Autonomous and tunable control of synthetic receptor activation using microRNAs

Mammalian cells continuously sense and respond to their environment to maintain homeostasis and function. Intracellular signaling often begins across membranes, with receptors serving as key mediators of cell communication. Early on, scientists realized that the functionality of receptors could be harnessed to sense and correct disease state, leading to the development of the first synthetic receptors (Kuwana, 1987). Since then, the synthetic receptor toolbox has expanded, and numerous synthetic receptors demonstrated diagnostic and therapeutic potential (Teng, 2024). However, such cell-based therapeutic approaches have raised safety and toxicity concerns, primarily associated with off-target responses and persistent receptor activation (Sun, 2018). Despite advances mitigating off-target effects (Fedorov, 2013) and enabling temporal and dosage control (Wu, 2015; Rodgers, 2016; Cho, 2018), no platform currently allows for autonomous control of receptor activation. Therefore, this project aims to establish autonomous control over receptor expression and activation through the engineering of an intracellular feedback loop.

Recent studies have shown that endogenous Notch signaling is regulated by microRNA (miRNA) interference, controlling Notch expression levels (Roese-Koerner, 2016). Inspired by this mechanism, we developed a platform incorporating a negative feedback loop between the synthetic Notch (synNotch) receptor (Morsut, 2016) and a post-transcriptional synNotch inhibitor, miRNA. In detail, mammalian cells are lentivirally transduced to express a synNotch receptor that, upon activation by a membrane-bound eGFP ligand expressed by a sender cell, induces expression of a tBFP reporter and a synthetic orthogonal miRNA (miR-FF3) (Rinaudo, 2007). The synNotch receptor gene is engineered such that its transcript contains complementary binding sites that are recognized and targeted by miR-FF3, closing the negative feedback loop between receptor and miRNA, enabling repression of synNotch expression (Figure 1). Preliminary data show that our system provides tunable and autonomous regulation of synNotch receptor expression and activation, demonstrating its potential for enhanced control over synthetic receptor function.