A new modified DNA enzyme that targets influenza virus A mRNA inhibits viral infection in cultured cells

Takahashi, Hitoshi; Hamazaki, Hiroyuki; Habu, Yuichiro; Hayashi, Mieko; Abe, Takayuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi

doi:10.1016/s0014-5793(04)00073-0

Cited by 57 publications

(36 citation statements)

References 39 publications

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“…The currently available influenza drugs provide only partial therapeutic or prophylactic protection, and viral drug-resistance is a worsening problem (de Jong et al, 2005;Kiso et al, 2004;Le et al, 2005;Ludwig et al, 2003;McKimm-Breschkin, 2000). A variety of nucleic-acid based approaches have been reported as promising against influenza A virus (Abe et al, 2001;Ge et al, 2003Ge et al, , 2004aMcKimm-Breschkin, 2000;Plehn-Dujowich & Altman, 1998;Tado et al, 2001;Takahashi et al, 2004;Tompkins et al, 2004) and it appears that sequence-specific intervention in the influenza virus life cycle represents a promising avenue for drug development. Here, we show that PPMO targeting viral polymerase genes provide a useful strategy to inhibit highly pathogenic influenza virus infection in vivo.…”

Section: Discussionmentioning

confidence: 99%

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

Gabriel

Nordmann

Stein

et al. 2008

Journal of General Virology

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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.

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Section: Discussionmentioning

confidence: 99%

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

Gabriel

Nordmann

Stein

et al. 2008

Journal of General Virology

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“…DNAzymes (46), oligoaptamers (45), and short interfering RNA (10,12) have all been documented to have antiviral activity in cell culture, and short interfering RNA has been documented to have antiviral activity in mice (11,47), against FLUAV. Intravenous delivery in mice of a liposome-encapsulated antisense phosphorothioate oligonucleotide with sequence complementary to the translation start site region of PB2 mRNA reduced FLUAV titers in lung tissue and significantly increased overall survival rates (26,27).…”

mentioning

confidence: 99%

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

Pastey

Kobasa

et al. 2006

Antimicrob Agents Chemother

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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.

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“…Several studies have also been conducted on 10-23 Dz to inhibit the replication of many RNA viruses such as the Japanese encephalitis virus [10], hepatitis [16,26], HIV [12], and influenza [27]. When an influenza virion enters the host cell, the acidic environment of the endosomal lumen activates the M2 ion channel of the virus and conducts protons across the viral membrane as a prerequisite for release of genetic material to the cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

Potent Intracellular Knock-Down of Influenza A Virus M2 Gene Transcript by DNAzymes Considerably Reduces Viral Replication in Host Cells

et al. 2015

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Influenza A virus has been known to be an important respiratory pathogen and cause of several epidemics and devastating pandemics leading to loss of life and resources across the globe. The M2 ion channel protein is highly conserved and essentially required during the trafficking, assembly, and budding processes of virus, thus an attractive target for designing antiviral drugs. We designed several 10-23 DNAzymes (Dz) targeting different regions of the M2 gene and analyzed their ability to specifically cleave the target RNA in both cell-free system as well as in cell culture using transient transfections. Dz114, among several others, directed against the predicted single-stranded bulge regions showed 70% inhibition of M2 gene expression validated by PCR and Western blot analysis. The activity was dependent on Mg(2+) (10-50 mM) in a dose-dependent manner. The mutant-Dz against M2 gene showed no down-regulation thereby illustrating high level of specificity of designed Dz114 towards the target RNA. Our findings suggest that Dz may be used as potential inhibitor of viral RNA replication and can be explored further for development of an effective therapeutic agent against influenza infection. These catalytic nucleic acid molecules may further be investigated as an alternative to the traditional influenza vaccines that require annual formulation.

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A new modified DNA enzyme that targets influenza virus A mRNA inhibits viral infection in cultured cells

Cited by 57 publications

References 39 publications

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

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

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

Potent Intracellular Knock-Down of Influenza A Virus M2 Gene Transcript by DNAzymes Considerably Reduces Viral Replication in Host Cells

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