MC09 cell genomic DNA indels compared With HEK293
Dramatic attention has been drawn to adenovirus as a gene delivery vehicle for cancer therapy recently, due to its capabilities of annihilating tumor cells and stimulating host immune response. However, few producer cell lines are perfect for adenoviral manufacture at clinical grade. In this study, we orchestrated a novel strategy to engineer and evolve HEK293 cells. Comprehensive genome editing by CRISPR-Cas9 technology not only modulated the cellular gene expression profile systemically that enhanced subgroup B and C adenovirus yields by 3~7 folds, but also substituted naive adenovirus sequence in HEK293 cells with a curated E1 transgene, drastically suppressing the production of RCA that is pathogenic in clinical trials. The E1 transgene introduced by CRISPR was able to stably express during cell culture. Moreover, preincubation of the candidate cell clones in bioreactor under serum-free condition arranged robust training, thus ensuing a directed evolution towards suspension adaptation. Altogether, we managed to derive a novel clone MC09 harboring both safety and potency for scalable adenoviral production at GMP grade, which would probably satisfy the prerequisite of large-scale adenovirus production for clinical applications.