AAV Assembled Capsids Are Produced in Cells Blocked fromFrom 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. Overall design: 11 RNA samples were used for RNA sequencing. All RNA extractions were performed on paraformaldehyde-fixed and antibody-stained cells. 2 RNA samples were isolated from un-transfected, fixed and stained 293SF cells, 3 from mock-transfected, fixed and stained cells and 6 from cells that were sorted into 2 populations after transfection, fixing and staining, one producing virus and the other not, after transfection, fixing and staining.