title: In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes.
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.
authors.affiliation: Informatics and Systems Department, Biomedical Informatics and Chemo-Informatics Group, Centre of Excellence for Advanced Sciences (CEAS), Division of Engineering Research, National Research Centre, Cairo, Egypt.|Centre for Informatics, Nile University, Shiekh Zayed City, Egypt.|Yousef-Jameel Science and Technology Research Centre, American University in Cairo, New Cairo, Egypt.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: ElHefnawi
authors.affiliation: Hepatitis Research Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.
authors.forename: TaeKyu
authors.initials: T
authors.lastname: Kim
authors.affiliation: Yousef-Jameel Science and Technology Research Centre, American University in Cairo, New Cairo, Egypt.
authors.forename: Mona A
authors.initials: MA
authors.lastname: Kamar
authors.affiliation: Hepatitis Research Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.
authors.forename: Saehong
authors.initials: S
authors.lastname: Min
authors.affiliation: Yousef-Jameel Science and Technology Research Centre, American University in Cairo, New Cairo, Egypt.
authors.forename: Nafisa M
authors.initials: NM
authors.lastname: Hassan
authors.affiliation: Biology Department, American University in Cairo, New Cairo, Egypt.|Immunology Department, Theodor Bilharz Research Institute, Giza, Egypt.
authors.forename: Eman
authors.initials: E
authors.lastname: El-Ahwany
authors.affiliation: Hepatitis Research Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.
authors.forename: Heeyoung
authors.initials: H
authors.lastname: Kim
authors.affiliation: Yousef-Jameel Science and Technology Research Centre, American University in Cairo, New Cairo, Egypt.|Biology Department, American University in Cairo, New Cairo, Egypt.
authors.forename: Suher
authors.initials: S
authors.lastname: Zada
authors.affiliation: Biology Department, American University in Cairo, New Cairo, Egypt.|Faculty of Biotechnology, Misr University for Science and Technology, 6th of October City, Egypt.
authors.forename: Marwa
authors.initials: M
authors.lastname: Amer
authors.affiliation: Hepatitis Research Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.
authors.forename: Marc P
authors.initials: MP
authors.lastname: Windisch
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D001665:Binding Sites; D045744:Cell Line, Tumor; D003198:Computer Simulation; D017124:Conserved Sequence / Q000235:genetics*; D016679:Genome, Viral; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D000067556:Internal Ribosome Entry Sites / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D034741:RNA, Small Interfering / Q000627:therapeutic use*; D000067492:RNAi Therapeutics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012093:Replicon / Q000235:genetics; D015203:Reproducibility of Results; D013997:Time Factors; D017361:Viral Nonstructural Proteins; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D049452:Green Fluorescent Proteins; D000067556:Internal Ribosome Entry Sites; D008156:Luciferases; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase