Increased recombinant AAV production by HEK293 cells using small
molecule chemical additives
Abstract
Recombinant adeno-associated virus (rAAV) has established itself as a
highly efficacious gene delivery vector with a well characterised safety
profile allowing broad clinical application. Recent successes in
rAAV-mediated gene therapy clinical trials will continue to drive demand
for improved rAAV production processes to reduce costs. Here we
demonstrate that small molecule bioactive chemical additives can
significantly increase recombinant AAV vector production by HEK cells up
to 3-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective
histone deacetylase inhibitor) were identified as positive regulators of
rAAV8 genome titre in a microplate screening assay. Addition of
nocodazole to triple-transfected HEK293 suspension cells producing rAAV
arrested cells in G2/M phase, increased average cell volume, and reduced
viable cell density relative to untreated rAAV producing cells at
harvest.. Final crude genome vector titre from nocodazole treated
cultures was >2-fold higher compared to non-treated
cultures.. Further investigation showed nocodazole addition to cultures
to be time critical.Genome titre improvement was found to be scalable
and serotype independent across two distinct rAAV serotypes, rAAV8 and
rAAV9. Furthermore, a combination of M344 and nocodazole produced a
positive additive effect on rAAV8 genome titre, resulting in a 3-fold
increase in genome titre compared to untreated cells.