AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities.

Currently, the most common approach for manufacturing good manufacturing practice (GMP)-grade adeno-associated virus (AAV) vectors involves transiently transfecting mammalian cells with three plasmids that carry the essential components for production. Here, we developed an all-in-one, single-plasmid AAV production system, called AAVone, in which the adenovirus helper genes (E2A, E4orf6, and VA RNA), AAV packaging genes (rep and cap), and the vector transgene cassette are consolidated into a single compact plasmid with a 13-kb backbone. The AAVone system achieves a 2- to 4-fold increase in yields, exhibits low batch-to-batch variation, eliminates the need for fine-tuning the ratios of the three plasmids, and simplifies the production process, compared with the traditional triple-plasmid system. AAVs generated by the AAVone system show similar in vitro and in vivo transduction efficiency, but a substantial reduction in DNA impurities from plasmid bacterial backbones and a marked reduction in non-functional snap-back genomes. The AAVone system does not pose a risk for generating replication-competent AAV contaminants. Furthermore, the AAVone system requires significantly less DNAs for AAV production, while achieving favorable full:empty particle ratios and further reducing impurities. In summary, the AAVone platform is a highly efficient, straightforward, cost-effective, and highly consistent AAV production system, making it particularly suitable for manufacturing of GMP-grade AAV vectors.