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.