Recipients: Caleb Bashor, Laura Segatori
In recent decades, mammalian cells have emerged as the primary workhorses for the production of recombinant protein (rProtein) biopharmaceuticals. However, developing safe, efficient, and adaptable rProtein bioproduction pipelines remains a major outstanding challenge. While extensive research has gone into optimizing aspects of bioprocessing (e.g., bioreactor design, growth media formulation), enhancing the intrinsic bioproduction performance of mammalian cell lines using synthetic biology approaches remains underexplored. To date, most approaches have perturbed individual cellular components, resulting in marginal improvements in yield. Maximizing cell growth and productivity while mitigating stress caused by protein overproduction may require engineering multiple cellular subsystems, including those regulating metabolism, protein trafficking, and unfolded protein response. In this project, we will test this possibility using an ultra-high throughput library approach to screen combinations of engineered genetic perturbations—transgene overexpression, transcriptional up/down regulation using CRISPRa/CRISPRi, and shRNA knock down—that perturb regulatory targets both within and between subsystems. We will clone >105 multi-gene arrays and chromosomally integrate them at single copy into HEK293 cells, a human-derived cell line commonly used for bioproduction. We will use flow sorting to isolate cells based on intracellular accumulation of folded rProtein targets and markers of early and late apoptosis, followed by NGS to determine genotype. This approach will reveal combinations of elements that act synergistically across to promote rProtein production while maintaining viability. We anticipate that the experimental pipeline generated from this study will be applicable to other cell types relevant to biomanufacturing as well as to the ex vivo development of cell therapies.