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.