Comparative analysis of lipid Nanoparticle-Mediated delivery of CRISPR-Cas9 RNP versus mRNA/sgRNA for gene editing in vitro and in vivo

Feb 7, 2024European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V

Comparing lipid nanoparticle delivery of CRISPR gene editing as protein versus RNA in cells and living organisms

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mRNA Technology on OpenScience ↗PubMed ↗DOI ↗

Abstract

Lipid-nanoparticles delivering mRNA Cas9 achieved a 60% gene knock-out in liver cells of female Ai9 mice.

CRISPR-Cas9 can create targeted breaks in DNA, leading to gene knock-out or correction.
Lipid-nanoparticles encapsulating mRNA Cas9 showed smaller sizes and better protection against degradation compared to those containing Cas9-RNP.
Higher gene editing efficiencies were observed with mRNA Cas9 in both HEK293T and HEPA 1-6 cells.
Lipid-nanoparticles containing mRNA Cas9 were primarily located in the liver after administration, while those with Cas9-RNP were also found in the spleen and lungs.
Gene editing was confirmed only for LNP delivering mRNA Cas9 and sgRNA.

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The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.

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Comparative analysis of lipid Nanoparticle-Mediated delivery of CRISPR-Cas9 RNP versus mRNA/sgRNA for gene editing in vitro and in vivo

Abstract

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