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

Autor: Tae Kyu Kim, Marwa Amer, Mona Kamar, Saehong Min, Nafisa M. Hassan, Suher Zada, Marc P. Windisch, Mahmoud ElHefnawi, Eman El-Ahwany, Hee-Young Kim
Rok vydání: 2016
Předmět:
0301 basic medicine
RNA viruses
Small interfering RNA
Time Factors
lcsh:Medicine
Hepacivirus
Viral Nonstructural Proteins
Virus Replication
Biochemistry
0302 clinical medicine
RNA interference
Small interfering RNAs
Replicon
RNA
Small Interfering
lcsh:Science
Luciferases
Conserved Sequence
Pathology and laboratory medicine
Internal Ribosome Entry Site
Multidisciplinary
Hepatitis C virus
Medical microbiology
Enzymes
Nucleic acids
RNA silencing
Genetic interference
030220 oncology & carcinogenesis
Viruses
Epigenetics
Pathogens
Oxidoreductases
Luciferase
Research Article
Genotype
Recombinant Fusion Proteins
Trans-acting siRNA
Green Fluorescent Proteins
Genome
Viral
Biology
Internal Ribosome Entry Sites
Transfection
Research and Analysis Methods
Antiviral Agents
Microbiology
RNAi Therapeutics
03 medical and health sciences
Cell Line
Tumor
Virology
Microbial Control
Genetics
Gene silencing
Humans
Computer Simulation
Non-coding RNA
Molecular Biology Techniques
Molecular Biology
Medicine and health sciences
Pharmacology
Binding Sites
Biology and life sciences
Flaviviruses
lcsh:R
Organisms
Viral pathogens
RNA
Reproducibility of Results
Proteins
Viral Replication
Hepatitis viruses
Gene regulation
Microbial pathogens
030104 developmental biology
Enzymology
Nucleic Acid Conformation
lcsh:Q
Gene expression
Antimicrobial Resistance
Zdroj: PLoS ONE
PLoS ONE, Vol 11, Iss 7, p e0159211 (2016)
ISSN: 1932-6203
Popis: 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.
Databáze: OpenAIRE
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