Molecular Therapy: Methods & Clinical Development (Jan 2016)

Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

  • Bryan A Piras,
  • Jason E Drury,
  • Christopher L Morton,
  • Yunyu Spence,
  • Timothy D Lockey,
  • Amit C Nathwani,
  • Andrew M Davidoff,
  • Michael M Meagher

DOI
https://doi.org/10.1038/mtm.2016.15
Journal volume & issue
Vol. 3, no. C

Abstract

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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.