High-cell-density production of adeno-associated viral vector serotype 6
by triple transfection in suspension HEK293 cell cultures
Abstract
The use of adeno-associated viruses (AAV) as vectors for gene and cell
therapy has risen considerably in recent years. Consequently, the amount
of AAV vectors required during the validation and clinical trials has
also increased. AAV serotype 6 (AAV6) is well-documented for its
efficiency in transducing different cell types and has been successfully
used in gene and cell therapy protocols. However, the number of vectors
required to effectively deliver the transgene to one single cell has
been estimated at 106 viral genomes (VG). Overall, this means that
large-scale production of AAV6 is needed. Suspension cell-based
platforms are currently limited to low-cell-density productions,
hindering the potential of this production process to increase yields.
Here, we investigate the improvement of the production of AAV6 at higher
cell densities. The production was performed by transient transfection
of HEK293SF cells. When the plasmid DNA is provided on a cell basis, the
production can be carried out at medium cell density without effects on
cell-specific titer or particle functionality, resulting in titers above
1010 VG/mL. Medium supplementation alleviated the cell density effect,
in terms of VG/cell, at high-cell-density productions. On the other
hand, the cell-specific functional titer was not maintained, and further
studies are necessary to understand the observed limitations. The
medium-cell-density production method reported here lays the foundation
for large-scale process operations, potentially solving the current
vector shortage in AAV manufacturing.