Neural Stem Cell-Derived Extracellular Vesicles Improve CNS Delivery and Therapeutic Efficacy of Antisense Oligonucleotide in Amyotrophic Lateral Sclerosis.

Antisense oligonucleotides (ASOs) hold clinical promise for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Yet, their efficacy is constrained by the need for invasive delivery, poor central nervous system (CNS) penetration, inefficient cellular uptake and rapid clearance. Here, we developed neural stem cell-derived extracellular vesicles (NSC-EVs) as natural carriers to enhance CNS targeting and therapeutic efficacy of ASO-based treatments for ALS. Using a passive loading method, therapeutic ASOs were efficiently associated with NSC-EVs (NSC-EV-ASO). Compared with free ASOs, NSC-EVs- ASOs exhibited markedly enhanced neuronal uptake in vitro and increased CNS accumulation and neuronal localization in vivo following intracerebroventricular, intranasal, or intravenous administration. Functionally, NSC-EV-ASOs reduced the mutant SOD1 protein expression and the pathogenic hexanucleotide repeats in the C9orf72 loci in ALS cellular models. In the ALS mouse model, intranasal administration of NSC-EV-ASOs more efficiently delayed disease onset, improved motor performance, preserved motor neurons and attenuated astrocyte activation during the study period compared with free ASOs. These findings provide proof-of-concept evidence supporting the potential of NSC-EV as a promising platform that not only enhances CNS delivery of ASOs but also contributes to overall therapeutic activity, offering a synergistic strategy to advance nucleic acid therapeutics for ALS and potentially other neurological diseases.