Encapsulating extracellular vesicles with a minimal RISC complex as novel gene silencing tool

Gene silencing modalities including small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) have prospered in both fundamental research and clinical translations in recent years, with delivery platform being one of the key elements for success. Extracellular vesicles (EVs) as natural carriers for cell-cell communication have been engineered in a variety of ways as delivery platform for gene silencing, yet facing limited efficiency and reproducibility. In this study, we developed a new strategy for engineering EVs as gene silencing tool. A minimal RNA-induced silencing complex (RISC), composing of modified Argonaute 2 (AGO2) protein and specially designed guide strand RNAs, were encapsulated into EVs and elicited prominent EGFP silencing in proof-of-concept study. This modular EVs platform, which we named as minRISC-EVs, efficiently silenced iNOS expression in M1 macrophages as well as STAT6/A20 expression in M2 macrophages, enabling macrophages polarization towards desired directions. The macrophage modulating ability was further validated in vivo , as minRISC-EVs against iNOS alleviated mice lung inflammation in lipopolysaccharide (LPS)-induced acute lung injury model, and minRISC-EVs against STAT6/A20 inhibited B16F10 tumor progression in the tumor xenograft model. In summary, minRISC-EVs can be utilized as novel gene silencing tool, and hold great promise for clinical translation in the future.