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 > 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.
Farson
Deborah
D
Cell Genesys, Inc., 500 Forbes Blvd., South San Francisco, CA 94080, USA. Debbie.Farson@cellgenesys.com
Harding
Thomas C
TC
Tao
Lucy
L
Liu
Jun
J
Powell
Sandra
S
Vimal
Vishalini
V
Yendluri
Satyasri
S
Koprivnikar
Kathryn
K
Ho
Kenneth
K
Twitty
Christopher
C
Husak
Paul
P
Lin
Andy
A
Snyder
Richard O
RO
Donahue
Brian A
BA
D000256:Adenoviridae / Q000254:growth & development*; D019149:Bioreactors; D002460:Cell Line; D002986:Clinical Trials as Topic; D016895:Culture Media, Serum-Free; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D013048:Specimen Handling
D016895:Culture Media, Serum-Free