An integrated ultrasound-guided focused ultrasound system enables spatiotemporal control of thermal gene activation in engineered immune cells

<b>Rationale:</b> Thermal gene switches (TGSs), engineered into cells, allow controlled gene expression upon heat stimulation, making it a promising tool for therapeutic applications. Their clinical translation, however, has been hindered by the lack of thermal activation platforms that can locally deliver heat and provide safe and accurate temperature control. Existing approaches are limited by poor delivery and localization of heat deep inside the body, reliance on exogenous agents, or the lack of integrated image guidance. To address these challenges, we developed a non-invasive system that combines real-time imaging with mild hyperthermia for reliable and localized activation of TGSs in deep tissue. <b>Methods:</b> We developed a dual-mode ultrasound-guided focused ultrasound (USgFUS) system using a single phased-array imaging transducer for both imaging and heating. The system integrates B-mode imaging and thermal strain imaging (TSI) for real-time anatomical guidance and temperature estimation. We validated the imaging performance both <i>in vitro</i> and <i>in vivo</i> settings and assessed focused ultrasound (FUS)-induced TGS activation of genetically engineered Jurkat T cells <i>in vitro</i> and <i>in vivo</i>. <b>Results:</b> The USgFUS system achieved high-resolution and high-contrast B-mode imaging, and it induced localized heating within temperature window of 39-43 °C, consistently within the mild hyperthermia range. TSI accurately estimated temperature elevation during FUS with 0.8 °C mean absolute error. <i>In vitro</i>, FUS heating increased transgene expression in TGS-engineered Jurkat T cells by ~150-fold compared to unheated controls, with negligible viability loss. <i>In vivo</i>, USgFUS selectively activated TGS in tumor-bearing mice, yielding a significant increase in transgene expression compared to unheated controls. <b>Conclusion:</b> This study introduces a dual-mode USgFUS system designed for non-invasive TGS activation. The system integrates local mild hyperthermia with real-time anatomical guidance and temperature monitoring using a standard clinical imaging probe. The results collectively demonstrate strong performance in preclinical models and engineered cells, enabling safe, spatiotemporally precise thermal gene regulation. Ultimately, our platform provides a foundation for future advancements in gene therapy, immunomodulation, and other biomedical applications.