Cholesterol-tuned lipid nanoparticles for plasmid DNA delivery and CRISPR-Cas9 gene editing

Abstract

Lipid nanoparticles (LNPs) have been extensively optimized for the cytoplasmic delivery of mRNA therapeutics, as evidenced by the clinical success of mRNA-based vaccines. However, the efficacy of LNPs for plasmid DNA delivery remains limited, because DNA must penetrate both the cellular membrane and the nuclear envelope to exert any therapeutic effect, which is an inherently more challenging intracellular trafficking requirement that current LNP formulations have not yet managed to resolve. Thus, this study aimed to systematically optimize the lipid composition of DNA-loaded LNPs (dLNPs) by varying the levels of cholesterol, ionizable lipid, and helper lipid across multiple formulations. Among the tested formulations, dLNP35-C56, comprising 35% ionizable lipid and 56% cholesterol, exhibited superior transfection efficiency, excellent serum stability, and favorable cytocompatibility, thereby identifying dLNP35-C56 as the optimal formulation for plasmid DNA delivery. To validate the therapeutic applicability, dLNP35-C56 was employed to deliver CRISPR-Cas9 components targeting the KRAS G12D oncogenic mutation, resulting in markedly enhanced mutation-selective editing efficiency and substantially reduced off-target editing in wild-type cells compared with conventional Lipofectamine-mediated delivery. These findings establish dLNP35-C56 as a potent and selective non-viral delivery platform for CRISPR-based cancer gene therapy.