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  • Title: Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors.
    Author: Farson D, Harding TC, Tao L, Liu J, Powell S, Vimal V, Yendluri S, Koprivnikar K, Ho K, Twitty C, Husak P, Lin A, Snyder RO, Donahue BA.
    Journal: J Gene Med; 2004 Dec; 6(12):1369-81. PubMed ID: 15538729.
    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.
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