CRISPR/Cas9 Genome Editing Using Lipid Nanoparticles for Hepatocellular Carcinoma Therapy

Hepatocellular Carcinoma (HCC) is the 6th most common cancer and one of the most lethal worldwide. According to predictions, HCC mortality will reach 1 million annual deaths by 2030. As of today, HCC remains resistant to most therapies, including new immunotherapy regimens, which commonly achieve only a few months of progression-free survival. Therefore, new therapeutic approaches are necessary, with gene editing being a promising direction. Gene editing is a method in which engineered nucleases are utilized to induce double-strand breaks at specific genomic loci to introduce genomic modifications. In the case of cancer, CRISPR/Cas9 can be used to knock out a critical gene and lead to cancerous cells` death. In this work, multiple genes were tested as potential targets, with all sgRNAs leading to high levels of editing. The screening process highlighted that PLK1 is the best candidate as it showed ~65% reduction in viability following the editing process. Next, various lipid nanoparticle formulations were tested to determine the best one at delivering mRNA to the tumor cells via GFP measurements. The chosen formulation was then used to co-encapsulate Cas9 mRNA with PLK1-targeting sgRNA, showing above 90% editing and above 90% reduction in viability in HCC cells, highlighting the system’s potential. Next, when testing LNPs’ biodistribution in an orthotopic xenograft model, the LNPs showed ~12% penetration into the tumor. Finally, we are set to start a final efficacy experiment in an animal model to test both editing of the tumor and the effects on tumor development.