Morpholino oligomers targeting the PB1 and NP genes enhance the survival of mice infected with highly pathogenic influenza A H7N7 virus

Abstract

Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded nucleic acid-analogue antisense agents that enter cells readily and can reduce gene expression by steric blocking of complementary RNA (cRNA) sequences. Here, we tested a panel of PPMO designed to target conserved sequences in the RNA genome segments encoding polymerase subunits of a highly pathogenic mouse-adapted influenza A virus (SC35M; H7N7). Three PPMO, targeting the translation start site region of PB1 or NP mRNA or the 39-terminal region of NP viral RNA (vRNA), potently inhibited virus replication in MDCK cells. Primer extension assays showed that treatment with any of the effective PPMO led to markedly reduced levels of mRNA, cRNA and vRNA. Initially, the potential toxicity of a range of intranasally administered PPMO doses was evaluated, by measuring their effect on body weight of uninfected mice. Subsequently, a non-toxic dosing regimen was used to investigate the effect of various PPMO on SC35M infection in a mouse model. Mice administered intranasal treatment of PPMO targeting the PB1-AUG region or NP vRNA, at 3 mg per dose, given once 3 h before and once 2 days after intranasal infection with 10¾LD 50 of SC35M, showed a 2 log 10 reduction of viral titre in the lungs and 50 % survival for the 16 day duration of the experiment, whereas the NP-AUG-targeted PPMO treatment resulted in 30 % survival of an otherwise lethal infection. These data suggest that PPMO provide a useful reagent to investigate influenza virus molecular biology and may constitute a therapeutic strategy against highly pathogenic influenza viruses.

Inhibition of Influenza Virus Infection in Human Airway Cell Cultures by an Antisense Peptide-Conjugated Morpholino Oligomer Targeting the Hemagglutinin-Activating Protease TMPRSS2

“…Two studies have detailed in vivo inhibition of influenza A virus infections by PPMO targeting viral RNAs encoding the polymerase subunits PB1 and NP, administered by intranasal instillation. Although relatively high levels of PPMO were required to achieve inhibition of virus production in the cell culture experiments here, Gabriel and coworkers (13) found that a dose of only 150 ng PPMO/kg suppressed viral titers in the lungs of mice and provided protection to 50% of mice challenged with a lethal H7N7 infection. Lupfer et al (26) showed that the peptide component of PPMO was necessary for efficacy against H3N8 influenza virus in mice, as PMO (without a peptide conjugate) were ineffective.…”

“…PPMO have shown considerable antiviral activity against a number of positive-and negative-strand RNA viruses in both cell cultures and murine experimental systems (reviewed in reference 35). Against influenza A viruses, PPMO targeting highly conserved sequences of PB1 and NP RNAs had potent and specific antiviral activity against multiple subtypes in experimentally infected cell cultures and mice (13,15,26).…”

Chemical Modification of Oligonucleotides for Therapeutic, Bioanalytical and other Applications

“…Work carried out by the Glazer group used bis-PNAs to target several polypurine sequences of the b-thalassemia mutation. Correction frequencies of up to 0.4 % for the splice-site mutation IVS2-1 were recorded by using a combination of bis-PNA and chloroquine treatment [54] NMA/2'-MOE PTEN [56] PMO LANA [57] HIV-1 PNA TAR [58] PNA PBS [59] LNA multiple [60] E. coli PNA and PMO acpP [61,62] PNA 23S rRNA [63] PNA and LNA RNase P [64] SARS-CoV PMO multiple [65] HCV PNA IRES [66] 2'-OMe E1 RNA [67] BMD/DMD PMO dystrophin gene [68,69] alphavirus PMO AUG translation [55] prions PS prion protein [70,71] b-thalassemia PNA IVS2-1 [72] AT PMO ATM [73] influenza A PMO PB1 and -2, NP [74,75] hypercholesterolemia LNA miR-122 [76] ChemBioChem www.chembiochem.org during the cells' S phase. Furthermore, sequences targeted with distances of several hundred base pairs away from the mutation still showed activity; this shows that further understanding of the mechanism of triplex stimulated recombination is still required.…”

Limited Compatibility of Polymerase Subunit Interactions in Influenza A and B Viruses

“…Primer extensions were performed at 45°C for 1 h. The NP and NA primers described in Ref. 33 were used. Transcription products were analyzed on 6% polyacrylamide gels containing 7 M urea in Tris-borate-EDTA buffer and detected by autoradiography.…”