Manufacture of adeno-associated virus vectors by a novel human-derived cell line HAT and comprehensive evaluation of the vectors

Recombinant adeno-associated viruses (rAAVs) are prominent vectors in gene therapy. However, efficient, low-cost manufacture of the high-quality rAAVs that are necessary for clinical success remains challenging. Here, we report the manufacture of rAAVs using a novel human-derived suspended cell line, human amniotic epithelial cell line for gene and cell therapy (HAT). The transfection conditions for HAT were optimized for rAAV2, 5, and 9, with their titer and full particle ratio (EF ratio) as critical quality attributes. The EF ratios in HAT cell lysate for rAAV2, rAAV5, and rAAV9 were 46%, 17%, and 76%, respectively, which were all higher than the values from human embryonic kidney 293 (HEK293) cells. After purification of HAT-cell-produced rAAV9 by affinity and anion exchange chromatographies, the EF ratio reached 97%. Culture of rAAV9 in HAT cells in a 2-L bioreactor produced 7.7 × 1014 vector genomes/L with an EF ratio of 89%. Comprehensive characterizations of purified rAAVs showed comparable quality attributes between the adeno-associated viruses (AAVs) produced by HAT and HEK293 cells. The in vitro and in vivo potencies of HAT- and HEK293-cell-produced rAAVs were similar, except for rAAV5, for which the HAT-cell-produced vector had higher potency. This study shows the potential of HAT cells for use in the manufacture of high-quality rAAVs.