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.