Engineered Cas9 exosome vesicles as a novel gene editing tool for targeted ASPN editing in osteoarthritis.

CRISPR-Cas9, an innovative genome-editing technique, holds immense promise in therapeutic applications; nevertheless, the lack of effective delivery methods for in vivo gene editing limits its utility in osteoarthritis (OA) treatment. Recently, exosomes, naturally derived nanosized vesicles secreted by cells, have attracted significant attention as potential vehicles for therapeutic cargo delivery. This study proposes a bioinspired engineered exosome-mediated CRISPR/Cas9 delivery platform for targeted editing of the Asporin (ASPN) gene as a potential precision therapy for OA. Specifically, chondrocyte affinity peptide (Cap)-modified MSC-derived exosomes were employed as natural, biocompatible carriers to deliver CRISPR/Cas9 components specifically to OA-affected chondrocytes, thereby achieving precise and efficient ASPN knockout. Flow cytometry analysis confirmed a modification efficiency of 79.1% for Cap, while the encapsulation efficiency of the ASPN-Cas9 plasmid into exosomes reached 9.5% ± 0.6%. Both in vivo and in vitro investigations revealed that this delivery approach markedly improved cellular uptake and gene-editing efficacy, achieving a substantial reduction of ASPN expression by 61.7%. This, in turn, alleviated ferroptosis, improved mitochondrial function, reduced chondrocyte senescence, inhibited inflammation, and enhanced the cartilage microenvironment. Altogether, these findings strongly suggest the promising therapeutic efficacy of this method in OA models, emphasizing its potential as a precise gene-targeting therapeutic intervention for OA.