title: Selection, optimization, and pharmacokinetic properties of a novel, potent antiviral locked nucleic acid-based antisense oligomer targeting hepatitis C virus internal ribosome entry site.
abstract: We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC(50)]) and cytotoxic (50% cytotoxic concentration [CC(50)]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC(50) = 4 nM) and low cytotoxicity (CC(50) >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.
authors.affiliation: Internal Medicine, Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, United Kingdom.
authors.forename: Carl
authors.initials: C
authors.lastname: Laxton
authors.forename: Kevin
authors.initials: K
authors.lastname: Brady
authors.forename: Sterghios
authors.initials: S
authors.lastname: Moschos
authors.forename: Paul
authors.initials: P
authors.lastname: Turnpenny
authors.forename: Jaiessh
authors.initials: J
authors.lastname: Rawal
authors.forename: David C
authors.initials: DC
authors.lastname: Pryde
authors.forename: Ben
authors.initials: B
authors.lastname: Sidders
authors.forename: Romu
authors.initials: R
authors.lastname: Corbau
authors.forename: Chris
authors.initials: C
authors.lastname: Pickford
authors.forename: E J
authors.initials: EJ
authors.lastname: Murray
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000493:pharmacokinetics* / Q000494:pharmacology*; D002460:Cell Line; D016174:Hepacivirus / Q000187:drug effects*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics* / Q000494:pharmacology*; D012270:Ribosomes / Q000821:virology*
chemical_list: D000998:Antiviral Agents; D016376:Oligonucleotides, Antisense