Specific Inhibition of Influenza Virus RNA Polymerase and Nucleoprotein Gene Expression by Liposomally Encapsulated Antisense Phosphorothioate Oligonucleotides in MDCK Cells

Abstract

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza A virus replication in MDCK cells. Liposomally encapsulated and free antisense S-ODNs with four target sites (PB1, PB2, PA and NP genes) were tested for their abilities to inhibit virus-induced cytopathogenic effects in a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the site around the PB2 AUG initiation codon showed highly inhibitory effects. In contrast, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with that directed to the PB2 target site. The liposomally encapsulated antisense S-ODNs exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas free antisense S-ODNs were observed to inhibit viral adsorption to MDCK cells.Liposomal preparations of oligonucleotides facilitated their release from endocytic vesicles, and thus cytoplasmic and nuclear localization was observed. The activities of the antisense S-ODNs were effectively enhanced by using the liposomal carrier. Interestingly, the liposomally encapsulated FITC-S-ODN-PB2-as accumulated in the nuclear region of MDCK cells. However, weak fluorescence was observed within the endosomes and the cytoplasm of MDCK cells treated with the free antisense S-ODNs. The cationic lipid particles may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in vitro or in vivo.

Antisense oligonucleotides targeting the RNA binding region of the NP gene inhibit replication of highly pathogenic avian influenza virus H5N1

“…They are potentially useful therapeutic agents for the treatment of viral diseases [13]. AS ODNs are effective against the influenza virus in vitro and in vivo by targeting the PA, PB2, NS1 and PB1 genes; these target sites are mainly focused on the conserved AUG initiation codon of the influenza virus [14][15][16][17]. There are no current studies of AS ODNs on the NP gene of H5N1 avian influenza.…”

Inhibition of influenza virus replication in cultured cells by RNA‐cleaving DNA enzyme

“…Because our data indicated that antisense phosphothioate oligonucleotides targeting AUG initiation codon sequences of PB2 mRNA effectively inhibited the influenza virus RNA polymerase activity [9–11], and influenza virus replication in infected cells [12–14] and mice [15], we tested the DNA enzyme which carries 10‐23 catalytic sequence flanked with complementary sequences around the AUG initiation codon of PB2 mRNA (Table 1).…”

“…To date, the application of antisense phosphothioate oligonucleotides to inhibit the replication of various viruses has been of great success [1]. We have demonstrated that liposome‐encapsulated antisense phosphothioate oligonucleotides complementary to PB2‐AUG and PA‐AUG initiation codons inhibited the influenza virus RNA polymerase activity [9–11], and influenza virus replication in MDCK cells [12–14] and in mice [15]. In this paper, we describe the inhibition of influenza virus replication in cultured cells by RNA‐cleaving DNA enzyme which was designed from 10‐23 catalytic motif targeting the PB2‐AUG initiation codon, and compare the results with those for antisense phosphothioate oligonucleotides complementary to the same position [15].…”

Anti-influenza drugs and neuraminidase inhibitors

“…An antisense phosphorothioate oligonucleotide directed against the initiation codon of the influenza virus PB2 mRNA has been shown to inhibit virus replication in MDCK cells in a sequence specific manner, with an IC so of 0.15 11M when encapsulated in cationic liposomes. Free oligonucleotides could interfere with virus attachment in a sequence independent manner, with ICsos between 1 and 311M [45]. Protective efficacy of a liposomal preparation was also demonstrated in the mouse model after intravenous administration of 40 mg/kg twice daily for 5 days.…”