Extracellular vesicles as vehicles for small non-coding RNA therapeutics: standardization challenges for clinical translation.

Aim: Extracellular vesicles (EVs) have emerged as promising vehicles for the delivery of small non-coding RNAs (sncRNAs); however, their clinical translation is hindered by the lack of standardized manufacturing methods, RNA loading protocols, and dosing strategies in both preclinical and clinical settings. This review aims to analyze the current landscape of EV-based RNA therapeutics to identify key trends and discrepancies, providing insight for the clinical development of future sncRNA-loaded EVs. Methods: PubMed and Google Scholar were used to identify 74 published articles using cell-derived EVs loaded with sncRNA. EV source, EV surface modifications, type of loaded RNA, loading methods, and dosages used in preclinical studies were quantitatively analyzed to identify trends and discrepancies. Results: Most studies utilize naïve EVs derived from stem or immortalized cells, with electroporation and donor cell transfection being the predominant RNA loading strategies. EV loading and dosage schemes in preclinical studies are mainly based on protein content, while only a minority of studies use particle number. More generally, the variability in measurement units reflects the absence of standardized guidelines for both RNA loading and treatment dosing, generating variability and challenges in comparing results across studies. Conclusion: Reliable dosing strategies are extremely important for determining the therapeutic potential of EVs in preclinical settings and ensuring clinical translatability. However, a standardized framework for EVs as robust platforms for RNA delivery remains to be established. We underscore the critical need for universal quantification methods, standardized measurement units, and reproducible protocols for EV production and application.