A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes

Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typi-cally, three plasmids are used to encode the necessary compo-nents to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alterna-tive arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full cap-sids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual-transfection system. Furthermore, pOXB dual-and tri-ple-transfection-based vectors performed consistently in vivo. The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

Automated summary

""Transient transfection using plasmid DNA is a common method for producing adeno-associated virus (AAV) vectors. A dual-plasmid system, pOXB, has been developed with an alternative sequence arrangement, which results in significantly increased AAV vector productivity and full capsid packaging compared to the traditional triple transfection method. This system has shown reproducibility across different AAV genomes and capsid serotypes, and it is scalable at a bioreactor scale of 50-L. The pOXB dual-plasmid system offers significant process gains while maintaining the flexibility of transient transfection."