Delineation factors governing production of multi-component cytokines in clinical grade cells

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Mammalian cells can produce therapeutic molecules known as biologics. Biologics can be used to treat cancer, auto-immune disorders and infectious viral disease. This project will address critical gaps in the ability engineer mammalian cells. These gaps limit cell therapy applications where cells are delivered to specific disease sites in the body in order to facilitate local biologic production. New methods to engineer clinical-grade cells for these types of applications will be developed. Complex biologics that are composed of multiple protein complexes will be the engineered products of these cells. Undergraduates, especially those from underrepresented minorities, will be trained through participation in the research.The main goals of this project are to identify and develop biosynthetic routes to circumvent bottlenecks that limit the secretion levels of fully-bioactive macromolecules. Multi-fragment, pro-inflammatory cytokines and clinical-grade cell lines will be used as model biologic and cell platforms. Protein expression will be manipulated precisely to identify bottlenecks that affect cytokine synthesis and heterodimerization. Multiple-gene manipulation strategies will be used to modulate the expression of additional genes in order to probe downstream secretion bottlenecks. Processes of particularly interest are proteolytic protein processing, folding, and vesicular trafficking. These experiments will test the overarching hypothesis that maximized secretion of multi-fragment proteins requires precision multi-gene translational control combined with the upregulation of multiple, downstream effector proteins along the secretion pathway. Engineered cells will be encapsulated in biopolymers. Their secretion stability and robustness will be evaluated. The extent to which these changes are linked to endoplasmic reticulum driven unfolded protein responses (UPR) is of primary interest.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.