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 μg/day) partially protected mice from WNV disease, with minimal to no PPMO-mediated toxicity. A higher treatment dose (300 μg/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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 2470-2482 |
| Number of pages | 13 |
| Journal | Antimicrobial Agents and Chemotherapy |
| Volume | 51 |
| Issue number | 7 |
| DOIs | |
| State | Published - Jul 2007 |
| Externally published | Yes |
Fingerprint
ASJC Scopus subject areas
- Pharmacology (medical)
Cite this
In vitro resistance selection and in vivo efficacy of morpholino oligomers against west nile virus. / Deas, Tia S.; Bennett, Corey J.; Jones, Susan A.; Tilgner, Mark; Ren, Ping; Behr, Melissa J.; Stein, David A.; Iversen, Patrick L.; Kramer, Laura D.; Bernard, Kristen A.; Shi, Pei-Yong.
In: Antimicrobial Agents and Chemotherapy, Vol. 51, No. 7, 07.2007, p. 2470-2482.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - In vitro resistance selection and in vivo efficacy of morpholino oligomers against west nile virus
AU - Deas, Tia S.
AU - Bennett, Corey J.
AU - Jones, Susan A.
AU - Tilgner, Mark
AU - Ren, Ping
AU - Behr, Melissa J.
AU - Stein, David A.
AU - Iversen, Patrick L.
AU - Kramer, Laura D.
AU - Bernard, Kristen A.
AU - Shi, Pei-Yong
PY - 2007/7
Y1 - 2007/7
N2 - 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 μg/day) partially protected mice from WNV disease, with minimal to no PPMO-mediated toxicity. A higher treatment dose (300 μg/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.
AB - 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 μg/day) partially protected mice from WNV disease, with minimal to no PPMO-mediated toxicity. A higher treatment dose (300 μg/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.
UR - http://www.scopus.com/inward/record.url?scp=34447263267&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34447263267&partnerID=8YFLogxK
U2 - 10.1128/AAC.00069-07
DO - 10.1128/AAC.00069-07
M3 - Article
C2 - 17485503
AN - SCOPUS:34447263267
VL - 51
SP - 2470
EP - 2482
JO - Antimicrobial Agents and Chemotherapy
JF - Antimicrobial Agents and Chemotherapy
SN - 0066-4804
IS - 7
ER -