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.