Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection

Abstract

Neurological disorders are often debilitating conditions with no cure. The majority of current therapies are palliative rather than disease-modifying; therefore, new strategies for treating neurological disorders are greatly needed. mRNA-based therapeutics have great potential for treating such neurological disorders; however, challenges with delivery have limited their clinical potential. Lipid nanoparticles (LNPs) are a promising delivery vector for the brain, given their safer toxicity profile and higher efficacy. Despite this, very little is known about LNP-mediated delivery of mRNA into the brain. Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain; greater than half of the entire striatum and hippocampus was found to be penetrated along the rostro-caudal axis by direct intracerebral injections of MC3 LNP mRNAs. MC3 LNP Cre mRNA successfully transfected cells in the striatum (∼52% efficiency) and hippocampus (∼49% efficiency). In addition, we demonstrate that MC3 LNP Cas9 mRNA/Ai9 sgRNA edited cells in the striatum (∼7% efficiency) and hippocampus (∼3% efficiency). Further analysis demonstrates that MC3 LNPs mediate mRNA delivery to multiple cell types including neurons, astrocytes, and microglia in the brain. Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.

Figure 1.. The scheme of the procedure.

(A) Schematic showing the injection procedure. MC3 LNPs containing either Cre mRNA or Cas9 mRNA/Ai9 sgRNA injected into the brain were able to deliver mRNA and induce gene recombination or editing in Ai9 mice. (B) Left: Scheme of the injection site for MC3 LNP Cre mRNA and controls (saline or Cre mRNA) in either the striatum or hippocampus of adult Ai9 mice. Saline, Cre mRNA (0.125 μg μL⁻¹ or 0.250 μg μL⁻¹) or MC3 LNP Cre mRNA (0.125 μg μL⁻¹ or 0.250 μg μL⁻¹) was injected into the striatum or hippocampus of Ai9 mice. Right: Scheme of Cre-mediated recombination of tdTomato gene. (C) Left: Schematic showing the injection site for MC3 LNP Cas9 mRNA/Ai9 sgRNA and controls (saline or Cas9 mRNA) in either the striatum or hippocampus of adult Ai9 mice. Saline, Cas9 mRNA (0.125 μg μL⁻¹ or 0.250 μg μL⁻¹) or MC3 LNP Cas9 mRNA/Ai9 sgRNA (0.125 μg μL⁻¹ or 0.250 μg μL⁻¹) was injected into the striatum or hippocampus of Ai9 mice. Right: Scheme of Cas9-mediated editing of tdTomato gene.

Figure 2.. Post-synthesis stability of MC3 LNPs.

MC3 LNP Cre mRNA (0.125 μg μL⁻¹; stored at 4°C) was injected 24 hr, 48 hr and 1 month after the MC3 LNP synthesis into the striatum (Left) or hippocampus (Right) of Ai9 mice. The presence of tdTomato (red) expression and DAPI (blue) nuclear staining were analyzed 21 days after the MC3 LNP Cre mRNA injection. Scale bars represent 100 μm (Left) for whole brain images and 30 μm (Right) for higher magnification images, respectively.

Figure 3.. Transfection distance and efficiency after MC3 LNP-mediated Cre mRNA delivery in striatum and hippocampus.

(A, B) Transfection distance and total tdTomato⁺; DAPI⁺ cells in striatal injected samples (A), and hippocampal injected samples (B). (C, D) Quantification of tdTomato⁺; DAPI⁺ cells in every 12th brain slice in striatal injected samples (C), and hippocampal injected brain samples (D). Saline (n = 3 mice), low dose of Cre mRNA (0.125 μg μL⁻¹; Cre mRNA 1X; n = 2 mice), high dose of Cre mRNA (0.250 μg μL⁻¹; Cre mRNA 2X; n = 2 mice), low dose of MC3 LNP Cre mRNA (0.125 μg μL⁻¹; MC3 Cre mRNA 1X; n = 3 mice), and high dose of MC3 LNP Cre mRNA (0.250 μg μL⁻¹; MC3 Cre mRNA 2X; n = 2 mice).

Figure 4.. MC3 LNPs can deliver Cre mRNA and enhance Cre-mediated recombination in mouse striatum and hippocampus.

(A) tdTomato (red) expression and DAPI (blue) nuclear staining were analyzed 21 days after the saline, Cre mRNA or MC3 LNP Cre mRNA injection in the striatum (Left), and the hippocampus (Right). Cre mRNA or MC3 LNP Cre mRNA was injected with two doses: 0.125 μg μL⁻¹ (1X) or 0.250 μg μL⁻¹ (2X). The scale bar represents 30 μm. (B, C) Quantification of the percentage of tdTomato⁺ cells among DAPI⁺ cells in the striatum (permutation one-way ANOVA: F_(4,55) = 129.20, P < 0.001) (B), and the hippocampus (permutation one-way ANOVA: F_(4,55) = 110.80, p < 0.001) (C). All data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001 by post hoc permutation t-test. Post hoc p values were calculated between Cre mRNA (0.125 μg μL⁻¹ or 0.250 μg μL⁻¹) and MC3 LNP Cre mRNA (0.125 μg μL⁻¹ or 0.250 μg μL⁻¹) as well as between dosages 0.125 μg μL⁻¹ vs. 0.250 μg μL⁻¹ within Cre mRNA or MC3 LNP Cre mRNA. Saline (n = 15 images from 3 mice), low dose of Cre mRNA (0.125 μg μL⁻¹; Cre mRNA 1X; n = 10 images from 2 mice), high dose of Cre mRNA (0.250 μg μL⁻¹; Cre mRNA 2X; n = 10 images from 2 mice), low dose of MC3 LNP Cre mRNA (0.125 μg μL⁻¹; MC3 Cre mRNA 1X; n = 15 images from 3 mice), and high dose of MC3 LNP Cre mRNA high dose (0.250 μg μL⁻¹, MC3 Cre mRNA 2X; n = 10 images from 2 mice).

