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.