Development and characterization of DNAzyme candidates demonstrating significant efficiency against human rhinoviruses - 19/09/18

Doi : 10.1016/j.jaci.2018.07.026

Daniel P. Potaczek, MD, PhD ^a,^b,^c,^{∗
These authors contributed equally to this work.}, Sebastian D. Unger ^a,^b,^{∗
These authors contributed equally to this work.}, Nan Zhang, MD, PhD ^b,^d, Styliani Taka, PhD ^b,^e, Sven Michel, PhD ^f, Nesibe Akdağ, BSc ^a,^b, Feng Lan, PhD ^b,^d, Markus Helfer, PhD ^f, Christoph Hudemann, PhD ^a,^b, Markus Eickmann, PhD ^g, Chrysanthi Skevaki, MD, PhD ^a,^b, Spyridon Megremis, PhD ^b,^h, Anne Sadewasser, PhD ^f, Bilal Alashkar Alhamwe, BSc ^a,^b, Fahd Alhamdan, BSc ^a,^b, Mübeccel Akdis, MD, PhD ^b,^i,^j, Michael R. Edwards, PhD ^b,^k,^l, Sebastian L. Johnston, PhD ^b,^k,^l, Cezmi A. Akdis, MD ^b,^i,^j, Stephan Becker, PhD ^g, Claus Bachert, MD, PhD ^b,^d, Nikolaos G. Papadopoulos, PhD ^b,^e,^h, Holger Garn, PhD ^a,^b,^{‡
These authors contributed equally to this work.}, Harald Renz, MD ^a,^b,^⁎,^{‡
These authors contributed equally to this work.}
^a Institute of Laboratory Medicine, member of the German Center for Lung Research (DZL), Universities of Giessen and Marburg Lung Center (UGMLC), and the inVIVO Planetary Health, Group of the Worldwide Universities Network (WUN), Marburg, Germany
^g Institute of Virology, Philipps-University Marburg, Marburg, Germany
^b PreDicta Consortium
^c John Paul II Hospital, Krakow, Poland
^d Upper Airway Research Laboratory, Department of Otorhinolaryngology, Ghent University Hospital, Ghent, Belgium
^e Allergy and Clinical Immunology Unit, 2nd Pediatric Clinic, National and Kapodistrian University of Athens, Athens, Greece
^f Secarna Pharmaceuticals GmbH, Planegg, Germany
^h Division of Infection, Inflammation and Respiratory Medicine, University of Manchester, London, United Kingdom
ⁱ Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland
^j Christine Kühne-Center for Allergy Research and Education (CK-CARE), Davos, Switzerland
^k Airway Disease Infection Section, National Heart and Lung Institute (NHLI), Imperial College London, London, United Kingdom
^l Medical Research Council (MRC) and Asthma UK Centre in Allergic Mechanisms of Asthma, London, United Kingdom

^∗Corresponding author: Harald Renz, MD, Institute of Laboratory Medicine, Philipps-University Marburg, Baldinger Straße, 35043 Marburg, Germany.Institute of Laboratory MedicinePhilipps-University MarburgBaldinger StraßeMarburg35043Germany

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Abstract

Background

Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available.

Objective

We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study.

Methods

A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5′-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro–expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell–based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo–cultured human sinonasal tissue.

Results

Screening phase–generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency.

Conclusions

Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials.

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Graphical abstract

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Key words : Allergy, antisense, antiviral, asthma, atopy, DNAzyme, exacerbation, human rhinovirus, RNA, therapy

Abbreviations used : ALI, COPD, CRE, CRSwNP, DMEM, NF-κB, RV, TCID₅₀, TCM, TLR9, UTR

Plan

Methods

In vitro and in silico testing

Exclusion of cytotoxic and off-target effects

Antiviral efficacy testing in living cells and tissue samples

Results

Molecular development of anti-RV DNAzyme candidates

Exclusion of cytotoxic and off-target effects of Dua-1-L12R9 and Dua-2-L10R11

Antiviral efficiency of Dua-1-L12R9 in living human cells/tissues

Discussion

Supported by the PreDicta EC FP7 Collaborative Project (260895; the PreDicta Consortium members); Universities Giessen and Marburg Lung Center (UGMLC; to H.R., H.G., D.P.P., and C.S.); the German Center for Lung Research (DZL; 82DZL00502/A2; to H.R., H.G., D.P.P., and C.S.); the Von Behring-Röntgen-Foundation (Von Behring-Röntgen-Stiftung; to H.R., H.G., and D.P.P.); the German Academic Exchange Service (DAAD; to B.A.A., personal reference no. 91559386); HessenFonds, World University Service (WUS); the Hessen State Ministry for Higher Education, Research and the Arts (HMWK; to F.A.); the German Research Council (DFG; SFB1021, project C04); a Chair from Asthma UK (CH11SJ; to S.L.J.); and the Medical Research Council (MRC) Centre grant (G1000758; S.L.J. and M.R.E).

Disclosure of potential conflict of interest: S. Michel, M. Helfer, and A. Sadewasser are or were employees of Secarna Pharmaceuticals GmbH. H. Garn and H. Renz have consultant arrangements with and have received stock/stock options from Sterna Biologicals GmbH and Secarna Pharmaceuticals GmbH. The rest of the authors declare that they have no relevant conflicts of interest.