• Authors: Piras, B. A., Drury, J. E., Morton, C. L., Spence, Y., Lockey, T. D., Nathwani, A. C., Davidoff, A. M., Meagher, M. M.
  • Year: 2016
  • Journal: Mol Ther Methods Clin Dev 3 16015
  • Applications: in vitro / DNA / PEIpro
  • Cell type: HEK-293T/17
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T/17, 293T/17


AAV was produced by two plasmid transfection using PEIpro 1 day after seeding cells at a density of 7.26 × 10^4 cells/cm². Cell cultures were maintained from between 3 and 7 days post-transfection. For cultures lasting longer than 3 days, additional media (50% of the original transfection volume) was added at day 3 to provide cells with additional nutrients.


With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.