Producing high-quantity and high-quality recombinant adeno-associated virus by low-cis triple transfection

Recombinant adeno-associated virus (rAAV)-based gene therapy is entering clinical and commercial stages at an unprecedented pace. Triple transfection of HEK293 cells is currently the most widely used platform for rAAV manufacturing. Here, we develop low-cis triple transfection that reduces the transgene plasmid usage by 10- to 100-fold, and overcomes several major limitations associated with standard triple transfection. This new method improves packaging of yield-inhibiting transgenes by up to 10-fold, and generates rAAV batches with reduced plasmid backbone contamination that otherwise cannot be eliminated in downstream processing. When tested in mice and compared with rAAV produced by standard triple transfection, low-cis rAAV shows comparable or superior potency, and results in diminished plasmid backbone DNA and RNA persistence in tissue. Mechanistically, low-cis triple transfection relies on the extensive replication of transgene cassette (i.e., ITR-flanked vector DNA) in HEK293 cells during production phase. This cost-effective method can be easily implemented and widely applicable to producing rAAV of high quantity, purity, and potency.