Preferential delivery of lipid-ligand conjugated DNA/RNA heteroduplex oligonucleotide to ischemic brain in hyperacute stage

Abstract

Antisense oligonucleotide (ASO) is a major tool used for silencing pathogenic genes. For stroke in the hyperacute stage, however, the ability of ASO to regulate genes is limited by its poor delivery to the ischemic brain owing to sudden occlusion of the supplying artery. Here we show that, in a mouse model of permanent ischemic stroke, lipid-ligand conjugated DNA/RNA heteroduplex oligonucleotide (lipid-HDO) was unexpectedly delivered 9.6 times more efficiently to the ischemic area of the brain than to the contralateral non-ischemic brain and achieved robust gene knockdown and change of stroke phenotype, despite a 90% decrease in cerebral blood flow in the 3 h after occlusion. This delivery to neurons was mediated via receptor-mediated transcytosis by lipoprotein receptors in brain endothelial cells, the expression of which was significantly upregulated after ischemia. This study provides proof-of-concept that lipid-HDO is a promising gene-silencing technology for stroke treatment in the hyperacute stage.

Keywords: drug delivery; gene-silencing efficacy; heteroduplex oligonucleotide; hyperacute ischemic stroke; lipoprotein receptor; receptor-mediated transcytosis.

Graphical abstract

Figure 1

Increased expression of Malat1 in neurons and ECs exposed to OGD and in the brain after pMCAO Measurement of Malat1 levels with qRT-PCR analyses in Neuro2A (A) and bEnd3 (B) cells 2 or 4 h after oxygen–glucose deprivation (OGD) (n = 3, mean values ±SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus controls; one-way ANOVA, followed by Dunnett’s post hoc test). (C) Malat1 levels increased in both the ipsilateral (ischemic) cortex and the contralateral non-ischemic cortex 4 days after pMCAO (n = 3, mean values ±SEM. ∗p < 0.05, ∗∗p < 0.01 versus sham-operated controls; two-way ANOVA, followed by Bonferroni’s post hoc test). ViewRNA in situ hybridization (ISH) for Malat1 RNA (red) in the cerebral cortexes of sham-operated controls (D, ipsilateral side) and pMCAO animals 4 days after pMCAO (E, ipsilateral ischemic cortex; F, contralateral non-ischemic cortex). ISH images are representative of three independent experiments. Scale bar in (D)–(F), 50 μm.

Figure 2

High gene-silencing efficacy of lipid-HDO on ischemic cortex lesion and ECs in the hyperacute phase after stroke qRT-PCR analyses of Malat1 RNA levels in the ischemic cortex (ipsilateral) (A) and the contralateral non-ischemic cortex (B) 3 days after i.v. administration (50 mg/kg) of PBS (vehicle controls), ASO, Toc-ASO, HDO, Toc-HDO, or Cho-HDO targeting Malat1, or shuffle Toc-HDO targeting a scrambled sequence of Malat1 (n = 3, mean values ±SEM. ∗∗∗p < 0.001 versus PBS controls, one-way ANOVA, followed by Dunnett’s post hoc test; †p < 0.05 versus ASO, two-tailed, unpaired t test; ‡p < 0.05 versus ASO, two-tailed, unpaired t test; §p < 0.05 versus HDO, two-tailed, unpaired t test). (C) qRT-PCR of measurement of Malat1 RNA levels in fractionated brain ECs purified from half-brain samples. Analyses were done 3 days after i.v. administration of Toc-HDO targeting Malat1 (50 mg/kg) or PBS alone, via the tail vein 3 h after pMCAO (n = 3, mean values ±SEM. ∗p < 0.05 versus PBS controls; two-way ANOVA, followed by Bonferroni’s post hoc test). (D) qRT-PCR analyses 3 days after i.v. administration of 0, 12.5, 25, or 50 mg/kg Toc-HDO targeting Malat1 shows dose-dependent reduction of gene silencing in both the ischemic cortex and the contralateral non-ischemic cortex (n = 3, mean values ±SEM. ∗∗∗p < 0.001 versus PBS controls; two-way ANOVA, followed by Tukey’s post hoc test). In situ hybridization for Malat1 RNA in the ischemic cortex (E) and the contralateral non-ischemic cortex (F) 3 days after i.v. administration of PBS (left) or Toc-HDO (right) 3 h after pMCAO. Scale bar in (E) and (F), 50 μm.

Figure 3

Quantification and distribution of i.v. administered oligonucleotides in the hyperacute phase after ischemic stroke (A and B) Alexa Fluor 647-labeled ASO or Toc-HDO targeting Malat1 (50 mg/kg) was injected into the tail vein immediately or 3 h after pMCAO surgery. (A) Concentrations of brain oligonucleotide content in the ischemic and contralateral non-ischemic cortexes 3 h after pMCAO surgery (n = 3, mean values ±SD. ∗∗∗p < 0.001 versus contralateral non-ischemic cortex; †††p < 0.001 versus ASO; two-way ANOVA, followed by Bonferroni’s post hoc test). (B) Concentrations of brain oligonucleotide content in the ischemic and contralateral non-ischemic cortexes 6 h after pMCAO surgery (n = 3, mean values ±SD. ∗∗∗p < 0.001 versus contralateral non-ischemic cortex; †††p < 0.001 versus ASO; two-way ANOVA, followed by Bonferroni’s post hoc test). Immunofluorescence images in the ischemic cortex (C, D, F, and G) and the contralateral non-ischemic cortex (E and H) following i.v. administration of Alexa Fluor 568-labeled ASO or Toc-HDO 6 h after pMCAO. Red, Alexa Fluor 568-labeled oligonucleotides (ASO or Toc-HDO). Scale bar in (C)–(H), 100 μm. Toc-HDO targeting Malat1 (50 mg/kg) was injected via the tail vein 3 h after pMCAO surgery. Shown are concentrations of Toc-cRNA (I) and DNA/LNA gapmer (ASO) (J) in the ischemic cortex and the contralateral non-ischemic cortex 6 h after pMCAO surgery, as measured by HELISA (n = 3, mean values ±SEM.∗p < 0.05 versus ASO; Student’s two-tailed t test).

