Adapting A Replication Competent Adeno-Associated Virus (rcAAV) Assay For Commercial Manufacturing

By Oxana M. Tsygankova, Ph.D., Leann Walsh, Lana Sweet, Brian Tomkowicz, Ph.D.

Recombinant adeno-associated virus (rAAV)-based gene therapies are widely used in clinical applications due to their non-integrating nature, unlike lentiviruses. Most rAAV manufacturing platforms rely on transient triple-transfection of plasmid DNA—typically in HEK-293 cells—to produce viruses that can infect but not replicate in humans. However, replication-competent AAVs (rcAAVs) can occasionally emerge, likely due to recombination events that place the rep and cap genes between ITRs on the cis-plasmid, enabling in vivo replication.

To address this risk, we developed a cell-based qPCR assay to monitor and quantify rcAAVs during manufacturing. This assay uses Rep-targeting primer/probe sets for test articles derived from transduced cells, and is validated with Cap-specific qPCR. It detects rcAAV events from AAV1 and AAV2 serotypes with a sensitivity of 10 infectious units. Integrating this assay into product release testing enhances patient safety by providing a rapid and reliable method to detect rcAAVs during commercial production.