Exosome-mediated delivery of CRISPR/Cas9 for targeting pancreatic cancer

CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. MD Anderson Cancer Center researchers report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. They show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, the researchers demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant Kras ^G12D oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.

CRISPR/Cas9–mediated gene editing suppresses oncogenic Kras^G12D in vitro

KPC689 cells were transfected with 5 μg plasmid DNA (Cas9/Kras^G12D gRNA1/2 with LentiCRISPR V2 or PX458 backbone, and the Cas9 vector controls) by Lipofectamine 2000 for 48 h. (A) Epifluorescence microscopy imaging was used to evaluate transfection efficiency of lipofectamine 2000 by using GFP/Cas9 vector control (PX458) plasmid. Scale bar, 100 μm. (B, C) Quantitative PCR was used to evaluate mRNA expression levels of cas9 (B) and Kras^G12D (C). (C) Data in (C) are normalized to 18s and untransfected control. One-way ANOVA with Tukey’s multiple comparisons test was used to evaluate mean differences among groups based on ΔC_T values. (D) T7/Surveyor assay was used to evaluate gene editing in genomic DNA of KPC689 cells following transfection with Lipofectamine after 48 h. All results are expressed as mean ± standard deviation. ***P < 0.001, ****P < 0.0001.