T cell engineering by targeted small extracellular vesicles delivering CRISPR/Cas9 system for PD-1 knock-out

Among the emerging cancer therapies, immunotherapy using T cell engineering stands out as one of the most promising approaches. While the clustered regularly interspaced palindromic repeats (CRISPR/Cas) system is widely recognized as one of the most common gene-editing system tools, the main challenge for in vivo delivery applications remains the lack of an efficient and safe intracellular carrier. Small extracellular vesicles (s-EVs), as natural nanoparticles with low immunogenicity, emerge as a promising candidate for delivering the CRISPR/Cas system. Two guide RNAs (gRNAs) targeting the programmed cell death (PD-1) gene were cloned into the PX-459 vector, and these CRISPR/Cas9 plasmids were loaded into targeted s-EVs by electroporation. Subsequently, T cells were exposed to these s-EVs, and the disruption to the PD-1 gene was examined using flow cytometry and sequencing. Flow cytometry results indicated that newly produced targeted s-EVs exhibited a higher uptake by T cells compared to non-targeted s-EVs. The successful loading of PX-459 plasmids into targeted s-EVs and their functional delivery to T cells were confirmed. Subsequent carboxyfluorescein succinimidyl ester (CFSE) staining and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that the knockout (KO) of the PD-1 gene in T cells significantly enhanced both their cytotoxicity and cytokine release compared to untreated T cells. Targeted s-EVs were used in the current study as a means to deliver the CRISPR/Cas9 system into T cells, enabling genetic modification of target cells. This approach holds the potential to improve and simplify immunotherapy. This study demonstrates a novel approach to genetic modification in T cells using targeted s-EVs containing the CRISPR/Cas9 gene-editing system. KEY POINTS: •CRISPR/Cas9 is a kind of powerful gene editing system in cancer therapeutics. •s-EVs can be applied as a biological carrier for CRISPR/Cas9 system. • T cell engineering-based immunotherapy is a promising form of anticancer therapy.