An optimized triple-plasmid system with enhanced viral and helper gene expression for improved recombinant adeno-associated virus production

Recombinant adeno-associated virus (rAAV) is a preferred gene therapy vector due to its safety and efficacy. The predominant production method relies on a transient expression system utilizing mammalian cells, though low production efficiency and high dosage requirements increase costs and limit large-scale applications. The triple-plasmid system forms the basis of rAAV transient expression, where the genetic elements and their configurations significantly impact production efficiency. Here, we developed two novel triple-plasmid transient expression systems, AAV-RepE4 and AAV-RepDBP, leveraging insights into essential viral gene elements and helper components. The rAAV titer of HEK293F cells transfected with the AAV-RepE4 system increased by 2.6-fold compared to the traditional triple-plasmid system, and its single-cell packaging capacity improved by 2.2-fold. Notably, the AAV-RepE4 system exhibited higher expression levels of rep52/rep40 and a relatively moderate upregulation of rep78/rep68. This optimization facilitated viral genome synthesis and packaging while supporting favorable host cell conditions. These findings offer valuable insights into the characteristics of the viral vector production process and offer theoretical guidance and new strategies for optimizing gene therapy vector manufacturing.