Efficient Suppression of Hepatitis C Virus Replication by Combination Treatment with miR-122 Antagonism and Direct-acting Antivirals in Cell Culture Systems.

Fanwei Liu,Kazuhisa Murai,Takayoshi Shirasaki,Seishi Murakami,Masao Honda,Minkyung Yi,Masaya Funaki,Hong Tang,Shuichi Kaneko,Tetsuro Shimakami

doi:10.1038/srep30939

Abstract

Direct-acting antivirals (DAAs) against Hepatitis C virus (HCV) show effective antiviral activity with few side effects. However, the selection of DAA-resistance mutants is a growing problem that needs to be resolved. In contrast, miR-122 antagonism shows extensive antiviral effects among all HCV genotypes and a high barrier to drug resistance. In the present study, we evaluated three DAAs (simeprevir, daclatasvir, and sofosbuvir) in combination with anti-miR-122 treatment against HCV genotype 1a in cell cultures. We found that combination treatments with anti-miR-122 and a DAA had additive or synergistic antiviral effects. The EC50 values of simeprevir in simeprevir-resistant mutants were significantly decreased by combining simeprevir with anti-miR-122. A similar reduction in EC50 in daclatasvir-resistant mutants was achieved by combining daclatasvir with anti-miR-122. Combination treatment in HCV-replicating cells with DAA and anti-miR-122 sharply reduced HCV RNA amounts. Conversely, DAA single treatment with simeprevir or daclatasvir reduced HCV RNA levels initially, but the levels later rebounded. DAA-resistant mutants were less frequently observed in combination treatments than in DAA single treatments. In summary, the addition of miR-122 antagonism to DAA single treatments had additive or synergistic antiviral effects and helped to efficiently suppress HCV replication and the emergence of DAA-resistant mutants.

Highlights

NS5A inhibitor RAVs are particular examples[9]
locked nucleic acid (LNA)–anti-miR-122 has been added to the cell culture medium in previous studies[22], we transfected it into cells because transfection is a more effective way to suppress Hepatitis C virus (HCV) replication[14]
We tested the effects of a non-relevant single-stranded RNA oligonucleotide as the LNA-RNA-Control on HCV replication

Summary

Introduction

NS5A inhibitor RAVs are particular examples[9]. RAVs can interfere with subsequent interferon-free DAA therapies because there are a limited number of DAA classes and RAVs sometimes show cross-drug resistance to the same class. Anti-miR-122 therapy is expected to show universal antiviral effects on all HCV genotypes because miR-122 binding sites at the 5′UTR are highly conserved among all HCV genotypes[18]. It provides a high barrier to the emergence of resistance because miR-122–binding negative HCV mutants are highly unfit in HCV cell culture systems[19]. Miravirsen (SPC3649) is a 15-base oligonucleotide with locked nucleic acid (LNA) modification complementary to part of miR-122 It is one of the most developed anti-miR-122 therapies and has been intravenously administered to African green monkeys[20]. When miravirsen was administered to HCV-infected chimpanzees, it effectively suppressed HCV RNA levels without any evidence of resistant mutants[17]. We explored whether these two antiviral therapies could compensate for each other’s weaknesses and effectively suppress HCV replication in HCV cell culture systems

Methods

Results

Conclusion