Optimizing GPC3 gene editing in liver cancer: A comparison of Cas9 RNP and mRNA nanoliposome delivery systems

Primary liver cancer ranks as the third leading cause of cancer-related deaths worldwide and has one of the lowest survival rates among all cancers. Late-stage detection and resistance to first-line therapies underscore the urgent need for novel treatment approaches. Glypican-3 (GPC3), a heparan sulfate membrane proteoglycan that is often overexpressed in hepatocellular carcinoma (HCC), is associated with a worse prognosis. As it is absent from healthy adult liver, it makes an attractive therapeutic target. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing offers a versatile method to disrupt genes overexpressed in cancer, such as GPC3. The purpose of this study was to compare the efficiency of nanoliposomes carrying CRISPR-Cas9 ribonucleoproteins (RNPs) versus nanoliposomes carrying Cas9 mRNA for GPC3 targeting liver cancer cells. Nanoliposomes were designed using various lipid formulations and loaded either with Cas9-GFP RNPs or Cas9-GFP mRNA. We performed liposome-mediated Cas9-GFP RNP and mRNA internalization experiments in liver cancer cells, followed by fluorescence microscopy to compare delivery efficiency, while particle size distribution was determined using Dynamic Light Scattering (DLS). GPC3 knockout efficiency was evaluated using immunoblotting. Preliminary observations indicate that both RNP-loaded and mRNA-loaded nanoliposomes successfully enter liver cancer cells, with notable differences in internalization and intracellular GFP distribution between distinct lipid compositions. Initial comparisons also suggest that further optimization is required to achieve effective GPC3 protein reduction. This study demonstrates that both Cas9 RNP and Cas9 mRNA-loaded nanoliposomes show potential for delivering CRISPR components to liver cancer cells, with each approach offering distinct advantages for gene silencing. These results provide the bases for future therapeutic experiments in liver cancer mouse models.