Figure 4

Distribution of i.v.-administered Toc-HDO on brain ECs and neurons in ischemic cortex (A and B) Orthogonal reconstruction from a confocal z series represented as if viewed in the x–z (top) and z–y (right) panels, showing colocalization of Alexa Fluor 568-labeled Toc-HDO (red, 6 h after pMCAO) and CD31 (green) or NeuN (green) signals in the ischemic cortex. Blue, Hoechst 33342; scale bar, 100 μm. (C) In the protocol, 50 mg/kg Toc-HDO targeting Malat1 was injected i.v. 3 h after pMCAO. (D) PBS control image was obtained 3 h after i.v. administration. (E–H) Representative images show phosphorothioate-positive signals in the ischemic cortex 3, 9, 24, or 72 h after i.v. administration of 50 mg/kg Toc-HDO targeting Malat1 3 h after pMCAO. ECs lining microvessels, green arrows; neuron-like cells, blue arrows. Scale bars in (D)–(F), 10 μm.

Figure 5

Upregulation of lipoprotein receptors during the hyperacute phase after ischemic stroke Measurement of LDLR (A), SRB1 (B), and LRP1 (C) mRNA levels by qRT-PCR analyses in the ischemic cortex and contralateral cortex in sham-operated mice and 3 h, 6 h, and 3 days after pMCAO (n = 3, mean values ±SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 versus sham controls in the ischemic cortex; ‡p < 0.05 versus sham controls in the contralateral non-ischemic cortex; two-way ANOVA followed by Dunnett’s post hoc test; ††p < 0.01, †††p < 0.001 compared between the ischemic cortex and its contralateral non-ischemic cortex; two-way ANOVA, followed by Bonferroni’s post hoc test). Confocal immunofluorescence double-labeling images with antibodies against LDLR (red, D), SRB1 (red, E), and the endothelial-specific marker CD31 (green) in the ischemic cortex and contralateral non-ischemic cortex 6 h after induction of pMCAO in mice. (F) Confocal immunofluorescence double-labeling images with antibodies against LRP1 (red) and the neuronal cell marker NeuN (green) in the ischemic cortex and contralateral non-ischemic cortex 6 h after induction of pMCAO in mice. Scale bars in (D)–(F), 50 μm. qRT-PCR analyses of Malat1 RNA levels in the ischemic cortex (G) and in the contralateral non-ischemic cortex (H) in WT mice or LDLR KO mice 3 days after i.v. administration. PBS (vehicle controls) or 50 mg/kg of Toc-HDO targeting Malat1 was injected into WT or LDLR KO mice via the tail vein 3 h after pMCAO (n = 3, mean values ±SEM. ∗∗p < 0.01 versus PBS controls; †p < 0.05 compared between WT mice and LDLR KO mice; two-way ANOVA, followed by Bonferroni’s post hoc test).

Figure 6

Toc-HDO targeting Malat1 exacerbated ischemic damage and neurological functions after pMCAO Representative cresyl violet staining of serial coronal sections 4 days after pMCAO in the PBS (A), shuffle Toc-HDO (B), and Toc-HDO (C) groups. The infarction area is traced by a red line. (D) Total infarction volume measured in eight coronal sections in each group 4 days after pMCAO (n = 4, mean values ±SEM. ∗∗p < 0.01 versus PBS controls, one-way ANOVA followed by Dunnett’s post hoc test). (E) CBF in the ischemic cortex in each group 4 days after pMCAO (n = 4, mean values ±SEM. ∗p < 0.05 versus PBS controls, one-way ANOVA followed by Dunnett’s post hoc test). (F) As assessed by the EBST, animals displayed more frequent turns toward the contralateral side (right) after pMCAO. Animals in the Toc-HDO group showed significantly less recovery of right-biased body swing rate than did the PBS controls (∗p < 0.05 versus PBS controls; repeated-measures ANOVA, followed by Tukey’s post hoc test).

Figure 7

Modulation of proliferating ECs and degenerating neurons in ischemic cortex under silencing of Malat1 Representative images show microvessel staining with anti-CD31 (A) and FluoroJade-C (FJ-C; green) (B) in the ischemic cortex 4 days after i.v. administration of PBS, shuffle Toc-HDO, or Toc-HDO. Scale bars, 50 μm. Bar graphs show average number of Ki-67+/CD31+ cells per section (C, ∗p < 0.05 versus PBS controls), average area of microvessels per section (D, ∗∗∗p < 0.0001 versus PBS controls), and average number of FJ-C-positive cells per section (E, ∗∗∗p < 0.0001 versus PBS controls) in the ischemic cortex in each group (n = 4, mean values ±SEM; one-way ANOVA followed by Dunnett’s post hoc test).

Figure 8

Microarray analysis expression profiles of mRNAs in mice from the sham group and from the ischemic groups given i.v. PBS or Toc-HDO targeting Malat1 (A) Venn diagram of significantly upregulated mRNAs (fold change ≥2) in the sham-operated control group and in the ischemic groups given i.v. PBS or Toc-HDO. (B) Heatmap obtained by hierarchical clustering analysis shows differential expression of mRNAs; n = 3 per group. Blue and red represent low and high expression levels, respectively. Each column represents a single group, and each row represents a single mRNA.