Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model

Figures

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  Figure 1. Dose-dependent knockdown of ANGPTL3 gene expression by miAngA to N (A and B) Luciferase activity in Huh7 cells co-transfected with LucAng and miAng or miSCR plasmids, and (C) endogenous ANGPTL3 mRNA in Huh7 cells transfected with miAng or miSCR plasmids. For (A) and (B), the relative mean (±SD) luciferase activity was calculated as the ratio between RL and FL activities (n = 3–5). For (C), ANGPTL3 mRNA levels in miAng-transfected cells are expressed as a percentage mean (±SD) relative to miSCR-transfected Huh7 cells (set to 100%, n = 2). FL, firefly luciferase; Luc, luciferase; miRNA, microRNA; RL, Renilla luciferase; SCR, scrambled control.

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  Figure 2. Vector DNA, Angptl3 mRNA and ANGPTL3 protein quantification in liver or plasma of WT mice treated with vehicle, rAAV5-miAngE or rAAV5-miSCR (A) Vector DNA copy numbers in liver tissue, (B) correlation between Angptl3 mRNA and mature 23 nucleotide miAngE expression levels, and (C) changes in plasma ANGPTL3 protein levels over time in WT mice injected with vehicle control; low-, moderate-, or high-dose rAAV5-miAngE; or rAAV5-miSCR. Low dose, 1 × 1013 gc/kg; moderate dose, 5 × 1013 gc/kg; high dose, 2.5 × 1014 gc/kg; control, treatment-naïve plasma. VCN, vector copy number, are shown as genome copies per μg genomic DNA. GC, genome copies; rAAV5, recombinant adeno-associated virus serotype 5; WT, wild type. For (B), Pearson r was computed for X vs. every Y dataset. Pearson correlation calculations were based on the assumption that both X and Y values were sampled from populations that follow a Gaussian distribution. Two-tailed p value and 95% confidence intervals were used (GraphPad Prism 8.0.0). For (C), significant changes vs. vehicle control were determined using one-way ANOVA, Bonferroni post-test (GraphPad Prism 8.0.0). Mean is (± SD). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. LLOQ,…

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  Figure 3. Vector DNA, Angptl3 mRNA and ANGPTL3 protein quantification in liver and/or plasma of dyslipidemic APOE∗3-Leiden.CETP mice treated with vehicle, rAAV5-miSCR or rAAV5-miAngE (with or without atorvastatin) (A) Vector DNA copy numbers in liver, (B) percent change in liver Angptl3 mRNA levels relative to vehicle control, (C) changes in liver ANGPTL3 protein levels vs. vehicle or miSCR controls, and (D) changes in plasma ANGPTL3 protein levels vs. vehicle or miSCR controls in dyslipidemic APOE∗3-Leiden.CETP mice injected with moderate-high dose rAAV5-miAngE or rAAV5-miSCR, with or without atorvastatin. For (A)–(C), analyzed samples are vehicle group n = 20, miSCR group n = 15, miAngE group n = 15, miSCR + statin n = 15, miAngE + statin n = 15. For (D), week 0 analyzed samples vehicle group n = 8, miSCR group n = 15, miAngE group n = 15, miSCR + statin n = 15, miAngE + statin n = 15; for (D), week 2–16 analyzed samples vehicle group n = 14 (except week 16 n = 9), miSCR group n = 9, miAngE group n = 9, miSCR + statin n = 9, miAngE + statin n = 9. The p values for (C) and (D) were calculated using one-way ANOVA, Bonferroni post-test. Moderate-high dose,1 × 1014 gc/kg of rAAV5-miAngE or…

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  Figure 4. Plasma total cholesterol and triglycerides levels in dyslipidemic APOE∗3-Leiden.CETP mice treated with vehicle, rAAV5-miSCR or rAAV5-miAngE (with or without atorvastatin) Changes over time in mean (±SD) plasma levels of (A) total cholesterol and (B) triglycerides in dyslipidemic APOE∗3-Leiden.CETP mice injected with vehicle control, moderate-high dose rAAV5-miAngE, or rAAV5-miSCR, with and without atorvastatin. The p values were calculated using one-way ANOVA, Bonferroni post-test. Moderate-high dose (1 × 1014 gc/kg) of rAAV5-miAngE or rAAV5-miSCR. Atorvastatin dose 45 mg/kg rAAV5, recombinant adeno-associated virus serotype 5. Mean is (±SD). ∗p < 0.05 miAngE vs. vehicle; ∗∗p < 0.01 miAngE vs. vehicle; ∗∗∗p < 0.001 miAngE (up to week 12) and miAngE + atorvastatin vs. vehicle; ###p < 0.001 miAngE and miAngE + atorvastatin vs. miSCR;##p < 0.01 miAngE and miAngE + atorvastatin vs. miSCR;δp < 0.05 miSCR+ atorvastatin vs. vehicle; δδδp < 0.001 miSCR+ atorvastatin vs. vehicle; γp < 0.05 miSCR+ atorvastatin vs. miSCR;γγp < 0.001 miSCR+ atorvastatin vs. miSCR.

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  Figure 5. Lipid profiles for cholesterol and triglycerides in dyslipidemic APOE∗3-Leiden.CETP mice treated with vehicle, rAAV5-miSCR or rAAV5-miAngE (with or without atorvastatin) Lipid profiles for (A) cholesterol and (B) triglycerides in dyslipidemic APOE∗3-Leiden.CETP mice 16 weeks after treatment with vehicle control, moderate-high dose rAAV5-miAngE or rAAV5-miSCR, with and without atorvastatin. For (A) and (B), dots represent the mean value of the group per fraction (n = 9 per treatment group). Moderate-high dose (1 × 1014 gc/kg) of rAAV5-miAngE or rAAV5-miSCR. Atorvastatin dose 45 mg/kg. Tables in (A) and (B) give the area under the curve for fractions 3–8 VLDL, 9–17 LDL, and 18–24 HDL at t = 16 for indicating their cholesterol and triglycerides content, respectively, for each experimental treatment. HDL, high-density lipoprotein; LDL, low-density lipoprotein; rAAV5, recombinant adeno-associated virus serotype 5; TG, triglyceride; VLDL, very low-density lipoprotein.

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  Figure 6. Atherosclerotic lesion area and lesion severity in dyslipidemic APOE∗3-Leiden.CETP mice treated with vehicle, rAAV5-miSCR or rAAV5-miAngE (with or without atorvastatin) (A) Total atherosclerotic lesion area, (B) severity of atherosclerotic lesions, and (C) number of total lesions per cross-section in dyslipidemic APOE∗3-Leiden.CETP mice 16 weeks after treatment with vehicle control or moderate-high dose rAAV5-miAngE or rAAV5-miSCR, with or without atorvastatin treatment. Severity of atherosclerotic lesions is expressed as a percentage of all segments showing undiseased tissue or type I–V lesions. Representative pictures of a fatty streak, and mild and severe lesions are shown (original magnification ×20). Scale bar, 200 μm. Pink-purple are smooth muscle cells; yellow is connective tissue/collagen; white/translucent are lipid droplets in macrophages and cholesterol clefts. Statistical analyses were performed using a Kruskal-Wallis test followed by individual Mann-Whitney tests for group comparisons. Moderate-high dose (1 × 1014 gc/kg) of rAAV5-miAngE or rAAV5-miSCR. Atorvastatin dose 45 mg/kg rAAV5, recombinant adeno-associated virus serotype 5. Mean is (±SD). ∗p ≤ 0.05 vs.…