Engineering mammalian gene activity sensors-actuator devices Cellular devices that generate user-defined outputs in response to environmental cues hold unprecedented opportunities for modern medicine through the development of living designer systems that detect and correct human pathologies. These sensor-actuator devices are currently based on cell surface sensing capabilities, mostly achieved by rewiring native ligand-receptor interactions or evolving non-native receptors linked to signal transduction systems. The performance of receptor-based sensors depends ultimately on the signal transduction mechanism embedded in the receptor system, however, and may not accurately recapitulate the physiologic response to the biomarker input. Cellular physiological states are determined by complex mechanisms that integrate signals associated with different quantitative features of extracellular and intracellular cues and provide blueprints to regulate the levels, states, and dynamics of gene expression. Regulation of gene expression thus ultimately determines cell functionality during physiological and pathological processes and is constantly and dynamically modulated to respond to environmental as well as intracellular stimuli. We thus envisioned a novel class of cellular devices that actuate user-defined biomolecular programs in response to the detection of the device’s physiological state achieved through real-time monitoring of the activity of chromosomal genes. These gene activity sensor-actuator devices are based on innovative tools recently developed by our groups for designing orthogonal systems that (i) link output expression to chromosomal genes, thereby recapitulating complex mammalian regulatory processes with high fidelity, and (ii) amplify the signal output with high resolution of the input dynamics, thereby recapitulating dynamic behaviors with superior sensitivity. To generate robust sensor-actuator devices that translate detection of gene expression signatures into user-defined outputs, we will explore the design rules of sense-and-respond mechanisms for linking detection of gene activity to output production (Aim 1), translate gene activity into precisely modulated delays in output production (Aim 2), self- adjust output production in response to output-induced modulation of gene activity (Aim 3). This approach is expected to create a paradigm shift in the way we design cellular devices that sense and respond to the environment, as it will provide a strategy to engineer cells to sense virtually any cellular process associated with a transcriptional response, eliminating the need to rewire ligand-receptor interactions or evolve synthetic receptor-based devices. Results from this study will generate design rules of cellular devices that sense gene activity with high dynamic resolution, enabling the development of cell-based diagnostics and therapeutics for a diverse range of applications.

RELEVANCE TO PUBLIC HEALTH The proposed research is relevant to public health because understanding how to engineer cells that sense the environment and respond with high sensitivity is ultimately expected to ameliorate the diagnosis and treatment of a wide range of clinical indications, including diabetes, cancer, infectious diseases, and genetic disorders, thereby effectively improving human health.