Synthetic circuit technologies for precision control and signal processing in mammalian cells

PROJECT SUMMARY Synthetic gene circuits that enable precise, customizable control of gene regulation and human cell function can address unmet needs in biology, medicine, and synthetic biology, ranging from next-generation cell-based therapies to programming tissue function. However, the lack of engineering toolkits that provide robust in vivo activity, clinically-suitable compositions, and easy implementation for implementing gene circuits in biomedically- relevant human cell types has drastically limited our ability to realize these important biomedical goals. To overcome these limitations, our collaborative team recently established a powerful set of toolkits that enable the construction of “clinically-optimized” synthetic circuits for (FDA-approved) drug-inducible and cell-autonomous control of therapeutic human cell function. Using these toolkits, we already succeeded in engineering advanced control of primary human T cells, such as programming T cells to recognize multi-faceted features of target cells and launch complex, multi-tiered therapeutic actions – foundational work that serves as the basis of a recently launched clinical trial for next-generation cellular therapy. In this project, we will expand our gene circuit engineering capabilities in critical new directions – toward engineering precise control of human macrophage and coordinated multi-immune cell functions – through the development of monocyte/macrophage-optimized toolkits. Macrophages are functionally versatile cell types that play central homeostatic roles in nearly all tissues. Additionally, macrophages have a number of unique properties that make them promising tools for potential cell- based therapies, including their natural ability to migrate into tumors and their ability to phagocytose cells and interface with the adaptive immune system through antigen presentation. However, engineering macrophage function through synthetic biology-based methods remains nascent and technologically underdeveloped. We will address this unmet need through three independent Aims. First, we will develop macrophage-optimized toolkits of synthetic humanized transcription factors and transcriptional receptors for programmed gene regulation, enabling the construction of circuits for localized, signal-dependent production of immune effectors and payloads in vitro and in vivo. Second, we will establish a novel, high-throughput pipeline to generate and screen engineered macrophage receptors with customizable phagocytic and immune activation functions. Finally, we will demonstrate novel circuit types that allow macrophages to coordinate their responses with endogenous and/or engineered T cells to drive synergistic and amplified immune functions, thus establishing the basis for engineering multicellular immune networks. Together, this work and our toolkits, which will make freely available to the academic scientific community, will substantially expand the capabilities of mammalian cell engineering and usher in an exciting era of macrophage cell-based biotechnologies with broad biomedical and therapeutic applicability.