In vitro resistance selection and in vivo efficacy of morpholino oligomers against West Nile virus

We characterize the in vitro resistance and demonstrate the in vivo efficacy of two antisense phosphorodiamidate-morpholino-oligomers (PMO) against West Nile virus (WNV). Both PMOs were conjugated with an Arg-rich peptide (PPMO). One 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'CSI PPMO). The 3'CSI PPMO displayed a broad-spectrum of anti-flavivirus activity, suppressing WNV, Japanese encephalitis virus, and St. Louis encephalitis virus by 3-5 logs in cell culture, likely due to absolute conservation of the 3'CSI PPMO-targeted sequences across these viruses. Selection and sequencing of PPMO-resistant WNV showed that the 5'end PPMO-resistant viruses contained 2-3 mismatches within the PPMO-binding site, whereas the 3'CSI PPMO-resistant viruses accumulated mutations outside the PPMO-targeted region. Mutagenesis of a WNV infectious clone demonstrates that the mismatches within the PPMO-binding site are 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, is the determinant for 3'CSI PPMO resistance. In a mouse model, both the 5'end and 3'CSI PPMOs (100 and 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 modification of the peptide conjugate composition to reduce its toxicity, yet maintain its ability to effectively deliver PMO into cells, could improve PMO-mediated therapy.