Discussion
Improving the yield of intact, genome-containing rAAV particles during
viral vector production is a critical step to reducing overall
production costs. Here we describe a simple and robust method by which
viral vector titre, a fundamental critical quality attribute of viral
vector production, may be quickly improved in an established and
previously optimised suspension culture system. Small molecule enhancers
of recombinant protein expression have been extensively used across a
wide range of mammalian cell systems to improve transient production
performance[8]–[10],[22],[39],[55],[56].
The ease of use and low cost of small molecule enhancers (particularly
for chemicals with pronounced biological activity at low dosages) makes
them an attractive solution to improving rAAV yield.
Investigation of the cellular mechanisms underpinning increased viral
genome titre point to the arrest of rAAV producing cells in mitosis soon
after PEI-mediated transient transfection of rAAV producing plasmids.
Prior studies have shown a strong preference for wildtype AAV
replication within the G2/M phase of the cell cycle[57],[58]. Nocodazole has been shown to
significantly improve transient transfection efficiency in CHO
cells[39] which may stem from increased nuclear
permissibility of transfection complexes due to the breakdown of the
nuclear membrane during mitosis. A reduction in cell proliferation
caused by nocodazole addition may also benefit viral production by
reducing plasmid copy number dilution and maintaining mRNA transcript
levels [9]. A recent study utilising a
CRISPR-mediated genome wide screening strategy identified two target
genes (ITPRIP and SKA2) that when modulated in cells increased rAAV
genome titre and improved full/empty capsid ratios, with both target
genes (strongly, in the case of SKA2) associated with cell cycle
modulation [59]. Further to this, a proteomic
study of HEK cells during AAV5 production highlighted a number of
proteins involved in cell cycle and proliferation as being strongly
downregulated during production [60]. The
molecular effects of cell cycle arrest within the G2/M phase on rAAV
production are unknown, but there is a potential correlation between the
loss of essential nucleolar functions and the nucleolar localization of
viral proteins, while the volume increase could possibly minimize
crowding effects due to accumulation of viral
proteins[61]. This apparent link between the cell
cycle and AAV production, and the abundance of cell cycle modulating
molecules, necessitates further investigation into the use of cell cycle
modulators for both improving rAAV production yields and ultimately
improving our understanding of the underlying biological processes
governing rAAV production.
The apparent important temporal aspect of cell cycle regulation within
the production process may also provide avenues for non-chemical
interventions to improve vector yield as this relationship becomes
better understood. Whilst we have not investigated the mechanism behind
M344-mediated titre enhancement, HDAC6 (of which M344 is a selective
inhibitor) has been shown to bind to, and regulate clearance of,
ubiquitinated proteins via induction of the heat-shock cellular
response[62]. AAV capsid proteins are a known
target of ubiquitination post-viral entry[63], and
the ubiquitin-proteasome pathway (UPP) has been suggested to play an
active role in AAV capsid monomer degradation[64],
therefore we posit a link between M344-mediated UPP dysregulation and
increased crude viral titre. Due to the robustness of the results
between small-scale plate-based cultures and larger scale shake flasks,
we believe that the screening process shown here could be further scaled
down and automated to increase throughput, due to the availability of
instrumentation that can rapidly and accurately dispense very small
volumes of drugs into culture. As minimal volumes are required for ddPCR
analysis of genome titre, identification of novel enhancers of rAAV
could be rapidly incorporated into existing rAAV production platforms
with minimal changes to existing protocols.
In summary, we show that the use of readily available small molecule
enhancers can significantly improve rAAV production yield. We show that
small molecule enhancers of rAAV production are amenable to optimisation
in an existing suspension HEK293 cell system and that positive hits from
initial small-scale screening of enhancer molecules can translate to
larger scale production platforms. We also show that increased titre
resulting from nocodazole treatment is consistent across two different
serotypes, suggesting broad applicability in rAAV manufacturing.