Extracellular Vesicle-Mediated U1 snRNA Delivery Restores Aberrant Pre-mRNA Splicing in Human Cells.

Splicing defects represent a significant class of human genetic disorders, yet strategies to directly correct aberrant splice-site recognition remain limited. The small nuclear RNA (snRNA) U1 plays a critical role in pre-messenger RNA splicing by base-pairing with the conserved 5' splice-site 'GU' dinucleotide. Disruption of this interaction can lead to abnormal splicing or frameshift mutations, contributing to disease pathology. Extracellular vesicles (EVs) can transport small molecules to cells for therapeutic applications. Here, U1 snRNA-overexpressing HEK293T cells were used to generate approximately 120 nm-diameter U1 snRNA-enriched EVs, whose purity and content were confirmed by exosomal marker Western blots and reverse transcription-quantitative PCR. When HeLa cells expressing a β-globin minigene bearing a β-thalassaemia-like 5' splice-site mutation were treated with U1-snRNA-enriched EVs, they corrected up to sixty percent of normal exon-intron junction recognition in a dose-dependent manner. Recovery was abolished by heat or RNase treatment, suggesting that intact vesicular RNA cargo was essential for activity. These findings provide the first demonstration that EVs can transport spliceosomal snRNAs capable of reconstituting splice-site recognition in recipient cells and introduce a novel class of RNA-based therapeutics that exploit the natural cargo-shuttling capacity of EVs to correct splicing defects associated with genetic disease.