| Engineered cell-based therapies represent a promising emerging strategy for treating diseases, with the potential to enable both personalized treatments and therapeutics that carry out functions beyond the reach of existing drugs and delivery technologies. The most successful example to date, cancer immunotherapies, has utilized engineered T cells, which naturally detect and destroy foreign or damaged cells, to treat blood-based cancers, such as leukemia and lymphoma. While these therapies have demonstrated some clinical successes, controlling or modulating the activity of these therapies post-implantation remains both attractive, to enhance the potency and lifetime of the therapies, and challenging, as the tools necessary for sensing combinations of environmental cues and engineering customizable cellular programs or circuits are rather limited. Specifically, there is a need to develop tools that enable bioengineers to precisely program cells to sense and respond to molecules in the environment outside of the cell, which carry important information about the physiological state and local environment, but are not transported across the cell membrane. Toward this goal, the Leonard lab has developed a platform for engineering novel protein biosensors, termed Modular Extracellular Sensor Architecture (MESA), to detect exclusively extracellular cues. MESA comprises a platform for engineering novel receptors which, upon ligand-binding and dimerization, release a sequestered transcription factor to regulate expression of an "output" gene or genes within the cell.;Here, I present three areas of development of this technology: (1) demonstrating a liganddependent signaling mechanism for both small molecule and soluble cues (2) rewiring cellular input-output by coupling the sensing of biologically relevant extracellular signals to modulation of endogenous gene expression and (3) integrating ligand-specific MESA receptors with intracellular gene circuits to enable the cell to "process" multiparametric environmental cues. Since this was the first time anyone had attempted to build a receptor/signaling system from scratch, I first designed, tested, and built a series of receptors responsive to a small molecule, rapamycin, and this prototype receptor achieved 15-fold inducible signaling in the presence of the ligand vs. in the absence of ligand, which is on par with or superior to the performance of many natural receptors. I then extended upon this initial finding to engineer MESA receptors responsive to novel inputs, such as cytokines, using scFv antibody fragments as ligand binding domains, and combined this innovation with an intracellular system developed for activating endogenous genes using the dCas9 protein and small RNAs provided in trans. Because this platform utilizes modular, engineerable domains for ligand binding (scFv antibody fragments) and output (programmable transcription factors based upon Cas9), this approach may be readily extended to rewire cellular input-output behavior for novel inputs and outputs. Moreover, I have integrated an intracellular logical processing circuit with MESA receptors, thereby enabling multiplexed MESA signaling within a single cell. By developing a general methodology for implementing MESA receptors in conjunction with intracellular genetic circuits, it is possible to design customized functional programs with the potential to improve both the safety and efficacy of existing cell-based therapies. Overall, this work lays the foundation for leveraging MESA receptors for a range of applications including cell-based therapies and fundamental biological research. |
