Downloaded from www.microbiologyresearch.org by IP: 54.224.212.167 On: Mon, 08 Aug 2016 23:23:35 Morpholino oligomers targeting the PB1 and NP genes enhance the survival of mice infected with highly pathogenic influenza A H7N7 virus Gu ¨ lsah Gabriel, 1 3 Alexandra Nordmann, 1 David A. Stein, 2 Patrick L. Iversen 2 and Hans-Dieter Klenk 1 Correspondence Gu ¨ lsah Gabriel guelsah.gabriel@path.ox.ac.uk 1 Institute of Virology, Philipps University Marburg, Germany 2 AVI BioPharma Inc., Corvallis, OR, USA Received 17 September 2007 Accepted 29 November 2007 Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded nucleic acid-analogue antisense agents that enter cells readily and can reduce gene expression by steric blocking of complementary RNA (cRNA) sequences. Here, we tested a panel of PPMO designed to target conserved sequences in the RNA genome segments encoding polymerase subunits of a highly pathogenic mouse-adapted influenza A virus (SC35M; H7N7). Three PPMO, targeting the translation start site region of PB1 or NP mRNA or the 39-terminal region of NP viral RNA (vRNA), potently inhibited virus replication in MDCK cells. Primer extension assays showed that treatment with any of the effective PPMO led to markedly reduced levels of mRNA, cRNA and vRNA. Initially, the potential toxicity of a range of intranasally administered PPMO doses was evaluated, by measuring their effect on body weight of uninfected mice. Subsequently, a non-toxic dosing regimen was used to investigate the effect of various PPMO on SC35M infection in a mouse model. Mice administered intranasal treatment of PPMO targeting the PB1-AUG region or NP vRNA, at 3 mg per dose, given once 3 h before and once 2 days after intranasal infection with 10¾LD 50 of SC35M, showed a 2 log 10 reduction of viral titre in the lungs and 50 % survival for the 16 day duration of the experiment, whereas the NP-AUG-targeted PPMO treatment resulted in 30 % survival of an otherwise lethal infection. These data suggest that PPMO provide a useful reagent to investigate influenza virus molecular biology and may constitute a therapeutic strategy against highly pathogenic influenza viruses. INTRODUCTION Influenza A viruses belong to the family Orthomyxoviridae and possess eight RNA genome segments (PB1, PB2, PA, NP, HA, M, NS and NA) of negative polarity, all of which are encapsidated by the nucleoprotein NP. All influenza virus RNA synthesis is carried out in the nucleus of the infected cell and is mediated by the viral polymerase subunits (PB1, PB2 and PA) and NP (Elton et al., 2006; Neumann et al., 2004). The influenza virus strain SC35 of the H7N7 subtype is highly pathogenic to chickens but has low pathogenicity in mice (Gabriel et al., 2005; Li et al., 1990). SC35 was passaged several times in mice to produce SC35M, which is highly pathogenic to mice (Gabriel et al., 2005; Scheiblauer et al., 1995). Recently, we showed that the high pathogenicity of SC35M is due primarily to mutations in the genes of the polymerase complex. Recombinant SC35 viruses with single SC35M-specific mutations in the PB1, PB2, PA and NP genes correlated with high polymerase activity in mammalian cells and high virulence in mice (Gabriel et al., 2005). Therefore, we considered it of interest to investigate the effects of reducing the levels of SC35M polymerase and NP components on SC35M pathogenicity to mice. There is increasing evidence that strains of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 and H7N7 subtypes have been transmitted directly from birds to humans (Claas et al., 1998; Fouchier et al., 2004; Subbarao et al., 1998), and pose a pandemic threat. As there is no vaccine currently available against these HPAIV subtypes, the development of a therapeutic strategy employing the targeting of viral gene sequence that is highly conserved between influenza A virus subtypes and strains is generally acknowledged as highly desirable. Phosphorodiamidate morpholino oligomers (PMO) are antisense agents that are structurally similar to single- stranded DNA. Each base is joined to a novel backbone consisting of a morpholine ring and phosphorodiamidate intersubunit linkage (Summerton & Weller, 1997). PMO 3Present address: Sir William Dunn School of Pathology, University of Oxford, UK. Supplementary material is available with the online version of this paper. Journal of General Virology (2008), 89, 939–948 DOI 10.1099/vir.0.83449-0 0008-3449 G 2008 SGM Printed in Great Britain 939