A new T-antigen negative HEK293 cell line with improved AAV productivity.

Viral vectors for gene therapy, such as recombinant adeno-associated viruses, are produced in human embryonic kidney (HEK) 293 cells. However, the presence of the SV40 T-antigen-encoding CDS SV40GP6 and SV40GP7 in the HEK293T genome raises safety issues when these cells are used in manufacturing for clinical purposes. We developed a new T-antigen-negative HEK cell line from ExcellGene's proprietary HEKExpress,® using the CRISPR-Cas9 strategy. We obtained a high number of clonally-derived cell populations and all of them were demonstrated T-antigen negative. Stability study and AAV production evaluation showed that the deletion of the T-antigen-encoding locus did not impact neither cell growth nor viability nor productivity. The resulting CMC-compliant cell line, named HEKzeroT,® is able to produce high AAV titers, from small to large scale.