Optimized Lipid Nanoparticle- based CRISPR-Cas9 Delivery for Enhanced Gene Therapy in Cancer

Abstract

The CINDELA technology uses the CRISPR-Cas9 system to target insertions and deletions (InDels) of cancer-specific genes and has great potential in cancer treatment leading to cell death [1]. However, due to its delivery efficiency limitation, it faces challenges to achieve high therapeutic impact. To address this, we employed lipid nanoparticles (LNPs) to safely and efficiently transport the Cas9 protein/gRNA complex into cancer cells. The LNPs consist of cationic/ionizable lipids, cholesterol, phospholipids, and PEG-lipids, providing biocompatibility, cargo protection, and enhanced cellular uptake, thus maximizing the efficiency of the delivery CRISPR- Cas9 system. We designed and optimized various LNP formulations based on the definitive screening design (DSD) and full factorial design (FFD). By adjusting the ratios of lipid components, we synthesized multiple LNP formulations, and their cytotoxicity in cancer cells was comparatively assessed. Through this, we found the optimal LNP composition for the CRISPR-Cas9 system using the cas9 protein. The findings of this study are expected to contribute to the understanding of the potential of LNP-based CRISPR-Cas9 delivery systems and the identification of optimized formulations for cancer gene therapy.