Inhibition of hepatitis C virus (HCV) replication by specific RNA aptamers against HCV NS5B RNA replicase

J Virol. 2013 Jun;87(12):7064-74. doi: 10.1128/JVI.00405-13. Epub 2013 Apr 17.

Abstract

This study identified specific and avid RNA aptamers consisting of 2'-hydroxyl- or 2'-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2'-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2'-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2'-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2'-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aptamers, Nucleotide / administration & dosage
  • Aptamers, Nucleotide / genetics*
  • Aptamers, Nucleotide / pharmacology
  • Binding, Competitive
  • Cell Line, Tumor
  • Hepacivirus / enzymology
  • Hepacivirus / genetics*
  • Hepacivirus / physiology
  • Hepatocytes / virology
  • Humans
  • Male
  • Mice
  • Mice, Inbred BALB C
  • RNA-Dependent RNA Polymerase / antagonists & inhibitors*
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism
  • SELEX Aptamer Technique
  • Viral Nonstructural Proteins / antagonists & inhibitors*
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism
  • Virus Replication / drug effects*
  • Virus Replication / genetics

Substances

  • Aptamers, Nucleotide
  • NS-5 protein, hepatitis C virus
  • Viral Nonstructural Proteins
  • RNA-Dependent RNA Polymerase