Comprehensive genome editing and signaling pathway engineering evolve potent and safe adenoviral producer cell

Dramatic attention has been drawn to adenovirus as a vehicle for vaccine and cancer therapy, due to the capabilities of annihilating tumor cells and stimulating host immune response. However, few producer cells are perfect for adenoviral manufacture. Herein, we orchestrate a strategy by combining homology-independent targeted integration and sgRNA library, which not only modulates the cellular gene expression profile systemically that enhance subgroup B/C adenovirus yields by 3 ~ 7 folds, but also substitutes naïve adenovirus sequence in HEK293 cells with a recombinant E1 transgene, drastically suppressing the production of clinically pathogenic replication-competent adenovirus. Moreover, directed evolution towards a serum-free gradient in bioreactor ensues an adaptive clone MC09, with both safety and potency, for scalable adenoviral manufacture at clinical grade. Molecularly, combined inhibition of carbon metabolism, apoptosis and lysosome pathways curtail adenoviral yield in MC09 cells, as demonstrated by triple knockdown of H6PD, Lamin A/C and caspase-3. Concomitantly, overexpression of these molecules further boosts biosynthesis of adenoviral vectors, highlighting a cooperative effect among multiple pathways to amplify adenovirus production. CRISPR-Cas9-based library screening and genome editing to generate a novel cell clone, MC09, with supreme adenovirus yield and negligible replication competent adenovirus production.