Exosomal delivery of CRISPR/Cas9 complexes for targeted editing of KRAS^G12D in pancreatic cancer: prerequisites for clinical surgical application

Abstract

Objective. To evaluate exosomes obtained from patients with ductal adenocarcinoma of the pancreas as carriers of CRISPR/Cas9 complexes for targeted editing of KRAS^G12D in pancreatic cancer and to test their stability, editing efficiency, and applicability for endoscopic or intraoperative administration.Materials and methods. Biological material obtained from 42 patients with pancreatic ductal adenocarcinoma included tumor tissue, ascites fluid, and peripheral blood. Primary cultures of pancreatic ductal adenocarcinoma and organoids were created from tumor tissue. Exosomes were isolated from ascites fluid and culture supernatants, purified, and loaded with Cas9/sgRNA complexes targeting the KRAS^G12D mutation. Vesicle characterization included nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Editing efficiency and functional changes were studied in pancreatic ductal adenocarcinoma cells obtained from patients. To assess surgical feasibility, an intra–tumor endoscopic ultrasound fine–needle injection model was used with the introduction of DiI–labeled exosomes into a collagen–Matrigel hydrogel block (3:1).Results. Exosomes were obtained with a high yield of (1.6 ± 0.3) × 10ïၨ particles/ml with a diameter of (102 ± 9) nm, more than 90% of which were effectively labeled. The loading efficiency of Cas9/sgRNA complexes was (47 ± 5)%. In pancreatic ductal adenocarcinoma cultures (n = 12), delivery via exosomes resulted in (31.4 ± 4.2)% indels at the KRAS^G12D locus and a (48 ± 6)% reduction in KRAS protein expression. The viability of functionally edited cells decreased by 35%, apoptosis increased almost threefold, and migration ability decreased by 45%. Combined use with gemcitabine further reduced the viability of functionally edited cells by 25%. In the model of intratumoral endoscopic ultrasound–guided fine–needle injection, exosomes remained stable – (102 ± 9) nm before injection and (104 ± 11) nm after injection (p > 0.05) and within 24 hours were diffusely distributed in the tissue by 3–4 mm, confirming their suitability for surgical use.Conclusions. Exosomes obtained from patients, a biocompatible platform for delivering CRISPR/Cas9 complexes to pancreatic ductal adenocarcinoma tumor cells, provide effective editing of KRAS^G12D and suppression of tumor activity. Validation of the intratumoral endoscopic ultrasound–guided fine–needle injection model demonstrates the feasibility of translating this strategy into clinical intratumoral treatment.