Ionizable lipid-assisted efficient hepatic delivery of gene editing elements for oncotherapy

Abstract

CRISPR/Cas9-based gene editing has emerged as a powerful biotechnological tool, that relies on Cas9 protein and single guided RNA (sgRNA) to edit target DNA. However, the lack of safe and efficient delivery carrier is one of the crucial factors restricting its clinical transformation. Here, we report an ionizable lipid nanoparticle (iLP181, p K a = 6.43) based on iLY1809 lipid enabling robust gene editing in vitro and in vivo . The iLP181 effectively encapsulate psgPLK1, the best-performing plasmid expressing for both Cas9 protein and sgRNA targeting Polo-like kinase 1 (PLK1). The iLP181/psgPLK1 nanoformulation showed uniformity in size, regular nanostructure and nearly neutral zeta potential at pH 7.4. The nanoformulation effectively triggered editing of PLK1 gene with more than 30% efficiency in HepG2-Luc cells. iLP181/psgPLK1 significantly accumulated in the tumor for more than 5 days after a single intravenous injection. In addition, it also achieved excellent tumor growth suppression compared to other nucleic acid modalities such as siRNA, without inducing adverse effects to the main organs including the liver and kidneys. This study not only provides a clinically-applicable lipid nanocarrier for delivering CRISPR/Cas system (even other bioactive molecules), but also constitutes a potential cancer treatment regimen base on DNA editing of oncogenes.

Carrier strategies boost the application of CRISPR/Cas system in gene therapy

“…Huang et al reported an ionizable LNP iLP181 of iLY1809 (p K a = 6.43) to deliver psgPLK1 plasmid for treating liver cancer. [ 173 ] iLP181 showed neutral zeta potential at pH 7.4 and can be protonated in the endosome of acid environment. Protonated lipids can destroy the stability of the endosomal membrane and promote the escape of nucleic acid molecules from the endosome to the cytoplasm.…”

mRNA vaccines for COVID-19 and diverse diseases

“…Cationic or ionizable lipids are the primary substance to deliver RNA such as (i) 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) [ 111 ], (ii) N-[1-(2,3-dioleoyloxy) propyl]- N , N , N -trimethylammonium chloride (DOTMA) [ 112 ], (iii) N1,N3,N5-tris(3-(didodecylamino)-propyl)benzene-1,3,5-tricarboxamide (TT3) [ 113 ], (iv) dilinoleylmethyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), (v) N , N -Dimethyl-2,3-bis[(9Z,12Z)-octadeca-9,12-dienyloxy]propan-1-amine (DLinDMA) [ 114 ] and (vi) 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP) [ 115 ]. At certain pH, these lipids are positively charged and thus feasible for the mRNA to interact electrostatically, and easily fused with cellular membrane [ 116 ]. After endocytosis, the ionizable cationic lipid becomes more positively charged due to proton-pump mediated pH reduction [ 117 ].…”

Research Progress on Gene Editing Based on Nano-Drug Delivery Vectors for Tumor Therapy

“…The PLK1 gene has also been targeted in related studies of liver cancer. Li et al constructed a proprietary ionizable lipid nanoparticle named iLP181 ( Li et al, 2022 ). Four plasmids containing PLK1-targeting Cas9 protein and sgRNA were designed.…”