Figure 5.. The cell type specificity after the injection of MC3 LNP Cre mRNA in the striatum and hippocampus of Ai9 mice.

(A, C) Immunostaining of tdTomato⁺ (red) and either NeuN⁺ (cyan), GFAP⁺ (green) or Iba1⁺ (green) cells 21 days after stereotaxic injection of MC3 LNP Cre mRNA (0.250 μg μL⁻¹) in the striatum (A), and the hippocampus (C). The scale bar represents 30 μm. (B, D) Quantification of tdTomato⁺; NeuN⁺, tdTomato⁺; GFAP⁺ or tdTomato⁺; Iba1⁺ cells among total tdTomato⁺ cells (%) in the MC3 LNP Cre mRNA (0.250 μg μL⁻¹)-injected striatum (B), and hippocampus (D). All data are presented as mean ± SEM. MC3 LNP Cre mRNA (0.250 μg μL⁻¹; n = 10 images from 2 mice).

Figure 6.. Transfection distance and efficiency after MC3 LNP-mediated Cas9 mRNA/Ai9 sgRNA delivery in striatum and hippocampus.

(A, B) Transfection distance and total tdTomato⁺; DAPI⁺ cells in striatal injected samples (A), and hippocampal injected samples (B). (C, D) Quantification of tdTomato⁺; DAPI⁺ cells in every 12th brain slice in striatal injected samples (C), and hippocampal injected samples (D). Saline (n = 3 mice), low dose of Cas9 mRNA/Ai9 sgRNA (0.125 μg μL⁻¹; Cas9 mRNA 1X; n = 2 mice), high dose of Cas9 mRNA/Ai9 sgRNA (0.250 μg μL⁻¹; Cas9 mRNA 2X; n = 2 mice), low dose of MC3 LNP Cas9 mRNA/Ai9 sgRNA (0.125 μg μL⁻¹; MC3 Cas9 mRNA 1X; n = 2 mice), and high dose of MC3 LNP Cas9 mRNA/Ai9 sgRNA (0.250 μg μL⁻¹; MC3 Cas9 mRNA 2X; n = 3 mice).

Figure 7.. MC3 LNPs can deliver Cas9 mRNA with Ai9 sgRNA to promote gene editing in mouse striatum and hippocampus in a dose-dependent manner.

(A) tdTomato (red) expression and DAPI (blue) nuclear staining were analyzed 21 days after the saline, Cas9 mRNA/Ai9 sgRNA or MC3 LNP Cas9 mRNA/Ai9 sgRNA injection in the striatum (Left), and the hippocampus (Right). Cas9 mRNA/Ai9 sgRNA or MC3 LNP Cas9 mRNA/Ai9 sgRNA was injected with two doses: 0.125 μg μL⁻¹ (1X) or 0.250 μg μL⁻¹ (2X). The scale bar represents 30 μm. (B, C) Quantification of the percentage of tdTomato⁺ cells among DAPI⁺ in the striatum (permutation one-way ANOVA: F_(4,55) = 23.36, p < 0.001) (B), and the hippocampus (permutation one-way ANOVA: F_(4,55) = 15.29, p < 0.001) (C). All data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001 by post hoc permutation t-test. Post hoc p values were calculated between Cas9 mRNA/Ai9 sgRNA (0.125 μg μL⁻¹ or 0.250 μg μL⁻¹) and MC3 LNP Cas9 mRNA/Ai9 sgRNA (0.125 μg μL⁻¹ or 0.250 μg μL⁻¹) as well as between dosages 0.125 μg μL⁻¹ vs. 0.250 μg μL⁻¹ within Cas9 mRNA/Ai9 sgRNA or MC3 LNP Cas9 mRNA/Ai9 sgRNA. Saline (n = 15 images from 3 mice), Cas9 mRNA/Ai9 sgRNA low dose (0.125 μg μL⁻¹; Cas9 mRNA 1X; n = 10 images from 2 mice), Cas9 mRNA/Ai9 sgRNA high dose (0.250 μg μL⁻¹; Cas9 mRNA 2X; n = 10 images from 2 mice), MC3 LNP Cas9 mRNA/Ai9 sgRNA low dose (0.125 μg μL⁻¹; MC3 Cas9 mRNA 1X; n = 10 images from 2 mice), and MC3 LNP Cas9 mRNA/Ai9 sgRNA high dose (0.250 μg μL⁻¹; MC3 Cas9 mRNA 2X; n = 15 images from 3 mice).

Figure 8.. The cell type specificity after the injection of MC3 LNP Cas9 mRNA with Ai9 sgRNA in the striatum and hippocampus of Ai9 mice.

(A, C) Immunostaining of tdTomato⁺ (red) and either NeuN⁺ (cyan), GFAP⁺ (green) or Iba1⁺ (green) cells 21 days after stereotaxic injection of MC3 LNP Cas9 mRNA/Ai9 sgRNA (0.250 μg μL⁻¹) in the striatum (A), and the hippocampus (C). The scale bar represents 30 μm. (B, D) Quantification of (B) tdTomato⁺; NeuN⁺, tdTomato⁺; GFAP⁺ or tdTomato⁺; Iba1⁺ cells among total tdTomato⁺ cells (%) in the MC3 LNP Cas9 mRNA/Ai9 sgRNA (0.250 μg μL⁻¹)-injected striatum (B), and hippocampus (D). All data are presented as mean ± SEM. MC3 LNP Cas9 mRNA/Ai9 sgRNA (0.250 μg μL⁻¹; n = 10 images from 2 mice).