Generation and characterization of a HEK293 cell line optimized for recombinant adeno-associated virus production

HEK293 is a preferred cellular platform to produce viral vectors including adeno-associated viruses (AAV). However, HEK293 cells were shown to be genomically unstable and many HEK293 cell lines having distinct genotypes and phenotypes have been reported. Here we generated a stable clonal cell line specifically selected for the optimal production of recombinant AAV (rAAV) by the triple plasmid transfection method. Initially over two thousand single cell clones were isolated from a HEK293 polyclonal cell line and evaluated for their growth profile in suspension, doubling time, ability to recover freeze-thaw cycles and transfection efficacy. A selection of clones that met these specific criteria were then screened for their ability to produce high rAAV titers by triple plasmid transfection, yielding one high-performing clone named NBX1P01. This clone was genomically characterized using optical genome mapping and whole genome sequencing and further evaluated for rAAV production capacity across different serotypes and genes of interest (GOI). NBX1P01 was shown to be genomically stable over 55 population doubling levels (PDL), highly transfectable and able to produce rAAV titers similar or higher than those produced by a commercially available HEK293 cell line using the same culture, transfection, harvest and quantification protocol. The ratio of full-to-empty rAAV particles produced by NBX1P01 was two-fold higher than those of the commercial cell line. Long-read sequencing of the encapsidated DNA from the NBX1P01-produced rAAV indicated high levels of genome integrity with minimal levels of contaminants. These results demonstrated the versatility of NBX1P01 cells and their ability to produce high-quality rAAV vectors.