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.