title: Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors.
abstract: 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.
authors.affiliation: Cell Genesys, Inc., 500 Forbes Blvd., South San Francisco, CA 94080, USA. Debbie.Farson@cellgenesys.com
authors.forename: Deborah
authors.initials: D
authors.lastname: Farson
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Harding
authors.forename: Lucy
authors.initials: L
authors.lastname: Tao
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Sandra
authors.initials: S
authors.lastname: Powell
authors.forename: Vishalini
authors.initials: V
authors.lastname: Vimal
authors.forename: Satyasri
authors.initials: S
authors.lastname: Yendluri
authors.forename: Kathryn
authors.initials: K
authors.lastname: Koprivnikar
authors.forename: Kenneth
authors.initials: K
authors.lastname: Ho
authors.forename: Christopher
authors.initials: C
authors.lastname: Twitty
authors.forename: Paul
authors.initials: P
authors.lastname: Husak
authors.forename: Andy
authors.initials: A
authors.lastname: Lin
authors.forename: Richard O
authors.initials: RO
authors.lastname: Snyder
authors.forename: Brian A
authors.initials: BA
authors.lastname: Donahue
mesh_terms: 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
chemical_list: D016895:Culture Media, Serum-Free