Effective prevention and therapy of experimental allergic asthma using a GATA-3– specific DNAzyme Serdar Sel, MD, Michael Wegmann, PhD, Tanja Dicke, MSc,

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Effective prevention and therapy of experimental allergic asthma using a GATA-3– specific DNAzyme Serdar Sel, MD, Michael Wegmann, PhD, Tanja Dicke, MSc, Sarper Sel, MD, Wolfgang Henke, PhD, Ali Ö. Yildirim, PhD, Harald Renz, MD, Holger Garn, PhD Journal of Allergy and Clinical Immunology Volume 121, Issue 4, Pages 910-916.e5 (April 2008) DOI: 10.1016/j.jaci.2007.12.1175 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Structure and in vitro cleavage activity of the DNAzyme gd21. A, DNAzyme gd21 with its target sequence in the GATA-3 mRNA. B, Gd21 and hgd40 cleave murine GATA-3 mRNA, but not the control DNAzyme ODNg. C, Gd21 cleaves GATA-3 but not T-bet, BDNF, or NGF mRNA. D, Gd21 has no unspecific cleavage activity on total RNA. Gd21 activity is time-dependent (E) and dose-dependent (F). Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Antisense molecules against GATA-3 inhibit cellular inflammatory response in the airways of allergic mice and improve AHR. A, Numbers of white blood cells were quantified in the BAL. B, Goblet cells were counted in lungs sections. C, IL-5, IFN-γ, and MCP-1 concentrations were analyzed in the BAL. D, Lung function was analyzed by head-out body plethysmography. E, Representative HE-stained airways are shown. nd, Not detectable; OVA, ovalbumin. Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Surfactant facilitates the uptake of gd21 into lung epithelial cells. The percentage of A549 cells transfected with fluorescence-labeled gd21 using Alveofact or DOTAP was determined by FACS analysis. DOTAP, N-[1-(2,3-dioleoyloxy)]-N, N, N-trimethylammonium propane. Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Dose-response relationship of the activity of the GATA-3 DNAzyme gd21 on parameters of an already established allergic inflammatory response. The total number of BAL eosinophils (A) and airway reactivity to methacholine (B) were determined. C, Representative HE-staining (upper panel) or PAS staining (lower panel) airways are shown. OVA, Ovalbumin. Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Gd21 inhibits cellular inflammatory response in the airways and improves AHR in a chronic asthma mouse model. A, Numbers of eosinophils were quantified in BAL fluid. B, Mucin volumes (Vmucin) were assessed morphometrically and related to basal membrane area (Sep). C, Lung function was analyzed by head-out body plethysmography. D, Representative lung histologies of PAS-stained airways are shown. E, Collagen fibrils are shown in representative lung sections. OVA, Ovalbumin. Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Antisense treatment protocols Antisense treatment protocols. BALB/c mice were sensitized to ovalbumin by intraperitoneal injections of ovalbumin adsorbed to alum (Al[OH]3) followed by inhalational challenges with an 1% ovalbumin aerosol to induce allergic airway inflammation. Treatment using antisense molecules (200 μg) was performed by i.n. application. Treatment was performed in an acute asthma model (A), during a secondary allergen response (B), only during a first set of 2 ovalbumin challenges (C), and in a chronic asthma model (D). Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Gd21 and hgd40 lead to a decrease of GATA-3 mRNA and to an increase of T-bet mRNA expression. EL-4, a murine T-cell line, was transfected with gd21 or hgd40 by using DOTAP. The relative expression of GATA-3 and T-bet mRNA was determined by using real-time PCR (LightCycler; Roche, Mannheim, Germany) after sample normalization using the housekeeping gene L32. The GATA-3 and T-bet mRNA expression values from DOTAP control were set at 100%. Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Gd21 reduces IL-5 and IL-13 and increases IFN-γ production in an acute model of allergic asthma. Cytokine levels were determined in cell-free BAL fluid. nd, Not detectable; OVA, ovalbumin. Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Hgd40—but not the control DNAzyme ODNg—inhibits airway inflammation in a mouse model of acute allergic asthma (Fig E1). A, Differential leukocyte counts were quantified in the BAL. B, Representative HE-stained airways are shown. nd, Not detectable; OVA, ovalbumin. Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Long-term effect of gd21 in a murine model of experimental asthma Long-term effect of gd21 in a murine model of experimental asthma. Ovalbumin (OVA)-sensitized mice were intranasally treated with gd21 during the first round of OVA challenges. After 2 weeks, a second set of challenges was performed without treatment. Shown are means ± SEMs of the total number of BAL eosinophils (A) and airway reactivity to methacholine (B). Journal of Allergy and Clinical Immunology 2008 121, 910-916.e5DOI: (10.1016/j.jaci.2007.12.1175) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions