Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors

Background One of the major limitations to the use of adeno-associated virus (AAV) vectors for gene therapy has been the difficulty in producing enough vector to supply a clinical trial. More than 20000 roller bottles may be required to generate AAV by the traditional transient transfection process to treat 50 patients. A scalable kAV producer cell line grown in serum-free media will meet the needs for the manufacture of AAV gene therapeutics. Methods A packaging cell line was generated by introducing the AAV rep and cap genes into A549 cells. From this packaging cell line. a number of producer cell lines were generated by infecting the packaging cell with the appropriate AAV vector. Producer cell lines were then adapted to serum-free suspension conditions for growth in bioreactors. Results We report here the development of six AAV producer cell lines that generate >10(4) particles/cell. The rAAV vector preparations from these cell lines have physical and functional characteristics similar to rAAV vectors prepared by transient transfection. To enable large-scale production, producer cell lines were adapted to serum-free suspension and we demonstrate production of AAV at the 15 L scale. In addition. vector preparations from these cell lines were shown to be free of wild-type AAV. Conclusions AAV producer cell lines can be readily scaled to meet the needs of clinical trials. One 500 L bioreactor of these producer cells can produce the equivalent of 2500 high capacity roller bottles or 25 000 T-175 tissue Culture flasks. Copyright (C) 2004 John Wiley Sons, Ltd.