Cell-based therapies are transforming medicine by using living cells as programmable therapeutic agents that can sense disease-associated signals and execute tailored responses. Central to this progress are synthetic receptors: engineered molecular systems that emulate and augment natural signalling processes by converting extracellular cues into programmable intracellular responses. Their development represents a pivotal advance in mammalian cell engineering and has opened new avenues for the field of cell-based therapeutics, including chimeric antigen receptor (CAR)-T cell therapies, which have shown remarkable efficacy against haematological malignancies. However, synthetic receptor-based cell therapies remain constrained by significant safety and efficacy challenges. Constitutive or excessive synthetic receptor activation can induce severe toxicities while also promoting premature exhaustion and reduced persistence of therapeutic cells. Despite advances mitigating these adverse effects, current solutions depend on exogenous inputs, which provide useful control but do not allow therapeutic cells to autonomously adapt their activity in response to antigen exposure. Therefore, this project aims to establish autonomous control over receptor expression and activation through the engineering of an intracellular negative feedback loop.

To implement antigen-responsive, self-limiting control, we engineered an autonomous regulatory gene circuit for synthetic Notch (synNotch) receptor activation using an orthogonal microRNA (miRNA)-mediated negative feedback mechanism in mammalian cells. In this design, synNotch receptor activation induces expression of a fluorescent reporter and a synthetic orthogonal miRNA, which in turn targets miRNA binding sites placed in the synNotch transcript. As a result, ligand recognition by the receptor not only activates a downstream response, but also initiates delayed post-transcriptional repression of the receptor itself, creating a self-limiting regulatory loop.

In this work, we demonstrate that this strategy enables tunable control over receptor expression by varying the number of miRNA target sites incorporated into the receptor transcript. We further show that synNotch activation induces functional synthetic miRNA expression, and that integration of the full feedback circuit autonomously attenuates receptor abundance after activation. Flow cytometry and live-cell imaging revealed that this feedback produces transient downstream signalling: reporter expression initially increases, peaks, and subsequently declines as the feedback loop engages. Finally, mathematical modelling qualitatively recapitulated the self-limiting response dynamics and supported the robustness of the circuit design.

Altogether, these results establish miRNA-mediated feedback as a compact and programmable strategy for engineering self-regulating synthetic receptors. By coupling antigen sensing to autonomous attenuation of receptor activity, this work provides a foundation for future synthetic receptor systems with improved controllability, offering potential relevance for safer and more predictable cell-based therapies.