title: In vitro resistance selection and in vivo efficacy of morpholino oligomers against West Nile virus.
abstract: We characterize in vitro resistance to and demonstrate the in vivo efficacy of two antisense phosphorodiamidate morpholino oligomers (PMOs) against West Nile virus (WNV). Both PMOs were conjugated with an Arg-rich peptide. One peptide-conjugated PMO (PPMO) binds to the 5' terminus of the viral genome (5'-end PPMO); the other targets an essential 3' RNA element required for genome cyclization (3' conserved sequence I [3' CSI] PPMO). The 3' CSI PPMO displayed a broad spectrum of antiflavivirus activity, suppressing WNV, Japanese encephalitis virus, and St. Louis encephalitis virus, as demonstrated by reductions in viral titers of 3 to 5 logs in cell cultures, likely due to the absolute conservation of the 3' CSI PPMO-targeted sequences among these viruses. The selection and sequencing of PPMO-resistant WNV showed that the 5'-end-PPMO-resistant viruses contained two to three mismatches within the PPMO-binding site whereas the 3' CSI PPMO-resistant viruses accumulated mutations outside the PPMO-targeted region. The mutagenesis of a WNV infectious clone demonstrated that the mismatches within the PPMO-binding site were responsible for the 5'-end PPMO resistance. In contrast, a U insertion or a G deletion located within the 3'-terminal stem-loop of the viral genome was the determinant of the 3' CSI PPMO resistance. In a mouse model, both the 5'-end and 3' CSI PPMOs (administered at 100 or 200 microg/day) partially protected mice from WNV disease, with minimal to no PPMO-mediated toxicity. A higher treatment dose (300 microg/day) caused toxicity. Unconjugated PMOs (3 mg/day) showed neither efficacy nor toxicity, suggesting the importance of the peptide conjugate for efficacy. The results suggest that a modification of the peptide conjugate composition to reduce its toxicity yet maintain its ability to effectively deliver PMO into cells may improve PMO-mediated therapy.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.
authors.forename: Tia S
authors.initials: TS
authors.lastname: Deas
authors.forename: Corey J
authors.initials: CJ
authors.lastname: Bennett
authors.forename: Susan A
authors.initials: SA
authors.lastname: Jones
authors.forename: Mark
authors.initials: M
authors.lastname: Tilgner
authors.forename: Ping
authors.initials: P
authors.lastname: Ren
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Behr
authors.forename: David A
authors.initials: DA
authors.lastname: Stein
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Iversen
authors.forename: Laura D
authors.initials: LD
authors.lastname: Kramer
authors.forename: Kristen A
authors.initials: KA
authors.lastname: Bernard
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003062:Codon; D017124:Conserved Sequence; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005260:Female; D066298:In Vitro Techniques; D007274:Injections, Intraperitoneal; D007279:Injections, Subcutaneous; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D009025:Morpholines / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D060172:Morpholinos; D009154:Mutation; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D012367:RNA, Viral / Q000235:genetics; D012641:Selection, Genetic; D014709:Vero Cells; D010948:Viral Plaque Assay; D014779:Virus Replication / Q000187:drug effects*; D014902:West Nile virus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development
chemical_list: D000998:Antiviral Agents; D003062:Codon; D009025:Morpholines; D060172:Morpholinos; D010455:Peptides; D012367:RNA, Viral; D001120:Arginine