A novel micelleplex for tumour-targeted delivery of CRISPR-Cas9 against KRAS-mutated lung cancer
Siyu Chen, Meriam Triki, Simone Pinto Carneiro, Olivia M. Merkel
Abstract
, indicating its ability to form stable micelles at low concentrations. C14-PEI efficiently encapsulates mRNA into micelleplexes through electrostatic interactions. When the mass ratio is 8 (w/w 8), the C14-PEI formulation exhibits conducive properties, which showed encapsulation efficiency of eGFP mRNA at 99% and led to a 130-fold increase in eGFP expression in A549 cells compared to untreated cells, demonstrating the robust delivery and expression capability of the micelleplexes. Importantly, toxicity tests using intracellular reduction of a tetrazolium salt revealed no significant cytotoxicity, underscoring the biocompatibility of C14-PEI. C14-PEI also shows high efficiency in co-encapsulating Cas9 mRNA and sgRNA, as confirmed by agarose gel electrophoresis. At an sgRNA to Cas9 mRNA molar ratio of 10, the micelleplexes successfully mediate the cutting of mutated KRAS with an indel efficiency exceeding 60%, as determined by the T7 Endonuclease I (T7EI) assay. Droplet digital polymerase chain reaction (ddPCR) further demonstrates that the gene editing efficiency, measured by edited gene copies, is 48.5% in the w/w 4 group and 37.8% in the w/w 8 group. Treatment with C14-PEI micelleplexes containing Cas9 mRNA and sgRNA targeting the KRAS G12S mutation significantly impairs the migration capability of A549 cells and increases apoptosis rates. These findings suggest that C14-PEI effectively disrupts KRAS signalling pathways, leading to reduced tumor cell proliferation and enhanced cell death.