Introduction
Recent regulatory approvals for
recombinant adeno-associated virus (rAAV) mediated gene therapy products
(Luxturna® in 2017 and Zolgensma® in 2019) - together with a significant
increase in the number of rAAV gene therapies in clinical
trials[1],[2] has highlighted the need for
improved production process technology with respect to titre, product
quality and cost. Indeed, the high cost of manufacturing rAAV gene
therapies to support the typically high therapeutic dosages used (e.g. 6
x 1013 – 2 x 1014 vector
genomes/kg[3]) has limited the economic viability
of AAV gene therapies despite evidence of promising clinical efficacy.
Therefore there is an urgent need for robust, intensified process
technology to support production of Good Manufacturing Practice (GMP)
quality material. As nearly all current rAAV production processes rely
on co-transfection of three plasmids encoding the necessary AAV and
helper genes into HEK293 cell hosts for transient production of rAAV
within 3-5 days[4], efficient use of plasmid DNA
(itself produced using a costly GMP-approved process) is also of
paramount importance.
rAAV is a complex macromolecular product requiring a diverse network of
cellular processes and molecular interactions to enable coordinated
cellular synthesis - host-cell proteins, transiently expressed viral
helper, capsid and replicase genes as well as the single stranded
therapeutic viral DNA payload itself [5]–[7].
Small molecule enhancers of recombinant protein production have
demonstrated efficacy in a wide variety of mammalian cell lines[3,10,11] and the addition of chemicals as diverse
as sodium chloride, sodium butyrate, and soy peptones have been shown in
previous studies to improve rAAV production yields[11]–[13]. Targeting of discrete pathways
involved in the replication, packaging, and trafficking of viral
particles by bioactive small molecule cell culture additives therefore
offers a simple and cost-effective way of increasing viral titre and
reducing overall production costs.
Here we show that microplate plate-based screening of chemical additives
can be used to rapidly identify positive effectors of rAAV synthesis in
HEK cells and that significant improvements in viral genome titre can be
obtained by subsequent optimisation of dosing regimen. Furthermore, we
also demonstrate that a combination of two functionally distinct small
molecule enhancers may act additively, resulting in a 3-fold increase in
viral vector titre (both increased genome titre and increased capsid
titre) to substantially improve the efficient use of transfected plasmid
DNA.