Ultrasound spatiotemporally enables prolonged therapeutic mRNA translation in engineered bacteria for enhanced cancer immunotherapy.

Rationale: Engineered bacteria have recently emerged as a novel and promising strategy for cancer immunotherapy. Nonetheless, precise spatiotemporal regulation of therapeutic gene expression within these bacteria is essential to optimize therapeutic efficacy while minimizing adverse effects. This study aims to develop a system for precise, ultrasound-driven regulation of gene expression in bacteria to enable targeted tumor therapy. Methods: A modular system (Stabilized Open RNA thermometer, SORT) was designed, comprising a modified RNA thermometer with QKI response elements (QRE), therapeutic coding sequences, and the RNA binding motif of QKI. As a proof-of-concept, the bacteria VNP20009 was engineered with plasmids expressing mutated IL-2 or soluble PD-1 (sPD-1) within the SORT cassette. Syngeneic tumor mouse models (4T1 breast cancer and A20 lymphoma) were used to assess bacterial accumulation, therapeutic protein expression, anti-tumor immunity, and toxicity. Results: Upon a single session of ultrasound irradiation, IL-2 or sPD-1 expression was efficiently and durably induced in the engineered VNP20009. In mouse tumor models, SORT-equipped VNP20009 accumulated in the tumor region and diminished from organs including the liver and lung. Ultrasound irradiation enabled the therapeutic protein (IL-2 or sPD-1) to be spatiotemporally switched-on within the tumor region. This localized expression resulted in robust activation of anti-tumor immunity alongside tolerable toxic effects. Conclusions: The modular SORT platform provides a refined approach for bacteria-based therapy, enabling spatiotemporal control of therapeutic gene expression. This system enhances anti-tumor efficacy while reducing off-target toxicity, representing a promising strategy for cancer immunotherapy.