To address the problem of poor asthma control due to drug resistance, an antisense oligonucleotide complementary to mmu-miR-145a-5p (antimiR-145) was tested in a house dust mite mouse model of mild/moderate asthma. miR-145 was targeted to reduce inflammation, regulate epithelial-mesenchymal transitions, and promote differentiation of structural cells. In addition, several chemical variations of a nontargeting oligonucleotide were tested to define sequence-dependent effects of the miRNA antagonist. After intravenous administration, oligonucleotides complexed with a pegylated cationic lipid nanoparticle distributed to most cells in the lung parenchyma but were not present in smooth muscle or the mucosal epithelium of the upper airways. Treatment with antimiR-145 and a nontargeting oligonucleotide both reduced eosinophilia, reduced obstructive airway remodeling, reduced mucosal metaplasia, and reduced CD68 immunoreactivity. Poly(A) RNA-seq verified that antimiR-145 increased levels of many miR-145 target transcripts. Genes upregulated in human asthma and the mouse model of asthma were downregulated by oligonucleotide treatments. However, both oligonucleotides significantly upregulated many genes of interferon signaling pathways. These results establish effective lung delivery and efficacy of locked nucleic acid/DNA oligonucleotides administered intravenously, and suggest that some of the beneficial effects of oligonucleotide therapy of lung inflammation may be due to normalization of interferon response pathways.
Muc5ac; RNA-seq; Staramine; TheraSilence; chemokine; cytokine; gene expression; locked nucleic acid.
Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.
Delivery of antimiR-145 ASO to Lung Tissues in a HDM Model of Mild/Moderate Asthma (A) The HDM asthma model was adapted from Collison et al. The protocol had 3 phases: (1) Three-day sensitization beginning on day 0 to promote atopy; (2) three doses of 2 mg/kg antimiR-145 or solvent control (5% dextrose in 0.9% saline) every other day beginning on day 13; and (3) four daily challenges with HDM extracts beginning on day 14. On day 18, blood was collected for antibody assays, bronchioalveolar lavage fluids were collected for immune cell analysis, and lung tissue was collected for histologic analysis and extraction of RNA and protein. Naive (unsensitized) animals were age matched and treated with saline instead of HDM extract. Serum HDM-specific serum IgG
1 and IgE were significantly increased after allergic sensitization. Total IgG and IgE were not significantly changed after HDM sensitization and challenge (data not shown). Data are mean ± SEM; Student’s t test; n = 5–7. (B) The distribution of antimiR-145 in lung tissue was determined by ISH. Sections of the left lung lobe of HDM-sensitized mice were probed with a DIG-labeled LNA/DNA oligonucleotide complementary to antimiR-145. Positive tissues were visualized with anti-DIG antibodies conjugated to AP and were counterstained with Nuclear Fast Red. Sections from a sensitized mouse treated with antimiR-145/TheraSilence are shown at 10×, 20×, and 40× magnifications (top panels). A lung section from a dextrose-treated, HDM-sensitized mouse (lower left panel) was negative, as was a section from a sensitized mouse treated with a nontargeting oligonucleotide (lower right panel). Images are representative examples of sections from n = 9–18 mice per treatment group. (C) Treatment with antimiR-145 reduced mature miR-145 levels in lung. qRT-PCR of miRNA-145 was performed on total RNA from mouse lungs. Sets of mice were unsensitized, sensitized with HDM and treated with dextrose (HDM), sensitized and treated with antimiR-145, or sensitized and treated with nontargeting control oligonucleotide. Statistical analysis was performed by one-way ANOVA with Dunnett’s post hoc test using the HDM-sensitized treatment as the reference group; n = 9–13.
AntimiR-145 Treatment Reduces Immune Cells in BALF Cells from BAL fluid (BALF) were counted by analytical flow cytometry. (A) The total number of CD45+ cells is shown. (B–D) Other panels show effects on eosinophils (B), neutrophils (C), and alveolar macrophages (D). Statistical hypothesis testing was performed with Kruskal-Wallis ANOVA and Dunn’s post hoc test using counts of HDM-sensitized treated animals as the reference. *p < 0.05; **p < 0.01; ***p < 0.001. n = 10–18.
AntimiR-145 Treatment Reduces Lung Tissue Inflammation (A) FFPE lung sections were stained with H&E to identify sites of inflammation (purple clusters). Representative micrographs for each treatment group. Volume density was calculated by measuring areas of inflammation and normalizing to lung lobe cross-sectional area. Data are indicated as geometric mean ± 95% CI. Statistical hypothesis testing was performed using Kruskal-Wallis ANOVA and Dunn’s post hoc test, with the HDM-sensitized treatment as the reference group. **p < 0.005; ***p < 0.001. n = 9–17. (B) Treatment with antimiR-145 reduces CD68+ tissue macrophages. FFPE lung lobe sections were stained with anti-CD68 antibody and counterstained with hematoxylin to identify tissue macrophages (brown). A representative micrograph for each treatment group is shown. CD68+ cells were counted in images covering an entire lung section for each mouse. The number of CD68+ cells was normalized to lung lobe cross-sectional area. Statistical hypothesis testing was performed with Kruskal-Wallis ANOVA and Dunn’s post hoc test. *p < 0.05; ***p < 0.01. n = 10–18. (C) Sirius red staining identifies eosinophils (bright pink with purple bilobular nucleus) at sites of lung inflammation. Eosinophils are indicated by red arrowheads in the enlarged inset. The number of eosinophils was counted in all fields of each lung section for each animal and normalized to lung lobe cross-sectional area. Statistical hypothesis testing was performed by Kruskal-Wallis ANOVA and Dunn’s post hoc test; ***p < 0.001. n = 3–18.
