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1.
Antivir Chem Chemother ; 17(5): 241-9, 2006.
Article in English | MEDLINE | ID: mdl-17176628

ABSTRACT

The RNA interference (RNAi) phenomenon is a recently discovered process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of mRNA containing the same sequence. We designed mammalian expression vectors that direct the synthesis of small interfering RNA (siRNA)-like transcripts and examined them for their siRNA-mediated gene interference targeting the env gene (NL4-3:7490-7508, E7490). We constructed siRNA expression vectors for two different strands (sense and antisense; tandem promoter) and for siRNA expressed from the short hairpin RNA (shRNA). The inhibition efficacy on HIV-1 replication differed between these two vectors. Notably, the shRNA vector pU6-env-shRNA inhibited p24 production more effectively than the tandem promoter expression vector pU6-env-siRNA. Furthermore, we examined the ability of lentiviral vectors expressing shRNA to suppress HIV-1 expression in HIV-1-infected SupT1 cells. The env-shRNA (E 7490) almost completely suppressed HIV-1 expression in infected cells for up to 15 days.


Subject(s)
Anti-HIV Agents/pharmacology , Gene Expression Regulation, Viral/drug effects , Gene Silencing/drug effects , HIV-1/drug effects , HIV-1/genetics , RNA, Small Interfering/pharmacology , Animals , COS Cells , Cell Line , Cells, Cultured , Chlorocebus aethiops , Genetic Vectors/pharmacology , Humans , Microbial Sensitivity Tests , Structure-Activity Relationship , Virus Replication/drug effects
2.
Article in English | MEDLINE | ID: mdl-16898417

ABSTRACT

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (> or = 90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490-7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.


Subject(s)
Anti-HIV Agents/pharmacology , Gene Expression Regulation, Viral/drug effects , Gene Silencing , HIV-1/genetics , RNA, Small Interfering/pharmacology , Transduction, Genetic , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Gene Silencing/drug effects , Genes, env/drug effects , Genes, env/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , HIV-1/drug effects , Humans , Microbial Sensitivity Tests
3.
Nucleic Acids Res ; 30(22): 4830-5, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12433985

ABSTRACT

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.


Subject(s)
Anti-HIV Agents/pharmacology , HIV-1 , Leukocytes, Mononuclear/virology , RNA, Double-Stranded/pharmacology , Animals , COS Cells , Cell Line , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression Regulation, Viral/drug effects , Genes, env , Genes, gag , HIV Core Protein p24/biosynthesis , HIV Core Protein p24/genetics , HIV Infections/prevention & control , HIV-1/genetics , HIV-1/metabolism , HeLa Cells , Humans , RNA Interference , RNA, Antisense/pharmacology , RNA, Viral/biosynthesis , Virus Replication/drug effects
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