Engineered microRNA feedback circuits enable tunable and autonomous control of synthetic receptor activity

Synthetic receptors, designed to emulate and augment natural signalling processes, are engineered molecular systems that transduce 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 show remarkable efficacy against haematological malignancies. However, synthetic receptor therapies remain constrained by significant safety and efficacy challenges. Constitutive or excessive synthetic receptor activation can induce severe toxicities while also promoting premature clearance of therapeutic cells. Despite advances mitigating these adverse effects, current solutions depend on exogenous inputs and consequently lack direct feedback mechanisms responsive to the presence of the targeted antigen. 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 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 fluorescent reporter and a synthetic orthogonal miRNA (miR-FF3). The synNotch receptor gene is engineered such that its transcript contains complementary binding sites that are recognized and targeted by miR-FF3, linking receptor activation to its own repression through post-transcriptional feedback, effectively concluding the envisioned negative feedback loop.

Using this circuit, we first demonstrated that the degree of synNotch repression is tunable, scaling with the number of miRNA target sites incorporated in the receptor transcript, enabling tunable control of receptor expression. Secondly, we showed using qPCR that synNotch activation can induce expression of functional synthetic miRNA. Finally, flow cytometry and live-cell imaging analyses revealed that the full genetic circuit autonomously attenuates synNotch expression following activation, resulting in transient, self-limiting downstream reporter expression that peaks before gradually declining as feedback engages. In addition, we developed a simple ordinary differential equation (ODE) model of the circuit that reproduces the key dynamic features of our system, reinforcing experimental observations with a quantitative framework.

Altogether, these results demonstrate that miRNA-based negative feedback enables autonomous and tunable regulation of synthetic receptor activity, providing a generalizable strategy to endow receptor systems with built-in homeostatic control, guiding the development of robust, self-regulating synthetic circuits, and laying a foundation for future therapeutic applications.