UC San Diego

With recent advancements in gene engineering, cellular functions have been utilized in therapeutics and diagnostics in an unprecedent amount. However, biological mechanisms for many cell activities remain unknown causing various adverse effects and overall lack of control. The potency of cell therapies such as CAR-T cell therapy leads to many on-target off-tumor effects due to lack of spatial control of CAR expression within the body. To better utilize cell function as a therapeutic, diagnostic, and overall engineering tool, this work aims to generate a genetic circuit capable of activating cell function with high spatial and temporal resolution. In utilizing clinical high-intensity focused ultrasound, localized heat can be harnessed to trigger a chemical cascade within the cell sustaining a specified gene function. Here, we demonstrate a proof of concept system in HEK293T cells where a short heat shock of 43C induces a 23% increase in the number of cells expressing a protein of interest under tightly controlled environmental conditions. We also demonstrate that 4-OHT induced ERT2 nuclear localization dominates the cytosolic retention of NFAT and a that a calcium stimulated split Cre system requires both N and C terminus of Cre as well as the calmodulin/M13 calcium induced dimerizer pair to produce functional Cre recombination. Lastly, this work characterizes the kinetics of nuclear import and export of a small NFAT library with mutations incurring variable affinities to calcineurin in an attempt to decrease system background.