In vitro resistance selection and in vivo efficacy of morpholino oligomers against West Nile virus.
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.
Deas
Tia S
TS
Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.
Bennett
Corey J
CJ
Jones
Susan A
SA
Tilgner
Mark
M
Ren
Ping
P
Behr
Melissa J
MJ
Stein
David A
DA
Iversen
Patrick L
PL
Kramer
Laura D
LD
Bernard
Kristen A
KA
Shi
Pei-Yong
PY
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
D000998:Antiviral Agents; D003062:Codon; D009025:Morpholines; D060172:Morpholinos; D010455:Peptides; D012367:RNA, Viral; D001120:Arginine