Optogenetically Regulated siRNA Synthesis and Outer Membrane Vesicle-Mediated Delivery by Engineered Bacteria

Outer membrane vesicles (OMVs), as promising delivery vectors, have been extensively explored for the delivery of nucleic acid-based drugs, particularly the RNA therapy. However, RNA-loaded OMVs delivery systems derived from in vivo engineered bacteria lack the controllable way for regulating target RNA synthesis. Constitutive RNA synthesis may not only exert excessive metabolic burden on engineered bacteria but also lead to unexpected RNA package and delivery, markedly compromising in vivo biomedical applications. Herein, we developed an optogenetically regulated small interfering RNA (siRNA) synthesis and delivery system based on engineered bacterial OMVs. A proof of concept was demonstrated for combining this optogenetic siRNA delivery system with photothermal therapy (PTT) to evaluate tumor thermal resistance modulation by using dual functional engineered strains. The first strain (opto-siTRPV1@OMVs BL-ves) was designed to secrete siRNA targeting transient receptor potential vanilloid subtype 1 (TRPV1) in response to green light stimulation, while a second melanin-producing strain (BL21-TYR) was established for efficient photothermal ablation under 808 nm near-infrared (NIR) laser irradiation. Experimental results showed that the combined therapeutic regimen demonstrated a preliminary tumor growth inhibition rate of approximately 63.06% in a murine model. Furthermore, this regimen remodeled the immunosuppressive tumor microenvironment (TME), activated antigen-presenting cells, promoted the infiltration of effector T cells, and thereby triggered robust anti-tumor immune responses. This study presents a novel strategy for the precise and regulated delivery of siRNA, and demonstrates the feasibility of optogenetically controlled siRNA production and OMV-mediated delivery in a laboratory setting.