Improvement in the suspension-culture production of recombinant adeno-associated virus–LacZ in HEK-293 cells using polyethyleneimine–DNA complexes in combination with hypothermic treatment

rAAV (recombinant adeno-associated virus) has become a very useful gene-delivery vector for gene therapy. However, it is very difficult to generate rAAV using triple transfection on a commercial scale, owing to its low transfection efficiency. An optimal procedure for transfection in suspension-culture mode was developed for rAAV-LacZ production in suspension-cultured HEK-293 (human embryonic kidney-293) cells mediated by PEI (polyethyleneimine)-DNA complexes in combination with transient severe hypothermia at 4 degrees C for 1 h in the present study (LacZ is the product of the reporter gene lacZ, which codes for beta-D-galactosidase). It showed that the PEI/DNA ratio, cell density at the beginning of transfection and cell-cycle arrest in G2/M-phase were key factors affecting suspension-culture triple-transfection efficiency and rAAV-LacZ productivity. After incubation at 4 degrees C for 1 h and re-warming at 37 degrees C for 18 h, HEK-293 cells at 1x10(6) cells/ml were transfected with PEI-DNA complexes at a PEI/DNA ratio of 5:1 (w/w) with final concentrations of 30 mug/ml 25 kDa linear PEI and 6 mug/ml plasmid DNA in culture. After 6 h incubation for transfection, an equal volume of medium was added to the culture for additional 48 h growth until harvest. Finally, the high transfection efficiency of some 75% and rAAV-LacZ titre of (7.48+/-0.59)x10(11) physical particles or 1.86+/-0.96x10(10) infectious particles were achieved in 250 ml shake flasks with 60 ml working volume, indicating a promising application for scale-up.