A mediator-free sonogenetic switch for therapeutic protein expression in mammalian cells.

An ultrasound-responsive transgene circuit can provide non-invasive, spatiotemporally precise remote control of gene expression and cellular behavior in synthetic biology applications. However, current ultrasound-based systems often rely on nanoparticles or harness ultrasound's thermal effects, posing risks of tissue damage and cellular stress that limit their therapeutic potential. Here, we present Spatiotemporal Ultrasound-induced Protein Expression Regulator (SUPER), a novel gene switch enabling mediator-free, non-invasive and direct regulation of protein expression via ultrasound in mammalian cells. SUPER leverages the mammalian reactive oxygen species (ROS) sensing system, featuring KEAP1 (Kelch-like ECH-associated protein 1), NRF2 (nuclear factor erythroid 2-related factor 2), and antioxidant response element (ARE) as its core components. We demonstrate that low-intensity (1.5 W/cm2, ∼45 kHz), brief (40 s) ultrasound exposure generates non-toxic levels of ROS, activating the KEAP1/NRF2 pathway in engineered cells and leading to the controlled expression of target gene(s) via a synthetic ARE promoter. The system exhibits robust expression dynamics, excellent reversibility, and functionality in various cell types, including human mesenchymal stem cell-derived lines (hMSC-TERT). In a proof-of-concept study, ultrasound stimulation of subcutaneously implanted microencapsulated engineered cells stably expressing the sonogenetic circuit in a type 1 diabetic mouse model triggered sufficient insulin production to restore normoglycemia. Our work highlights ultrasound's potential as a precise and non-invasive tool for advancing cell and gene therapies in personalized medicine.