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bioRxiv · 10.1101/2023.10.04.560774

Compact Programmable Control of Protein Secretion in Mammalian Cells

Vlahos, A. E.·Call, C. C.·Kadaba, S. E.·Guo, S.·Gao, X. J.
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Abstract

Synthetic biology has developed powerful tools to program complex behaviors, often using genetic control. Protein circuits offer a compact alternative, yet applications with intercellular signals often lack key regulatory capabilities and tunability. Here, we employ a parts-based engineering strategy to develop a single processing and output module for synthetic protein circuits, enabling complex logic, tunable sensitivity, and control over output magnitude. Using high-throughput assays, we systematically analyze the impact of human transmembrane domains on surface expression and circuit performance. We demonstrate the utility of these optimizations by encoding an open-loop circuit within translational delivery vectors, including viral and mRNA platforms, and validate its performance in vivo. Furthermore, we demonstrate multi-input logic and showcase a novel, protein-level NIMPLY gate to regulate CAR T-cell activation. Our modular design strategy provides new insights into domain-based protein engineering and establishes a versatile and complete protein-level platform to control intercellular signaling for translational cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/560774v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@4f96f7org.highwire.dtl.DTLVardef@1404cf5org.highwire.dtl.DTLVardef@7c948corg.highwire.dtl.DTLVardef@fcad06_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Vlahos, A. E., Call, C. C., Kadaba, S. E., Guo, S., Gao, X. J.. 2023-10-04. Compact Programmable Control of Protein Secretion in Mammalian Cells. https://doi.org/10.1101/2023.10.04.560774

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