neyns
B
B
Laboratory of Medical Oncology, Oncologisch Centrum, Akademisch Ziekenhuis, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
Vermeij
J
J
Teugels
E
E
De Rijcke
M
M
Hermonat
P
P
De Grève
J
J
D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D005814:Genes, Viral; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins