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In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes

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...

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Autores principales: ElHefnawi, Mahmoud, Kim, TaeKyu, Kamar, Mona A., Min, Saehong, Hassan, Nafisa M., El-Ahwany, Eman, Kim, Heeyoung, Zada, Suher, Amer, Marwa, Windisch, Marc P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956106/
https://www.ncbi.nlm.nih.gov/pubmed/27441640
http://dx.doi.org/10.1371/journal.pone.0159211
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author ElHefnawi, Mahmoud
Kim, TaeKyu
Kamar, Mona A.
Min, Saehong
Hassan, Nafisa M.
El-Ahwany, Eman
Kim, Heeyoung
Zada, Suher
Amer, Marwa
Windisch, Marc P.
author_facet ElHefnawi, Mahmoud
Kim, TaeKyu
Kamar, Mona A.
Min, Saehong
Hassan, Nafisa M.
El-Ahwany, Eman
Kim, Heeyoung
Zada, Suher
Amer, Marwa
Windisch, Marc P.
author_sort ElHefnawi, Mahmoud
collection PubMed
description 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.
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spelling pubmed-49561062016-08-08 In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes ElHefnawi, Mahmoud Kim, TaeKyu Kamar, Mona A. Min, Saehong Hassan, Nafisa M. El-Ahwany, Eman Kim, Heeyoung Zada, Suher Amer, Marwa Windisch, Marc P. PLoS One Research Article 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. Public Library of Science 2016-07-21 /pmc/articles/PMC4956106/ /pubmed/27441640 http://dx.doi.org/10.1371/journal.pone.0159211 Text en © 2016 ElHefnawi et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
ElHefnawi, Mahmoud
Kim, TaeKyu
Kamar, Mona A.
Min, Saehong
Hassan, Nafisa M.
El-Ahwany, Eman
Kim, Heeyoung
Zada, Suher
Amer, Marwa
Windisch, Marc P.
In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes
title In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes
title_full In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes
title_fullStr In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes
title_full_unstemmed In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes
title_short In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes
title_sort in silico design and experimental validation of sirnas targeting conserved regions of multiple hepatitis c virus genotypes
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956106/
https://www.ncbi.nlm.nih.gov/pubmed/27441640
http://dx.doi.org/10.1371/journal.pone.0159211
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