A synthetic signaling pathway engineering platform for creating precision cell-based sense-and-response devices

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SUMMARY Protein signaling networks are used by cells to sense, process, and respond to physical and molecular features in their external environment. Engineering artificial signaling networks that couple membrane receptor-mediated sensing of disease-associated signals to therapeutic responses could lead to breakthroughs in the development of dynamic cell-based therapeutic devices capable of autonomously detecting and treating disease. In contrast to native signaling networks, which rely on phosphorylation to transduce external signals, current approaches for constructing synthetic signaling networks in humans rely on nonnative regulatory mechanisms and operate on slow timescales or via single-turnover events. As a consequence, it is challenging to construct sense-and- respond programs that accurately couple environmental fluctuations to output response, or that can flexibly in- corporate diverse receptor-mediated inputs. The ability to engineer phosphorylation-based sense-and-response programs could enable functional behavior resembling native pathways, including rapid detecting and integration of extracellular signals. By enabling fine-tuned discrimination between different extracellular environments, such programs could enhance safety and efficacy profiles of cell-based therapies. In this project, we will establish a generalizable approach for engineering synthetic phosphorylation-based signaling in human cells, laying a foun- dation for next-generation cell therapy devices capable of sensing molecular cues associated with disease, and converting them into quantitatively defined therapeutic responses. To accomplish our goals, we will leverage a synthetic biology platform recently developed by our lab that enables bottom-up construction of synthetic phos- phorylation circuitry using engineered signaling proteins. As our preliminary work demonstrates, this platform can be used to create synthetic signaling pathways connecting receptor-mediated detection of extracellular mol- ecules to activation of downstream cellular processes (e.g., transcription). Here, we will investigate if this platform can be used to engineer sense-and respond program to treat inflammatory disease. Specifically, we will: 1) demonstrate the ability of synthetic pathways to be wired to receptors that sense diverse biomolecular cues associated with inflammation; 2) engineer signaling networks that integrate multiple signals and respond exclu- sively in the presence of defined combinations of inflammatory cues and; 3) test pathways in mesenchymal stromal cells (MSCs) to assess translatability of our platform. Our work will illuminate foundational principles for engineering synthetic signaling circuits and deliver a powerful technology platform for creating customized sense-and-respond programs that can precisely distinguish between features of healthy and diseased tissue. In addition to disease monitoring and diagnostic applications, these precision cell-based therapy devices could be used treat diseases ranging from inflammatory and autoimmune disorders, to tissue trauma and cancer.