This study describes a new AAV proviral plasmid for vector manufacturing that significantly decreases cross-packaged bacterial sequences; increases correctly packaged AAV payloads; and blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve safety of AAV vector products.

Adreno-associated viral vectors (AAV) are a leading delivery system for gene therapy in animal models and humans. With several FDA-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. As it stands, achieving 100% genome purity in AAV preps is currently impossible. High-quality AAV preps may contain a majority of correctly packaged genomes, but all of them have a small percentage of packaged DNA contaminants. This study focused on toxic DNA contaminants that can arise from AAV proviral plasmids and the raw materials required for manufacturing recombinant AAV in eukaryotic cells.

Typical AAV proviral plasmids are circular DNAs with one half containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and the other carrying prokaryotic sequences required for plasmid replication and selection in bacteria. The ITRs harbor AAV packaging signals, and any DNA placed between properly spaced ITR sequences can therefore be inserted into AAV capsids. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk.