Early addition of nocodazole enhances rAAV8 production in HEK293
cells
A recent study showed that cap gene expression, translation and
assembly of Cap proteins into empty AAV particles is a kinetically rapid
process[31]. ~80% of capsids are
assembled within the first 24 HPT whereas Rep mediated replication of
rAAV genome from the cis -ITR plasmid is slower, peaking after
capsid protein levels have plateaued due to the inhibitory action of Rep
binding to the packaging plasmid and inhibiting transcription or by
translational repression of cap mRNA. As capsid secretion from
the nucleus into the cytoplasm is independent of genome loading,
pre-assembled empty capsids can be secreted from the site of rAAV genome
loading, thus depleting the pool of empty capsids available to newly
replicated rAAV genome [31],[32]. Therefore,
we hypothesized that the effect of a given chemical effector may be
positive (or further enhanced), neutral, or negative with respect to the
timing of its deployment,
To further investigate the impact of nocodazole in enhancing rAAV
production, we deployed high dose nocodazole (4 µM) between 0 – 48 HPT
(Figure 2). Addition of 4 µM nocodazole immediately (0 HPT) and 4 HPT
resulted in a considerable reduction in VCD at 72 HPT of 74% and 64%
respectively, compared to rAAV8 producing cells without nocodazole
(Ctrl) (Fig. 2A). Also apparent at early addition timepoints was a
slight reduction in cell viability (p > 0.05; Fig. 2B) and
a significantly higher mean cell volume compared to cultures not treated
with nocodazole (up to 23%; Fig. 2C), consistent with previous work by
Tait et al., (2004) who observed increased cell size and decreased
viability in nocodazole treated CHO cells producing a recombinant
monoclonal antibody. Most strikingly, addition of nocodazole at 4 HPT
resulted in a 2.2-fold increase in rAAV8 genome titre at harvest
compared to control cells (Fig. 2D). Addition at the earlier timepoint
of 0 HPT was sub-optimal, with a 10% reduction in rAAV8 titer compared
to the titer of cells treated at 4 HPT (Figure 2D). We therefore
conclude that early addition (~4 HPT) of nocodazole to
rAAV producing cells is an effective positive mediator of rAAV
production in a small-scale, transient expression, suspension HEK cell
system.