AAV Assembled Capsids Are Produced in Cells Blocked From Cell Cycle Progression

Adeno-associated virus (AAV) is a promising delivery system for gene therapy. However, current manufacturing of AAV suffers from very low yields compared to other biotherapeutics. The AAV dose per patient ranges between 10¹¹and 10¹⁵ viral genomes (vg), requiring an average of 10 to 30 L production/dose. As a consequence, production costs are prohibitive for most indications. Our recent studies revealed that only 10% of the HEK293 cells that have received the AAV encoding DNA produce assembled AAV capsids. This observation prompts the question: Why would cells that have been successfully transfected, be unable to produce AAV. To answer this question, we undertook a detailed study to characterize the two sub-populations from the same transfection, the cells that were making assembled capsids and those that were not. We found that the two populations had distinct cell cycle profiles, with a block in cell cycle progression characterizing the producer population. RNA-seq analysis of the two populations reveals differences in the molecular pathways impacted and provides a basis for making changes to improve productivity.