Oligonucleotide Treatments Reduce Mucosal Metaplasia FFPE lung sections were stained with Periodic Acid Schiff stain to identify mucus-producing cells (magenta). (A) A representative micrograph for each treatment group illustrates staining in airways with a diameter of 150–200 μm. (B and C) Mucin density was measured by a point count method, and results were stratified by airway diameter: <100 μm (data not shown), 100–300 μm (B), 300–600 μm (C), and airways >600 μm (data not shown). Significant treatment effects were observed in airways with diameters between 100 and 600 μm. Statistical hypothesis testing was performed with Kruskal-Wallis ANOVA and Dunn’s post hoc test comparing all treatments to HDM-sensitized animals. *p < 0.05; **p < 0.01; ***p <0.005. n = 10–18 mice.
AntimiR-145 Upregulates mmu-miR-145a-5p Targets in HDM-Sensitized Mice Deseq2 analysis followed by gene set enrichment analysis (GSEA) was used to assess sequence-dependent effects of antimir-145 on mmu-miR-145a-5p target genes. Mouse miR-145a-5p targets were identified
in silico (DIANA-microT-CDS). (A) Expression of miR-145 target genes is reduced by HDM sensitization when compared to unsensitized controls. NES = −1.9, p < 0.01. n = 5 for unsensitized mice, and n = 10 for HDM-sensitized mice. (B) Expression of miR-145 target genes in lungs of HDM-sensitized mice is enhanced by antimiR-145 treatment (black circles); NES = 2.1, p < 0.001. n = 10 for HDM-sensitized and HDM-sensitized plus antimir-145 groups. A nontargeting LNA/DNA oligonucleotide did not significantly alter the expression of miR-145 target genes in HDM-sensitized mice (blue circles). n = 5 for nontargeting-oligonucleotide-treated mice.
HDM-Enhanced Expression of Asthma-Related Gene Expression Is Antagonized by antimiR-145 and a Nontargeting Oligonucleotide (A) Gene set enrichment analysis (GSEA) was performed on poly A RNA-seq data from whole lung samples. Left panel, asthma-related gene sets were compared between HDM-sensitized mice and sensitized mice treated with AM145 (blue bars). Asthma-related genes were also compared in HDM-sensitized mice versus sensitized mice treated with non-targeting oligonucleotide (yellow bars). Right panel, asthma-related gene expression in HDM-sensitized mice compared to age-matched unsensitized control mice (red bars). Normalized enrichment scores were significant for all gene sets; p < 0.001, n = 5-10. (B) Expression of human asthma biomarkers was assessed using rlog normalized counts from DESeq2. Data are expressed as mean counts +/− 95% confidence intervals; * − p < 0.05, One-way ANOVA on log transformed data, Dunn’s post hoc test with HDM-sensitized treatment as the reference group, n = 5-10.
AntimiR-145 Upregulates Sets of Interferon-Regulated Genes Gene set enrichment analysis (GSEA) was conducted to demonstrate differential gene expression in HDM-sensitized mice and HDM-sensitized mice treated with antimiR-145. HDM-sensitized mice served as the reference group for analysis of poly(A) RNA-seq data. A ranked set of differentially expressed genes (FDR < 0.2) was generated using DESeq2, and GSEA was performed using sets of interferon-regulated genes included in the Hallmark dataset of the MSigDB collection. (A) AntimiR-145 (black symbols) and nontargeting oligonucleotide (blue symbols) both significantly increased expression of most interferon-alpha-regulated genes (p < 0.001). (B) Treatments with antimiR-145 and nontargeting oligonucleotide also significantly enhanced interferon-beta-regulated genes (p < 0.001; n = 10 for each treatment).
Oligonucleotide Treatment Reduces Immune Cell Infiltration into BALF (A–D) Differential counts of immune cells in BAL fluid (BALF) were performed by flow cytometry: (A) total CD45+ cells, (B) eosinophils, (C) neutrophils, and (D) alveolar macrophages. Statistical hypothesis testing was performed by Kruskal-Wallis ANOVA and Dunn’s post hoc test. *p < 0.05; ***p < 0.005; ****p < 0.001. n = 10–18 mice. (E) Oligonucleotide treatment reduces tissue inflammation. Sections of FFPE lung were stained with H&E to identify sites of inflammation (purple clusters). A representative micrograph for each treatment group is shown. (F) Volume density of inflamed sites was quantified by measuring the area of each site divided by the total cross-sectional area of each lobe. Statistical hypothesis testing was performed with Kruskal-Wallis ANOVA and Dunn’s post hoc test using HDM-sensitized mice as the reference group. ***p < 0.005. n = 10–17.
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