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 (<i>E2A</i>, <i>E4orf6</i>, and <i>VA RNA</i>), AAV packaging genes (<i>rep</i> and <i>cap</i>), 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 <i>in vitro</i> and <i>in vivo</i> 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.