Antisense Oligonucleotides Targeting Influenza A Segment 8 Genomic RNA Inhibit Viral Replication

Abstract

Influenza A virus (IAV) affects 5%-10% of the world's population every year. Through genome changes, many IAV strains develop resistance to currently available anti-influenza therapeutics. Therefore, there is an urgent need to find new targets for therapeutics against this important human respiratory pathogen. In this study, 2'-O-methyl and locked nucleic acid antisense oligonucleotides (ASOs) were designed to target internal regions of influenza A/California/04/2009 (H1N1) genomic viral RNA segment 8 (vRNA8) based on a base-pairing model of vRNA8. Ten of 14 tested ASOs showed inhibition of viral replication in Madin-Darby canine kidney cells. The best five ASOs were 11-15 nucleotides long and showed inhibition ranging from 5- to 25-fold. In a cell viability assay they showed no cytotoxicity. The same five ASOs also showed no inhibition of influenza B/Brisbane/60/2008 (Victoria lineage), indicating that they are sequence specific for IAV. Moreover, combinations of ASOs slightly improved anti-influenza activity. These studies establish the accessibility of IAV vRNA for ASOs in regions other than the panhandle formed between the 5' and 3' ends. Thus, these regions can provide targets for the development of novel IAV antiviral approaches.

Keywords: antisense oligonucleotides; genomIc RNA; influenza RNA.

FIG. 1.

Targets of ASOs in predicted secondary structure of vRNA8 of influenza A/California/04/2009 (H1N1). Purple lines indicate regions targeted by ASOs. The nomenclature consists of vRNA8 nucleotide number complementary to the middle nucleotide of ASOs, length of ASOs, and containing modification: M = 2′-O-methyl-RNA and L = 2′-O-methyl-RNA with LNA. Note that relative to influenza A/Vietnam/1203/2004 (H5N1) (Supplementary Fig. S1) there is a 15 nucleotide insert between 614 and 615 so that the nucleotide numbers after 615 are larger by 15 relative to those for A/Vietnam/1203/2004 (H5N1). The numbering of vRNA8 is from its 5′ end. A consistent base pair is one where GU replaces a Watson–Crick pair or vice versa.

FIG. 2.

Antiviral activity of 230 nM ASOs in MDCK cells. Logarithm of average titer of influenza in CCS was analyzed by the immunofocus assay (FFU/mL): (A) for A/California/04/2009 after 36 h postinfection and (B) for B/Brisbane/60/2008 after 48 h postinfection. The mean was calculated from three independent experiments each containing three technical repeats. Numbers above bars indicate the log₁₀ differences between control virus titer from cells treated with Lipofectamine^® 2000 only (LPF) and with particular ASO added. Statistics were calculated using two-tailed T-test (*P < 0.05; **P < 0.01; ***P < 0.001). No statistically significant differences in virus titer were observed for influenza B/Brisbane/60/2008. CCS, cell culture supernatants; MDCK, Madin-Darby canine kidney.

FIG. 3.

Dose-dependent antiviral activity of ASOs against influenza A/California/04/2009 (H1N1) in MDCK cells. Logarithm of average titer of influenza A/California/04/2009 (H1N1) in CCS was analyzed after 36 h postinfection by immunofocus assay (FFU/mL). The mean was calculated from three independent experiments each containing three technical repeats. In this study, 230 and 115 nM concentrations of oligonucleotides were tested. Numbers above bars indicate the log₁₀ differences between control virus titer from cells treated with Lipofectamine 2000 only (LPF) and with a particular ASO added. Statistics for comparison to LPF and for effect of concentration were calculated using two-tailed T-test (*P < 0.05; **P < 0.01; ***P < 0.001 for comparison to LPF and ^#P < 0.05 for comparison of concentrations).

FIG. 4.

Antiviral activity of combinations of ASOs against influenza A/California/04/2009 (H1N1) in MDCK cells. Logarithm of average titer of influenza A/California/04/2009 in CCS was analyzed after 36 h postinfection by immunofocus assay (FFU/mL). The mean was calculated from three independent experiments each containing three technical repeats. In this study, 230 nM total concentration of oligonucleotides was tested with equal concentration of each ASO. Numbers above bars indicate the log₁₀ differences between control virus titer from cells treated with Lipofectamine 2000 (LPF) only and a particular ASO. Statistics were calculated for differences between 230 nM of 867-14L only and 230 nM total ASO concentration for combinations of ASOs using two-tailed T-test (*P < 0.05).