Using high-throughput expression and spatial proteomics to characterise remodelling of HEK293 cells during the production of recombinant adeno-associated virus (rAAV)-8

The potential of recombinant adeno-associated viruses (rAAVs) as therapeutic DNA delivery agents has been established with many rAAV-based gene therapies available on the market. Such rAAVs are most commonly manufactured via transient transfection of the necessary genetic components into HEK293 cells. Unfortunately, low yield and recovery during this process currently limits the wider application of rAAV delivery vehicles. Hence, to improve the manufacturing of rAAVs and ensure the long-term success of rAAV gene therapies will require a better understanding of the molecular mechanism(s) by which rAAVs are produced inside HEK293 cells.

In this project, rAAV8 was produced in HEK293-VP cells using a standard transient triple transfection method. The rAAV8 yield was similar to those previously reported with 2.64 x 10¹¹ viral genomes/mL at 96-hours post-transfection, as determined by qPCR. Using high-throughput mass spectrometry, the global abundance of cellular proteins was quantified and compared between control (mock transfected) and rAAV8-producing HEK293-VP cells at 48- and 96-hours post-transfection using a TMT workflow. By 48-hours post-transfection, 347 proteins were found to have significantly differential abundance in rAAV8-producing cells compared to control cells. This early response included the downregulation of cell cycle transition factors and DNA damage response proteins. At 96-hours post-transfection, 1,517 proteins displayed significant changes in abundance. Proteins with increased abundance were enriched for gene ontology terms relating to structural organisation of the cell whilst proteins relating to normal cellular metabolism were decreased in abundance. To further explore the effect of rAAV8-production on cellular organisation and protein localisation, global subcellular proteome maps were acquired for both mock transfected and rAAV8-producing HEK293-VP cells using the well-established Localisation of Organellar Proteins by Isotope Tagging after Differential Centrifugation (LOPIT-DC) method. By exploiting semi-supervised Bayesian classification methods, over 6,000 proteins were systematically localised to 12 distinct subcellular organelles and protein complexes in each condition. Comparison of these maps revealed that substantial subcellular remodelling takes place during rAAV8-production with over 50 proteins displaying differential localisation. The subcellular niches with the greatest degree of change were the nucleus, nucleolus and organelles of the secretory pathway. Further validation of these proteomic datasets using targeted assays and microscopy will provide insight into the key responses of HEK293-VP cells to rAAV8-production. Further, these datasets provide a rich resource for discovery of cellular factors which may promote or limit rAAV production for gene therapies.