Extracellular Vesicle-Associated miRNA in Multiple Sclerosis Subtypes: Differential Profiles in Secondary Progressive Disease and the Effect of One-Year Siponimod Treatment

Circulating extracellular vesicle-associated microRNAs (EV-miRNAs) are emerging as promising peripheral biomarkers in multiple sclerosis (MS). This prospective, observational pilot study was conceived as a hypothesis-generating investigation to characterize the expression profile of four candidate EV-miRNAs (miR-223-5p, miR-155-5p, miR-30a-5p, and miR-146a-5p) within an EV-enriched plasma fraction. The cohort comprised 16 patients with secondary progressive MS (SPMS) undergoing siponimod therapy, 13 age- and sex-matched healthy controls (HCs), and 7 patients with relapsing–remitting MS (RRMS) included as an exploratory comparator. Quantification was performed by quantitative real-time PCR employing the ΔΔC_t methodology, with miR-16-5p as the endogenous normalizer. Analyses were conducted cross-sectionally and longitudinally, the latter within a paired subgroup of 11 SPMS patients evaluated at baseline and after twelve months of uninterrupted treatment. Cross-sectional comparisons demonstrated a significant downregulation of EV-miR-223-5p in SPMS patients relative to HCs (fold-change [FC] = 0.26; FDR q = 0.026), whereas EV-miR-155-5p was significantly reduced in both the SPMS (FC = 0.35; FDR q = 0.033) and RRMS (FC = 0.28; FDR q = 0.046) cohorts compared with HCs. No significant intergroup differences were observed for EV-miR-30a-5p or EV-miR-146a-5p. Longitudinal assessment revealed no significant modulation of any target EV-miRNA following one year of siponimod therapy. These preliminary observations should be interpreted with caution, given the exploratory nature and modest cohort size. Importantly, the isolation of total plasma EVs does not permit resolution of the specific cellular provenance of the observed signals, nor does it capture their downstream functional consequences. Nevertheless, the selective downregulation of EV-miR-223-5p and EV-miR-155-5p may tentatively suggest candidate molecular signatures warranting further interrogation. Adequately powered studies incorporating cell-specific EV sorting and paired cerebrospinal fluid sampling will be required to substantiate these signals and clarify their potential utility in monitoring disease progression and therapeutic response in progressive MS.