Utilizing\nEngineered Bacteria as “Cell Factories” <i>In Vivo</i> for Intracellular RNA-Loaded Outer Membrane Vesicles’\nSelf-Assembly in Tumor Treatment

Delivery\nsystems play a crucial role in RNA therapy. However, the\ncurrent RNA delivery system involves complex preparation and transport\nprocesses, requiring RNA preassembly <i>in vitro</i>, transportation\nat low temperatures throughout, and possibly multiple injections for\nimproved therapeutic efficacy. To address these challenges, we developed\na simple and efficient RNA delivery system. This system only requires\nthe injection of engineered bacteria, which serve as <i>in vivo</i> “cell factories” for continuous production of the\ntarget RNA. The RNA can self-assemble with engineered bacteria’s\nouter membrane vesicles (OMVs), facilitating <i>in vivo</i> RNA delivery. Experimental results demonstrated that this system\nallowed effective delivery with excellent stability and continuity\nfor various types of RNA, including mRNA, miRNA, and siRNA. And the\nrelative abundance of target RNA in the OMVs was 10<sup>4</sup>–10<sup>7</sup> times higher than that in the mock group. We took the delivery\nof PD-L1 siRNA for tumor treatment as an example and found that this\nsystem could effectively downregulate the gene expression of PD-L1\nby approximately twofold. Notably, a single injection of engineered\nbacteria achieved a significant tumor suppression of 49.37% <i>in vivo</i>. This research provides promising insights into\nthe RNA delivery system for tumor therapy.