In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122

Abstract

Miravirsen is a β-D-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC50) of 0.67 μM. No cytotoxicity was observed up to the highest concentration tested (>320 μM) in different cell culture models, yielding a therapeutic index of ≥ 297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2'-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5' untranslated region (UTR) from cells passaged in the presence of up to 20 μM (40-fold the miravirsen EC50 concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5'UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. NCT01200420).

FIG 1

Miravirsen sodium (miravirsen) and the control compound SPC4729 both are 15-base oligonucleotides, comprising β-d-oxy-LNA and DNA monomers, respectively. Both molecules contain 8 LNA and 7 DNA nucleotides arranged in the sequences depicted. Capital letters denote LNA-modified nucleotides (^mC is LNA-5-methyl-cytidine), and lowercase letters denote DNA nucleotides. All of the internucleotide linkages, 14 in total, are phosphorothioate linkages, as indicated by the subscript s, and all LNAs are β-d-oxy-LNA, as indicated by the superscript o.

FIG 2

Analysis of the interaction of miravirsen with IFN-α2b (A), RBV (B), VX-222 (C), BMS-790052 (D), telaprevir (E), and 2′-MeC (F) by the method of Prichard and Shipman, Jr., using Macsynergy II, v1.0 (36). Eight successive 2-fold dilutions of miravirsen were evaluated alone or in all possible combinations with five successive 2- or 5-fold serial dilutions of a second compound. Calculated independent effects were subtracted from the observed combined effects. Volumes with positive values at the 95% confidence interval indicate synergy, while volumes with negative values indicate antagonism. Data are from one representative experiment. Conc., concentration.

FIG 3

HCV replicon genotype 1b cells were serially passaged in the presence of G418 alone (ctrl) or G418 with miravirsen (MIR), SPC4729 (oligonucleotide negative control), or telaprevir (TVR) for time periods of up to 35 days (TVR) or 148 days (MIR, SPC4729, or G418 alone) in fixed or escalating concentrations at a multiple (x) of the EC₅₀ concentrations of MIR (for MIR and SPC4729) or TVR. Colony formation, drug susceptibility assays, and nucleotide changes (*) were assessed throughout the study (d, days).

FIG 4

HCV replicon genotype 1b cells were passaged in the presence of G418 alone (medium control) or G418 with miravirsen, SPC4729 (oligonucleotide negative control), or telaprevir for 28 days in fixed concentrations at a multiple (X) of the EC₅₀ of miravirsen or telaprevir. Colony formation was assessed by staining surviving cells with crystal violet.

FIG 5

Subjects with chronic HCV genotype 1 infection were randomly assigned to receive five weekly subcutaneous injections of miravirsen at doses of 3 mg, 5 mg, or 7 mg per kilogram of body weight over a 29-day period (gray shading) (33). They were monitored for 18 weeks after randomization. Amplification and sequence analysis of the entire 5′UTR (nucleotides 1 to 341) were performed by 5′ RACE from six subjects (numbered 1 to 6) who experienced virologic rebound. Identification of the C3U nucleotide change (large open triangles) or wild-type sequences at position 3 (large open circles) are indicated. The lower limit of detection (LLOD) was 12 IU (1.08 log₁₀ IU per ml).