title: Morpholino oligomers targeting the PB1 and NP genes enhance the survival of mice infected with highly pathogenic influenza A H7N7 virus.
abstract: 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 3'-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 mug per dose, given once 3 h before and once 2 days after intranasal infection with 10xLD(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.
authors.affiliation: Institute of Virology, Philipps University Marburg, Germany.
authors.forename: Gülsah
authors.initials: G
authors.lastname: Gabriel
authors.affiliation: Institute of Virology, Philipps University Marburg, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Nordmann
authors.affiliation: AVI BioPharma Inc., Corvallis, OR, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Stein
authors.affiliation: AVI BioPharma Inc., Corvallis, OR, USA.
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Iversen
authors.affiliation: Institute of Virology, Philipps University Marburg, Germany.
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Klenk
mesh_terms: D000281:Administration, Intranasal; D000818:Animals; D000890:Anti-Infective Agents / Q000008:administration & dosage / Q000138:chemical synthesis / Q000627:therapeutic use* / Q000633:toxicity; D002460:Cell Line; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004334:Drug Administration Schedule; D004353:Drug Evaluation, Preclinical; D018390:Gene Targeting; D005814:Genes, Viral / Q000235:genetics; D053126:Influenza A Virus, H7N7 Subtype; D009980:Influenza A virus / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009025:Morpholines / Q000008:administration & dosage / Q000138:chemical synthesis / Q000627:therapeutic use* / Q000633:toxicity; D060172:Morpholinos; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000187:drug effects* / Q000235:genetics; D009976:Orthomyxoviridae Infections / Q000517:prevention & control*; D010455:Peptides / Q000008:administration & dosage / Q000138:chemical synthesis / Q000627:therapeutic use*; D016601:RNA-Binding Proteins / Q000187:drug effects* / Q000235:genetics; D014758:Viral Core Proteins / Q000187:drug effects* / Q000235:genetics; D014764:Viral Proteins / Q000187:drug effects* / Q000235:genetics / Q000633:toxicity
chemical_list: D000890:Anti-Infective Agents; D009025:Morpholines; D060172:Morpholinos; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010455:Peptides; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1