Specific Inhibition of Influenza Virus RNA Polymerase and Nucleoprotein Genes Expression by Liposomally Endocapsulated Antisense Phosphorothioate Oligonucleotides: Penetration and Localization of Oligonucleotides in Clone 76 Cells

Hatta, Toshifumi; Takai, Kazuyuki; Nakada, Susumu; Yokota, Tomoyuki; Takaku, Hiroshi

doi:10.1006/bbrc.1997.6185

Cited by 18 publications

(20 citation statements)

References 20 publications

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“…The results of the RNase protection assay essentially confirm the findings from the northern blot hybridization (Fig. 5) [39]. The PB2 mRNA was quantified by densitometric scanning.…”

supporting

confidence: 61%

“…Previously, we tested the inhibition of influenza virus RNA polymerase (PB1, PB2, PA) and nucleoprotein (NP) gene expression by circular dumbbell RNA/DNA chimeric oligonucleotides (CDRDON-Nu) with closed tetra-nucleotide loop structures, as determined by CAT protein expression (CAT activity), in the clone 76 cell line [39]. The in vitro activities of these oligonucleotides on the expression of the influenza A virus RNA polymerase and nucleoprotein genes were assessed on the basis of their inhibition of CAT protein expression with the CAT ELISA method.…”

mentioning

confidence: 99%

“…In the present study, we tested the inhibitory effects of an anti-S-ODN, as well as the CDRDON-Nu-PB2-as and Al-PB2-as with closed nucleotide and alkyl loop structures, in MDCK cells infected with influenza A virus (A/PR/8/34) at an m.o.i. of 0.01, using a cationic liposome (DOTAP) delivery system [39] [40]. To characterize the inhibitory effects of these oligonucleotides, we performed an Rnase-protection assay as an alternative method to quantitatively compare the mRNA levels.…”

mentioning

confidence: 99%

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Inhibition of Influenza Virus Replication in MDCK Cells by Circular Dumbbell RNA/DNA Chimeras with Closed Alkyl Loop Structures

Hamazaki¹,

Abe²,

Yamakawa³

et al. 2002

HCA

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Dedicated to Professor Wolfgang Pfleiderer on the occasion of his 75th birthdayWe have demonstrated that a new type of circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON) with two closed nucleotide or alkyl loop structures (hexa-ethylene glycol) inhibits influenza virus A replication in MDCK cells. The enzymatic synthesis of circular dumbbell RNA/DNA chimeric oligonucleotides was achieved by enzymatically ligating a self-complementary phosphorylated oligonucleotide with T4-RNA ligase. The CDRDON-Al, with two closed alkyl loop structures, showed higher nuclease resistance, hybridization, and cellular uptake than the anti-S-ODN and the CDRDON, with two closed nucleotide hairpinloop structures. The circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON-Al-PB2-as), containing an AUG initiation-codon sequence as the target of PB2, showed highly inhibitory effects on influenza A virus RNA expression. The limited toxicity of unmodified phosphodiester oligonucleotides and the sequencespecific binding to target mRNA indicate that circular dumbbell RNA/DNA chimeric phosphodiester oligonucleotides can be used with intact cells, and may prevent viral replication in culture.

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“…The results of the RNase protection assay essentially confirm the findings from the northern blot hybridization (Fig. 5) [39]. The PB2 mRNA was quantified by densitometric scanning.…”

supporting

confidence: 61%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Inhibition of Influenza Virus Replication in MDCK Cells by Circular Dumbbell RNA/DNA Chimeras with Closed Alkyl Loop Structures

Hamazaki¹,

Abe²,

Yamakawa³

et al. 2002

HCA

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“…These include small molecules, some as selective inhibitors of nucleic acid replication or protease function, and higher molecular weight agents, such as antisense molecules, the hairpin and other ribozymes, and vaccines (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). External guide sequences (EGSs) are oligoribonucleotides (16) that have also been used in conjunction with endogenous RNase P to diminish both the expression of herpes virus thymidine kinase in tissue culture and of several genes in Escherichia coli (4,5,(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P

Plehn-Dujowich

Altman

1998

Proc. Natl. Acad. Sci. U.S.A.

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The polymerase (PB2) and nucleocapsid (NP) genes encoded by the genome of inf luenza virus are essential for replication of the virus. When synthetic genes that express RNAs for external guide sequences targeted to the mRNAs of the PB2 and NP genes are stably incorporated into mouse cells in tissue culture, infection of these cells with inf luenza virus is nonproductive. Endogenous RNase P cleaves the targeted inf luenza virus mRNAs when they are in a complex with the external guide sequences. Targeting two different mRNAs simultaneously inhibits viral particle production more efficiently than does targeting only one mRNA.

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“…Liposome delivery of oligonucleotides to cells in vitro has been shown to reduce greatly the effective dose as compared to the use of the free oligonucleotide (Leonetti et al, 1989;Milhaud et al, 1991;Thierry & Dritschilo, 1992). We have tested the inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by antisense oligonucleotides and phosphorothioate oligonucleotides (S-ODNs), as determined by chloramphenicol acetyltranseferase expression in the clone 76 cell line (Hatta et al, 1997). Previously, we observed that the liposomally encapsulated S-ODNs and ODNs complementary to the sites around the influenza virus PB2 AUG and PA AUG initiation codons showed highly sequencespecific inhibitory effects.…”

mentioning

confidence: 99%

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

Abe

Suzuki

Hatta

et al. 1998

Antivir Chem Chemother

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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.

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Specific Inhibition of Influenza Virus RNA Polymerase and Nucleoprotein Genes Expression by Liposomally Endocapsulated Antisense Phosphorothioate Oligonucleotides: Penetration and Localization of Oligonucleotides in Clone 76 Cells

Cited by 18 publications

References 20 publications

Inhibition of Influenza Virus Replication in MDCK Cells by Circular Dumbbell RNA/DNA Chimeras with Closed Alkyl Loop Structures

Inhibition of Influenza Virus Replication in MDCK Cells by Circular Dumbbell RNA/DNA Chimeras with Closed Alkyl Loop Structures

Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P

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

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