Diosgenin enhances liposome-enabled nucleic acid delivery and CRISPR/Cas9-mediated gene editing by modulating endocytic pathways

Abstract

The CRISPR/Cas9 system holds great promise in treating genetic diseases, owing to its safe and precise genome editing. However, the major challenges to implementing the technology in clinics lie in transiently limiting the expression of genome editing factors and achieving therapeutically relevant frequencies with fidelity. Recent findings revealed that non-viral vectors could be a potential alternative delivery system to overcome these limitations. In our previous research, we demonstrated that liposomal formulations with amide linker-based cationic lipids and cholesterol were found to be effective in delivering a variety of nucleic acids. In the current study, we screened steroidal sapogenins as an alternative co-lipid to cholesterol in cationic liposomal formulations and found that liposomes with diosgenin (AD, Amide lipid: Diosgenin) further improved nucleic acid delivery efficacy, in particular, delivering Cas9 pDNA and mRNA for efficient genome editing at multiple loci, including AAVS1 and HBB, when compared to amide cholesterol. Mechanistic insights into the endocytosis of lipoplexes revealed that diosgenin facilitated the lipoplexes’ cholesterol-independent and clathrin-mediated endocytosis, which in turn leads to increased intracellular delivery. Our study identifies diosgenin-doped liposomes as an efficient tool to deliver CRISPR/Cas9 system.

Targeted delivery of miR-34a-5p by phenylborate-coupled polyethylenimide nanocarriers for anti-KSHV treatment

“…The nucleic acid delivery system based on a non-viral vector has many advantages such as low immunogenicity, good biocompatibility, high loading efficiency, and abundant sources ( Jiao et al, 2020 ; Jiang et al, 2022 ; Tong et al, 2023 ). These delivery carriers mainly include cationic polymers ( Van Bruggen et al, 2019 ; Feng et al, 2022 ), micelles ( Ivana et al, 2021 ; Winkeljann et al, 2023 ), liposomes ( Obuobi et al, 2020 ; Lohchania et al, 2022 ), dendrimers ( Choudhury et al, 2022 ; Liyanage et al, 2022 ), and nanoparticles (NPs) ( Vaughan et al, 2020 ; Sharma et al, 2022 ; Hu et al, 2023 ). Among them, cationic polymers have been widely studied and have a wide variety of controllable structure designs, which can form nanocomplexes by electrostatic interaction containing negative nucleic acids ( You et al, 2018 ), and then carry out in vivo and intracellular transport.…”