Development of Tunable Microenvironment-Responsive CAR T Cells Using Synthetic Gene Circuits to Enhance Potency and Safety - PROJECT SUMMARY Ovarian cancer is the most lethal malignancy of the female reproductive system, and despite advances in understanding of the disease, therapeutic options for treating this disease are limited. Synthetic biology affords the ability to engineer cell-based therapies, such as CAR T cells, that are programmable and targeted, and thus provide a pathway for overcoming barriers to the treatment of solid tumors. CAR T cells have demonstrated success in the treatment of hematological malignancies but have experienced challenges in their application to solid tumors due to the lack of specific tumor antigens present on solid tumor cells, as well as the presence of immunosuppressive microenvironments surrounding solid tumors. The hypoxic microenvironment ubiquitous to solid tumors can be used as a biomarker to restrict CAR expression within a tumor, improving the safety and specificity of solid tumor CAR T cell therapy, but so far this approach has only provided an effective anti-tumor response at levels of profound hypoxia. Thus, strategies are needed to provide specific, anti-tumor responses in tumors with modest levels of hypoxia that are not addressed by current approaches. To meet this need, we have developed a hypoxia biosensor (HBS) circuit enhanced by positive feedback motifs, enabling fast transcriptional output with minimal background signal. My objective is to engineer microenvironment-responsive CAR T cells (Tune-Up CARs) containing this HBS circuit to improve the safety and potency of solid tumor CAR T cell therapy for modestly hypoxic tumors. I hypothesize that the successful induction of this HBS circuit in the engineered T cells within the tumor microenvironment will enable CAR expression at modest levels of hypoxia and result in the tumor-localized delivery of immunostimulatory cytokines, inducing tumor regression. In the first Specific Aim of the proposal, I will functionally implement the HBS circuit in human primary T cells using transposon and lentiviral vectors to integrate the constructs into the T cell genome and evaluate their performance in vitro. The second Specific Aim will focus on evaluating the engineered Tune-Up CAR T cells to benchmark against current state-of-the-art technology in vivo. In the third Specific Aim, I will evaluate the anti- tumor function and specificity of Tune-Up CAR T cells in vivo in a translationally relevant ovarian cancer model. If successful, the proposed research will provide a new therapeutic technology for treating ovarian cancer and inform engineering of new classes of hypoxia-responsive cell-based therapies.
