Abstract

Harnessing rAAVs as viral vectors for therapeutic transgene delivery still requires improvements in yields and specificity to lower vector doses, and therefore manufacturing cost, as well as to improve patient safety. To this end, our research is focused on developing novel technologies to ensure manufacturing of high yielding rAAV particles using transient transfection, as well as enhancing viral particles quality and specificity. Here we present our state-of-the art approach to design new helper plasmids (pHelpers) with the aim of improving both the genomic titers and the infectivity (TU/mL) of the viral particle obtained from suspension cultures. We took the opportunity to exploit our proprietary DNA assembly method technology to explore the synergies of multiple genetic features modularly assembled in synthetic plasmids. We then explored how plasmid ratio optimization could further enhance AAV productivity through a Design of Experiment with a mixture design approach. This work led us to identifying plasmid ratios improving both VG titers and Full/Empty Ratios.