Enhanced\nExtracellular Vesicle Cargo Loading via microRNA\nBiogenesis Pathway Modulation

Extracellular vesicles (EVs) are physiological vectors\nfor the\nintercellular transport of a variety of molecules. Among these, small\nRNAs, and especially microRNAs (miRNAs), have been identified as prevalent\ncomponents, and there has thus been a robust investigation of EVs\nfor therapeutic miRNAs delivery. However, intrinsic levels of EV-associated\nmiRNAs are generally too low to enable efficient and effective therapeutic\noutcomes. We hypothesized that miRNA localization to EVs could be\nimproved by limiting competing interactions that occur throughout\nthe miRNA biogenesis process. Using miR-146a-5p as a model, modulation\nof transcription, nuclear export, and enzymatic cleavage steps of\nmiRNA biogenesis were tested for impact on EV miRNA loading. Working\nin HEK293T cells, various alterations in the EV biogenesis pathway\nwere shown to impact miRNA localization to EVs. The system was then\napplied in induced pluripotent stem cells (iPSCs), a more promising\nsubstrate for therapeutic EV production, and EVs were separated and\nassessed for anti-inflammatory efficacy <i>in vitro</i> and\nin a murine colitis model, where the preservation of function was\nvalidated. Overall, the results highlight necessary considerations\nwhen designing a cell culture system for the devoted production of\nmiRNA-loaded EVs.