In silico design and experimental validation of sirnas targeting conserved regions of multiple hepatitis c virus genotypes

Theodor Bilharz Research Institute

Bibliographic Information

Authors: ElHefnawi M.; Kim T.; Kamar M.A.; Min S.; Hassan N.M.; El-Ahwany E.; Kim H.; Zada S.; Amer M.; Windisch M.P.

Journal: PLoS ONE

Publisher: Public Library of Science

Publication Date: 21 July 2016

Volume / Issue: Volume 11 / Issue 7

Article No.: e0159211

ISSN: 19326203

DOI: 10.1371/journal.pone.0159211

Scopus: View on Scopus

PubMed: 27441640

Document Type: Article

Access: All Open Access; Gold Open Access; Green Open Access

Authors and Affiliations

ElHefnawi M., Informatics and Systems Department, Biomedical Informatics and Chemo-Informatics Group, Ctr. of Excellence for Adv. Sci. (CEAS), Division of Engineering Research, National Research Centre, Cairo, Egypt, Centre for Informatics, Nile University, Shiekh Zayed City, Egypt, Yousef-Jameel Science, Technology Research Centre, American University, Cairo, New Cairo, Egypt; Kim T., Hepatitis Research Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea; Kamar M.A., Yousef-Jameel Science, Technology Research Centre, American University, Cairo, New Cairo, Egypt; Min S., Hepatitis Research Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea; Hassan N.M., Yousef-Jameel Science, Technology Research Centre, American University, Cairo, New Cairo, Egypt; El-Ahwany E., Biology Department, American University, Cairo, New Cairo, Egypt, Immunology Department, Theodor Bilharz Research Institute, Giza, Egypt; Kim H., Hepatitis Research Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea; Zada S., Yousef-Jameel Science, Technology Research Centre, American University, Cairo, New Cairo, Egypt, Biology Department, American University, Cairo, New Cairo, Egypt; Amer M., Biology Department, American University, Cairo, New Cairo, Egypt, Faculty of Biotechnology, Misr University for Science and Technology, 6th of October City, Egypt; Windisch M.P., Hepatitis Research Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea

Abstract

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide interventions. © 2016 ElHefnawi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Keywords

Antiviral Agents; Binding Sites; Cell Line, Tumor; Computer Simulation; Conserved Sequence; Genome, Viral; Genotype; Green Fluorescent Proteins; Hepacivirus; Humans; Internal Ribosome Entry Sites; Luciferases; Nucleic Acid Conformation; Recombinant Fusion Proteins; Replicon; Reproducibility of Results; RNA, Small Interfering; RNAi Therapeutics; Time Factors; Viral Nonstructural Proteins; Virus Replication; messenger RNA; small interfering RNA; antivirus agent; green fluorescent protein; hybrid protein; internal ribosome entry site; luciferase; NS-5 protein, hepatitis C virus; viral protein; antiviral activity; Article; binding site; computer aided design; computer model; controlled study; drug design; drug efficacy; drug potency; drug targeting; genome analysis; Hepatitis C virus; measurement accuracy; nonhuman; prediction; replicon cell; RNA binding; RNA interference; RNA replication; validation study; virus genome; virus inhibition; virus mutant; conformation; drug effects; genetics; human; metabolism; reproducibility; time factor; tumor cell line

Citation Information

Scopus Citations: 30

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