Optimization of CRISPR/Cas9-loaded lipid nanoparticles as a potential gene editing tool for targeted delivery in lung cancer
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Date
2024-07-04
Authors
Brunetto Büttenbender, Mariana
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Abstract
Lung cancer remains one of the most severe and commonly diagnosed cancers worldwide, largely due to its poor prognosis and high mortality rates. Non-small cell lung cancer (NSCLC) is the most prevalent subtype, often associated with KRAS mutations, which are crucial targets for effective treatment. CRISPR-Cas9 systems offer an attractive and straightforward approach for directly editing the genome by forming an active, negatively charged ribonucleoprotein (RNP) complex of Cas9 protein and sgRNA. Lipid nanoparticles (LNP) have been extensively studied and demonstrated clinical relevance and effectiveness in delivering nucleic acid therapies in recent years, with the remarkable success of mRNA lipid-based vaccines for COVID-19. This study aimed to optimize CRISPR-Cas9 RNP-loaded LNPs for targeting KRAS mutations using versatile lipid nanoparticles (LNP) with different lipid compositions based on ionizable and cationic lipids. RNP-loaded LNPs were characterized by evaluating their physicochemical characteristics, encapsulation efficiency, and in vitro performance in KRAS-mutated A549 cells. The results indicated that formulations A2 and A6, with DOTAP concentration between 7.5% and 15%, achieved particle size and PDI below critical thresholds, exhibiting low dispersity, promising gene editing efficiency, and low cytotoxicity. Despite variations in lipid compositions, the best-performing formulations showed no significant differences in particle size, PDI and zeta potential, likely contributing to their comparable gene editing efficiency. Finally, these findings suggest that RNP can be efficiently delivered using lipid nanoparticles, supporting the development of effective CRISPR-Cas9 LNP-based delivery systems for potential personalized lung cancer treatment and targeted gene editing applications.
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Keywords
CRISPR-Cas9, Ribonucleoprotein, Non-small cell lung cancer, Lipid nanoparticles, Gene editing