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Inhibition of Multiple Subtypes of Influenza A Virus in Cell Cultures with Morpholino Oligomers

Qing Ge,^1, Manoj Pastey,^2, Darwyn Kobasa,^3, Piliapan Puthavathana,⁴ Christopher Lupfer,² Richard K. Bestwick,⁵ Patrick L. Iversen,⁵ Jianzhu Chen,¹ and David A. Stein^5*

Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,¹ Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon 97331,² Public Health Agency of Canada, Winnipeg, Manitoba, Canada,³ Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand,⁴ AVI BioPharma Inc., Corvallis, Oregon 97333⁵

Received 25 May 2006/ Returned for modification 7 July 2006/ Accepted 19 August 2006

Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMO) are single-stranded nucleic acid-like antisense agents that can reduce gene expression by sterically blocking complementary RNA sequence. P-PMO are water soluble and nuclease resistant, and they readily achieve uptake into cells in culture under standard conditions. Eight P-PMO, each 20 to 22 bases in length, were evaluated for their ability to inhibit influenza A virus (FLUAV) A/PR/8/34 (H1N1) replication in cell culture. The P-PMO were designed to base pair with FLUAV RNA sequences that are highly conserved across viral subtypes and considered critical to the FLUAV biological-cycle, such as gene segment termini and mRNA translation start site regions. Several P-PMO were highly efficacious, each reducing viral titer in a dose-responsive and sequence-specific manner in A/PR/8/34-infected cells. Two P-PMO, one designed to target the AUG translation start site region of PB1 mRNA and the other the 3'-terminal region of nucleoprotein viral genome RNA, also proved to be potent against several other FLUAV strains, including A/WSN/33 (H1N1), A/Memphis/8/88 (H3N2), A/Eq/Miami/63 (H3N8), A/Eq/Prague/56 (H7N7), and the highly pathogenic A/Thailand/1(KAN-1)/04 (H5N1). The P-PMO exhibited minimal cytotoxicity in cell viability assays. High efficacy by two of the P-PMO against multiple FLUAV subtypes suggests that these oligomers represent a broad-spectrum therapeutic approach against a high percentage of known FLUAV strains.

Published ahead of print on 11 September 2006.

These authors contributed equally to this work.

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