Project Summary Cell-based therapies have shown great potential for cancer treatment, but limitations in circuit design have prevented their applicability to diverse diseases. Current therapeutic cells only respond in a binary way to relatively high levels of a transient signal; further, gene therapies deliver only one genetic element to cells, which limits their abilities to perform complex functions and thus their applicability. Developing new types of cell systems that can respond to diverse biomarkers and enact multiple, independently regulated circuit elements is critical for expanding the scope of cell therapies. The work proposed here aims to design novel sensors, insulator elements, and computational models in the context of creating systems to modulate inflammation, primarily because chronic inflammation has a prominent role in the progression of multiple diseases. One aspect of the investigation will explore ways that cells can integrate inflammatory signals over extended time periods and how cells differentially respond based on the total signal experienced. The resulting engineered cell sensors could serve as a basis for developing therapies for chronic inflammation and other diseases with time-dependent biomarkers. Another aspect of the investigation will explore ways that genetic insulators and anti-silencing methods can be incorporated into synthetic circuits. Incorporating multiple, differentially-regulated genetic elements onto single circuits is challenging, as eukaryotic transcription factors can act across long distances. Determining a set of insulators that do not cross-react with others will help to create a generalizable framework for using insulators in complex genetic circuits, and help to enable the creation of more complex genetic circuits. Further, since epigenetic silencing decreases the activity and effectiveness of gene therapies over time, investigating ways to reduce silencing—through insulators and other genetic modifications—will help to create ways to improve the longevity of cell and gene therapies. The investigation will also use computational modeling to explore the interplay between responsive synthetic cells and pharmacokinetics. The model will incorporate inflammatory cell signaling, synthetic antibody production, and systems-level distribution to guide the design of therapeutic cells that modulate inflammation. Taken together, the proposed work will help to elucidate methods of cell sensing and gene regulation, which are critical steps for developing new classes of cell-based therapies.