Summary

The use of Lipid nanoparticles (LNP’s) to deliver CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) and sgRNA (single-guide RNA) gene-editing technologies have shown to efficiently target and permanently disrupt tumor survival genes without facing any genetic payload limitations. Thus, opening new avenues for cancer treatment and making the LNP delivery system the best choice today. In this study, Dr. Daniel Rosenblum et al. demonstrate the safety and efficacy of these lipid nanoparticles (LNPs) that use a novel amino-ionizable lipid to overcome the cargo limitations for genome editing and explain why LNP’s are the choice over the clinically approved MC3-cLNPs formulation. Some types of cancers are difficult to treat because of the high recurrence rate and potential drug-resistance development, highlighting the need for new therapeutic modalities in cancer therapy. However, Cas9 gene editing can potentially permanently disrupt tumor survival genes. However, current delivery systems for non-liver tissues and tumors only result in relatively low gene editing percentages. Therefore, higher tissue-specific targeting with sufficient editing efficiencies is needed to achieve therapeutic effects. Lipid nanoparticles are clinically approved nonviral nucleic acid delivery systems capable of delivering potentially large payloads (CRISPR/cas9 and the single-guide RNA (sgRNA)). Cationic ionizable lipids are the main component of LNPs that enable efficient cellular delivery and overcome payload limitations. 

Dr. Daniel Rosenblum et al. investigated a targeted nonviral LNP delivery system for therapeutic genome editing. For the LNP design, the scientific group used ionizable cationic lipids from a novel ionizable amino lipid library to co-encapsulate Cas9 mRNA and single-guide RNA (sgRNA). The library comprises a novel class of ionizable amino lipids based on hydrazine, hydroxylamine, and ethanolamine linkers with a linoleic fatty acid chain and amine head groups. In addition, the researchers Used the NanoAssemblr Spark™ microfluidic mixing device (Precision NanoSystems Inc.) to rapidly-produce low volume LNP formulations under controlled conditions with minimal waste.

Before the study, the scientists compared the CRISPR-LNP (cLNP) formulations using their own L8 ionizable lipid to the benchmark DLin-MC3-DMA (MC3), found in a clinically approved siRNA formulation for the delivery of Cas9 mRNA and a sgRNA. The formulations were evaluated for the in vitro gene knockout efficiency in human embryonic kidney (HEK) cells transgenically modified to express a green fluorescent protein (GFP). GFP fluorescence measurement determined the knockout efficiency. The scientist observed that although MC3-cLNPs were taken up efficiently, they did not reduce GFP expression at any concentration. Based on these data, the researchers chose L8-cLNPs over the benchmark DLin-MC3-DMA (MC3) for further study. To explore the potential of therapeutic genome editing, L8-cLNPs containing PLK1 sgRNA were evaluated as proof of concept. PLK1 is a kinase required for mitosis and lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells. sgPLK1-cLNPs effectively disrupted the targeted gene and caused cell cycle death and arrest. The data proved that cLNPs targeting the PLK1 gene could inhibit tumor growth and improve survival in two aggressive cancer models following single or double cLNP administrations. A thorough evaluation was conducted on cLNP for its therapeutic genome editing efficiency and its toxic and immunogenic nature. It was concluded that when administered systemically at therapeutically relevant doses, cLNP has the potential for a higher percentage of targeted gene editing without being toxic or immunogenic.

The study demonstrates the potential for targeted gene editing and delivery of novel therapeutics. The RNA-guided editing tool offers greater ease of customization and synthesis when compared to existing sequence-specific endonucleases while also being safe. Precision NanoSystems (PNI) has extensive experience and technologies for designing and generating sgRNA expressions either singly or in a multiplexed manner and offers optimized protocols for the delivery of Cas9-sgRNA components into desired human cells.