Small molecule additives can be combined to further enhance
rAAV8 production
In order to evaluate whether specific combinations of effectors could
act synergistically to further increase rAAV production, we utilized
nocodazole in combination with either z-VAD-fmk or M344 (see Figure 1).
M344 is a synthetic analogue of the anti-fungal drug Trichostatin A, an
inhibitor of Class I and IIB histone deacetylases, and recently used as
an enhancer of recombinant protein expression in mammalian cell
culture[10]. Z-VAD-fmk is a pan-caspase inhibitor[20]. Each chemical was added to rAAV8 producing
HEK293 cultures together with nocodazole at 4 HPT in 24-well
microplates. The inclusion of z-VAD-fmk in the initial screening
experiment was predicated on its anti-caspase activity, as we
hypothesised caspase mediated apoptosis - linked to rAAV production and
expression of AAV2 Rep proteins - would negatively affect final crude
titre[49],[50]. Additionally, caspase-mediated
apoptosis induced by nocodazole-mediated cell cycle dysregulation would
further increase cell death within the production cultures. Increased
cell viability and VCD in cultures treated with nocodazole/z-VAD-fmk
relative to untreated, nocodazole treated or nocodazole/M344 treated
cells suggests that apoptosis was reduced (Fig. 5A & B) but with an
unexpected reduction in crude genome titre (Fig. 5D). The observed
reduction may be a consequence of off-target induction of autophagy via
inactivation of n -glycanase 1 (NGLY1) that has been shown to
occur in HEK293 cells treated with z-VAD-fmk[51].
While the role of autophagy in rAAV production is undetermined,
inhibition of autophagy has been shown to increase recombinant protein
expression in CHO cells[52],[53] and
unintentional upregulation of autophagy may result in a reduction in
viral component proteins necessary for production of high viral titres.
Interestingly, the highest mean cell volume, which appeared to correlate
positively with genome titre, was measured in cells treated with both
nocodazole and z-VAD-fmk (Fig. 5C). This may be a result of reduced
apoptotic cell death allowing cell size to increase but with the caveat
that any benefit gained from this from a production perspective is
attenuated by the potential negative off-target effects of z-VAD-fmk.
We observed that the combination of 2.5 µM M344 and 4 µM nocodazole
produced an additive effect, increasing crude rAAV genome titre 2.6-fold
compared to untreated cultures, an improvement on nocodazole alone
(2-fold increase compared to untreated) (Fig 5D). This additive effect
was also observed in 30 mL shake flask cultures, whereby measurements of
VCD and viability closely replicated those observed in the screening
assay (Fig. 6A), together with an improvement in genome titre with
nocodazole/M344 from a 2.6-fold increase in the screening assay to a
3-fold increase compared to untreated cells in larger volume cultures
(Fig. 6B). AAV8 intact capsid-specific ELISA analysis of total capsid
titre showed a significant increase in intact capsids after addition of
nocodazole (4.3-fold increase) and nocodazole/M344 (11.3-fold increase)
compared to untreated controls (data not shown). We anticipate that rAAV
vector development could complement the process engineering strategy to
further maximize rAAV titer (e.g. high intact capsid levels are likely
to benefit from hybrid Rep with improved genome packaging
efficiency)[54] while combinatorial empirical
modeling will enable systematic determination of optimal chemical dosage
and timing.
Taken together, these data show that nocodazole, either alone or in
combination with select small molecules, can reproducibly boost both
genome and total viral particle titre in a transient suspension HEK293
rAAV production system and that it is both scalable and applicable to
production of rAAVs derived from two phylogenetically distinct and
clinically translatable pseudotyped capsids.