Bioinformatic Analysis of the Genetic Basis of Differential Adeno-Associated Virus Production Capability of 293 Variants.

Human embryonic kidney 293 (HEK 293) cells are the main producer cell line for recombinant adeno-associated virus (rAAV) production. However, AAV vector yields among 293 clones vary considerably. To elucidate the biological basis for these differences, whole genomes of an adherent and a suspension 293 cell clone with high-yield rAAV were sequenced using nanopore technology. All 293 cell derivative lines showed a twofold copy number gain at the adenoviral integration site across, suggesting a genome duplication event. To our surprise, the two high-producer clones, despite having been separately developed, are biologically closely grouped together as compared to other commonly used 293 clones. Their genomes contain a similar adenoviral gene integration region, which likely leads to high expression of proteins that facilitate AAV replication and packaging. Thus, genome duplication in the adenovirus integration locus may be a key factor affecting AAV production yield.