Combined Plasmid Redesign and Transfection Optimization Significantly Increases Upstream AAV Titers While Maintaining Vector Quality and In Vivo Potency

A high manufacturing cost of goods (CoG) remains a critical barrier to the broad clinical adoption of gene therapies and is driven in part by limited productivity in adeno-associated virus (AAV) manufacturing. Here, we report an optimized AAV production process developed to markedly increase upstream titers while preserving vector quality and potency. The process combines a redesigned plasmid system, an optimized plasmid ratio, and a novel synthetic transfection reagent and was benchmarked against a conventional triple-plasmid/PEI MAX workflow. Across multiple AAV capsids and independent production runs, the optimized process reproducibly increased crude harvest titers by approximately 10- to 33-fold relative to the standard process, while maintaining key vector quality attributes. Notably, within the detection limits of the assay, rcAAV was undetectable at 1 × 1010 vg input with the optimized process, whereas the conventional triple-plasmid (with native Rep-Cap sequence)/PEI MAX workflow remained rcAAV-positive under identical conditions. Importantly, the in vivo potency was comparable to that of vectors produced by the conventional process. These results position our optimized AAV production process as a promising strategy to materially reduce AAV manufacturing CoG per patient.