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 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, essential molecules to a cell for therapeutic applications. Thus, EVs were transfected with a U1 small nuclear RNA expression construct, resulting in approximately 120 nm diameter vesicles whose identity and purity were confirmed by the expression of several exosomal markers. When applied to HeLa cells expressing a β-globin minigene bearing a β-thalassaemia-like 5′ splice-site mutation, U1-enriched EVs corrected up to sixty percent of normal exon–intron junction recognition in a dose-dependent manner. Recovery was abolished by heat or RNase treatment, confirming that intact vesicular RNA cargo was essential for activity. These findings provide the first demonstration that EVs can transport spliceosomal small nuclear RNAs capable of reconstituting splice-site recognition in recipient cells and introduce a new class of RNA-based therapeutics that exploit the natural cargo-shuttling capacity of EVs to correct splicing defects associated with genetic disease.