Utilizing Engineered Bacteria as "Cell Factories" <i>In Vivo</i> for Intracellular RNA-Loaded Outer Membrane Vesicles' Self-Assembly in Tumor Treatment.

Abstract

Delivery systems play a crucial role in RNA therapy. However, the current RNA delivery system involves complex preparation and transport processes, requiring RNA preassembly <i>in vitro</i>, transportation at low temperatures throughout, and possibly multiple injections for improved therapeutic efficacy. To address these challenges, we developed a simple and efficient RNA delivery system. This system only requires the injection of engineered bacteria, which serve as <i>in vivo</i> "cell factories" for continuous production of the target RNA. The RNA can self-assemble with engineered bacteria's outer membrane vesicles (OMVs), facilitating <i>in vivo</i> RNA delivery. Experimental results demonstrated that this system allowed effective delivery with excellent stability and continuity for various types of RNA, including mRNA, miRNA, and siRNA. And the relative abundance of target RNA in the OMVs was 10⁴-10⁷ times higher than that in the mock group. We took the delivery of PD-L1 siRNA for tumor treatment as an example and found that this system could effectively downregulate the gene expression of PD-L1 by approximately twofold. Notably, a single injection of engineered bacteria achieved a significant tumor suppression of 49.37% <i>in vivo</i>. This research provides promising insights into the RNA delivery system for tumor therapy.