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.