Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3

Abstract

Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9-based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.

Keywords: Angptl3; CRISPR-Cas9 mRNA delivery; genome editing; lipid nanoparticles.

Fig. 1.

Schematic illustration of LNP-mediated in vivo CRISPR-Cas9–based genome editing to induce loss-of-function mutations in Angptl3 to lower blood lipid levels. The Cas9 mRNA and Angptl3-specific sgRNA (sgAngptl3) are encapsulated in the LNP and delivered to the liver hepatocytes where they cleave the Angptl3 target locus, leading to reduced ANGPTL3 protein. Reduced ANGPTL3 level leads to a disinhibition of Lipoprotein lipase (LPL), which allows LPL to regulate the levels of therapeutically relevant circulating lipids such as LDL-C and TG.

Fig. 2.

Lipidoid nanoparticles synthesis. (A) Chemical structure of tail-branched bioreducible lipidoids. (B) Whole body luciferase bioluminescence intensity of bioreducible LNPs versus MC3 LNP measured in Balb/c mice (n = 3) at 6 h postadministration at an fLuc mRNA dose of 0.5 mg/kg. Formulation: lipid/cholesterol/DSPC/DMG-PEG = 50/38.5/10/1.5 (molar ratio), lipid/mRNA = 12.5/1 (weight ratio). (C) Size and distribution of 306-O12B LNPs formulated with fLuc mRNA measured by DLS.

Fig. 3.

Optimization of fLuc mRNA 306-O12B LNP formulations. (A) Chemical structure of three different phospholipids. Efficacy (B) and biodistribution (C) of fLuc mRNA LNPs formulated with Cholesterol, DMG-PEG, and different phospholipids (DSPC, DOPE, or DOPC). From top to bottom, luminescence signal is shown from the heart, liver, spleen, lungs, and kidneys of representative mice. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Formulation parameters of LNPs formulated with cholesterol, DOPC, and DMG-PEG at seven different mole ratios. (E) Whole body luciferase bioluminescence intensity of different formulations in Balb/c mice at 6 h postinjection at a fLuc mRNA dose of 0.5 mg/kg. (F) Whole body luciferase bioluminescence intensity 6 h postinjection of 0.5 mg/kg fLuc mRNA for LNPs formulated with cholesterol, DOPC, and DMG-PEG at a molar ratio of 50/38.5/10/1.5 with differing 306-O12B/mRNA weight ratios (n = 3).

Fig. 4.

306-O12B LNP-mediated significant levels of in vivo genome editing of Angptl3 in wild-type C57BL/6 mice. Indels percentage (A) and serum ANGPTL3 levels (B) following injections of 306-O12B LNP formulated with Cas9 mRNA and sgAngptl3 at a mass ratio of 2:1, 1:1.2, and 1:2 (n = 5). 306-O12B LNP was formulated at a molar ratio of [306-O12B:Cholesterol:DSPC:DMG-PEG] of [50:38.5:10:1.5] with a 7.5/1 weight ratio of 306-O12B/total RNAs.

Fig. 5.

306-O12B LNP is more efficient than MC-3 LNP in inducing loss-of-function mutations in Angptl3 through CRISPR-Cas9–based genome editing. (A) NGS analysis of the indels in liver and serum analyses of ANGPTL3 protein, TG, and LDL-C level of mice at day seven postadministrated with Cas9 mRNA and sgAngptl3 coloaded 306-O12B LNP at a total RNA dose of 3.0 mg/kg. MC-3 LNP was used as a positive control (n = 5 or 6). *P < 0.05, **P < 0.01, ***P < 0.001. (B) Editing frequencies of specific edited alleles in each treatment group. (C) Editing frequencies at nine top predicted off-target sites.

Fig. 6.

306-O12B LNP-mediated CRISPR editing remains durable after 100 d. (A) NGS analysis of the indels in liver and serum analyses of ANGPTL3 protein, TG, and LDL-C level of mice at day 100 postadministrated with Cas9 mRNA and sgAngptl3 coloaded 306-O12B LNP. (B) Serum levels of AST and ALT and TNF-alpha measured at day 100 postinjection. Female C57BL/6 mice were systemically injected with 306-O12B LNP coformulated with Cas9 mRNA and sgAngptl3 with a single dose at 1.0, 2.0, and 3.0 mg/kg of total RNA. Mice serum were collected at day 100 after injection (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.