Backbone-Minimised Nanoplasmid DNA Systems Enable High-Titre AAV Production in Suspension HEK293 Platforms

Background: Scalability and cost remain major manufacturing barriers limiting broad patient access to adeno-associated virus (AAV) gene therapies. While capsid engineering has advanced vector biology, comparatively fewer innovations have addressed fundamental upstream productivity constraints. Transient triple-plasmid transfection is still the dominant AAV production platform and relies on large bacterial backbone plasmids that impose DNA burden and contribute significantly to the cost-of-goods. Methods: In this study, we evaluated a compact NanoplasmidTM DNA system (Aldevron) as a structural redesign of the transfection substrate to enhance upstream productivity. Conventional pUC-based triple-plasmid systems were compared to fully substituted NanoplasmidTM equivalents across suspension HEK293 production platforms optimised via response surface Design of Experiments. Hybrid plasmid configurations were also constructed to assess component-level contributions. Results: Complete substitution with NanoplasmidTM resulted in up to a 10-fold increase in vector genome titre relative to conventional plasmids under matched conditions. Hybrid systems failed to recapitulate this improvement, demonstrating that full-system backbone minimisation is required to ensure high yield. Productivity gains were preserved across transfection reagents and suspension media. NanoplasmidTM deployment represents a scalable, capsid-independent upstream intensification approach that improves yield without altering capsid biology. Conclusions: Integration of this approach within a design-for-manufacturability framework offers a practical route to reducing bacterial plasmid elements increasing safety, enhancing process robustness, and improving economic feasibility of AAV therapeutics.