Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9

Hosseini, Elaheh Sadat; Nikkhah, Maryam; Hosseinkhani, Saman

Abstract

Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person's genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability. Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [ DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum. Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.

Keywords: cationic liposomes; cholesterol domains; PEGylation; Cas9/sgRNA delivery

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Transfection

Lipid-mediated nanoparticles are at the forefront of advancing gene editing technologies, particularly in the application of the CRISPR-Cas9 system. CRISPR-Cas9 is a revolutionary tool that allows scientists to make precise modifications to DNA, enabling the correction of genetic defects, the study of gene functions, and the development of new therapies for various diseases. However, delivering the CRISPR-Cas9 components efficiently and safely into target cells is a significant challenge. Lipid-mediated nanoparticles offer a promising solution for this challenge. These nanoparticles are tiny, lipid-based carriers designed to encapsulate and protect the CRISPR-Cas9 components during their journey through the body. The lipid layer not only shields the genetic material from degradation but also facilitates its entry into cells. This is achieved through the nanoparticles' ability to merge with cell membranes, allowing the CRISPR-Cas9 components to be released directly into the cellular environment where gene editing occurs. The use of lipid-mediated nanoparticles enhances the precision and efficiency of CRISPR-Cas9 delivery, reducing off-target effects and improving the overall success of gene editing. Additionally, these nanoparticles can be engineered to target specific cell types, minimizing potential side effects and increasing the therapeutic potential of gene editing. As research progresses, lipid-mediated nanoparticles are expected to play a crucial role in translating CRISPR-Cas9 gene editing from the laboratory to clinical applications, paving the way for innovative treatments for genetic disorders.