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 20 000 roller bottles may be required to generate AAV by the traditional transient transfection process to treat 50 patients. A scalable AAV 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 > 104 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 © 2004 John Wiley & Sons, Ltd.