Initial screening of chemical additives to increase AAV genome titre
To identify novel small molecule enhancers of rAAV production, a panel of 15 small molecule culture additives was chosen based on either their reported propensity to increase recombinant protein expression in mammalian cell systems, or their reported positive effects on rAAV transduction efficiency. Due to the diverse mechanisms involved in AAV vector production, the panel featured chemicals that exert their influence on recombinant protein expression via a broad range of functional mechanisms. Specifically, we tested chemicals reported to be “chemical chaperones” (TUDCA, TMAO, betaine)[15]–[17], cell cycle modulators (nocodazole, BI-2536)[18],[19], caspase inhibitors (z-VAD-fmk)[20], histone deacetylase inhibitors (NaBu, VPA, M344, apicidin)[10],[21]–[23], insulin-mimetics (lithium chloride and zinc sulphate)[24], and proteasome inhibitors (ONX0912, MLN9708, MG132)[25]–[27].
To enable multi-parallel screening of small molecule additives, we employed pre-optimized small-scale cultures (700 μL culture volume) in 24-well microplates that exhibited a similar rAAV production profile to shake flask cultures (data not shown). Triple transfection was performed using PEI in a serum-free medium, and chemical additives were introduced to rAAV8-producing suspension HEK293 cells at 24 HPT. Each additive was used at three different concentrations (Low, medium, high – values based on previous literature, see Table S1). Total cell culture was harvested at 72 HPT, cells were chemically lysed and the rAAV8 genome titre in the crude supernatant was measured by ddPCR. Crude genome titre in cells containing chemical additives was compared to that of untreated rAAV8 producing cells to determine changes to titre mediated by the small molecules. Several chemicals displayed a clear concentration-dependent impact on genome titre, both positively (z-VAD-fmk) and negatively (LiCl, ONX0912) (Fig. 1A). Several compounds with similar mechanistic properties showed a marked reduction in rAAV titre at the tested concentrations – most notably inhibitors of proteasomal function (ONX0912, MLN9708, MG132) and the reported chemical chaperones (TUDCA, TMAO, betaine). Notably, the screening showed that high relative dose nocodazole (4 µM) added to culture 24 HPT resulted in a 1.34-fold increase in rAAV8 titer compared to untreated control cells (p = 0.62, n = 2), while M344 appeared to be effective at low dose (2.5 µM) with a 1.36-fold increase in rAAV8 titer (p = 0.64, n = 2). Of note, nocodazole treatment increased mean cell volume at all three concentrations, an attribute that appears to correlate positively with measured genome titre (r2 = 0.79, p < 0.0001) (Fig. 1B) and has been shown in previous reports to be a major cellular determinant of recombinant protein productivity in Chinese hamster ovary (CHO) cells [28]–[30]. Nocodazole treated cells were found to be up to 27% larger than control cells. Nevertheless, this phenotypic variation was not observed with M344, in which the increase in titer was not accompanied by an increase in cell volume. It may therefore be inferred that these molecules modulate rAAV expression via distinct mechanisms independent of each other and that increased control of rAAV production may be achieved by the application of specific combinations of small molecule effectors.