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Results for “RNA Interference”

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PubMed2003

Inhibition of gammaherpesvirus replication by RNA interference.

RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.

Animals↗
PubMed2006

Tuning silence: conditional systems for RNA interference.

RNA interference (RNAi) has emerged as a powerful tool to downregulate the expression of specific genes. Drug-inducible systems allowing for conditional RNAi that offer the unique potential to modulate expression of virtually any endogenous gene in the cell have been recently developed. Their applications are very broad, ranging from basic studies of gene function to translational research including modeling of human diseases, analysis of potential side effects of candidate drugs, testing of gene-based therapies and loss-of-function screens. Here we summarize the state of the art of systems allowing for drug-controllable knockdown, and provide a description of their current and future applications.

Genetic Vectors↗
PubMed2006

Moment analysis for kinetics of gene silencing by RNA interference.

RNA interference (RNAi) was quantitatively evaluated from a kinetic viewpoint. A simple kinetic evaluation based on moment analysis was proposed, assuming suppression and recovery phases of gene expression. We defined the area under the curve of the inhibitory effect (AUC(IE)) as an index of the total intensity of RNAi and the mean response time of the inhibitory effect (MRT(IE)) as an index of its duration. The proposed kinetic analysis helps to understand the RNAi effect in a quantitative and time-dependent manner, which will be beneficial for designing RNAi-based gene silencing for both experimental and therapeutic purposes.

Animals↗
PubMed2003

Inhibition of virus replication by RNA interference.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene function by reverse genetics. Additionally, inhibition of virus replication by means of induced RNAi has now been reported for numerous viruses, including several important human pathogens such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, dengue virus, poliovirus and influenza virus A. In this review, we will summarize the current data on RNAi-mediated inhibition of virus replication and discuss the possibilities for the development of RNAi-based antiviral therapeutics.

Animals↗
PubMed2004

Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference.

RNA interference (RNAi) is a ubiquitous mechanism of eukaryotic gene regulation that can be exploited for specific gene silencing. Retroviruses have been successfully used for stable expression of short hairpin RNAs in mammalian cells, leading to persistent inhibition of gene expression by RNAi. Here, we apply retrovirus-mediated RNAi to epithelial Madin-Darby canine kidney cells, whose properties limit the applicability of other RNAi methods. We demonstrate efficient suppression of a set of 13 target genes by retroviral coexpression of short hairpin RNAs and a selectable marker. We characterize the resulting knockdown cell populations with regard to composition and stability, and examine the usefulness of proposed guidelines for choosing RNAi target sequences. Finally, we show that this system can be used to simultaneously target two genes, giving rise to double knockdowns. Thus, retrovirus-mediated RNAi is a convenient method for gene silencing in Madin-Darby canine kidney cells, and is likely to be applicable to virtually any mammalian cell.

Cell Polarity↗
PubMed2005

Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference.

RNA interference (RNAi)-mediated gene knockdown is a potent approach for studying gene function. We have previously reported a plasmid-based, tamoxifen-inducible gene knockdown system in cultured cells using a combined RNAi and Cre-LoxP system. Here, we validate this system in mouse and show that it can be used to suppress the expression of an endogenous gene (Fgfr2) with high efficiency. We show that transgenic mice carrying the U6-ploxPneo-Fgfr2 RNAi construct are normal, displaying Fgfr2 transcripts equivalent to those of wild-type controls, indicating that the U6 promoter is inactive in vivo due to the presence of the neo in the promoter. After excision of the neo by crossing with transgenic mice that express Cre in the mouse germline, the U6 promoter is activated, leading to over 95% reduction of Fgfr2 transcripts, and consequently, embryonic lethality. On the other hand, activation of the U6 promoter using transgenic mice that express Cre in the progress zone of the limb results in live mice with malformation of digits of both the forelimbs and hindlimbs. This method provides a fast, yet efficient way to decipher gene functions in vivo in a tissue-specific manner.

Animals↗
PubMed2005

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference.

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.

Animals↗
PubMed2006

Engineering novel traits in plants through RNA interference.

RNA interference (RNAi) is a homology-dependent gene silencing technology that involves double-stranded RNA directed against a target gene or its promoter region. Using hairpin constructs, double-stranded RNA can be expressed in plants relatively easily, enabling this technology to be applied to a wide range of species to silence the expression of both specific endogenous genes and genes of invading pathogens. RNAi has also been used to engineer metabolic pathways to overproduce secondary products with health, yield or environmental benefits. The application of tissue-specific or inducible gene silencing, with the use of appropriate promoters, and the ability to silence several genes simultaneously should enhance our ability to create novel traits in plants.

Gene Expression Regulation, Plant↗
PubMed2002

Lentiviral-mediated RNA interference.

RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA). These siRNAs are capable of binding to a specific mRNA sequence and causing its degradation. The recent demonstration of a plasmid vector that directs siRNA synthesis in mammalian cells prompted us to examine the ability of lentiviral vectors to encode siRNA as a means of providing long-term gene silencing in mammalian cells. The RNA-polymerase III dependent promoter (H1-RNA promoter) was inserted in the lentiviral genome to drive the expression of a small hairpin RNA (shRNA) against enhanced green fluorescent protein (EGFP). This construct successfully silenced EGFP expression in two stable cell lines expressing this protein, as analyzed by fluorescence microscopy, flow cytometry, and Western blotting. The silencing, which is dose dependent, occurs as early as 72 hr postinfection and persists for at least 25 days postinfection. The ability of lentiviruses encoding siRNA to silence genes specifically makes it possible to take full advantage of the possibilities offered by the lentiviral vector and provides a powerful tool for gene therapy and gene function studies.

Cell Line↗
PubMed2004

Knockdown of mouse adult beta-globin gene expression in MEL cells by retrovirus vector-mediated RNA interference.

RNA interference (RNAi) efficiently induces sequence-specific gene silencing in mammalian cells through short interfering RNA (siRNA) of 21-23 nucleotides synthesized in vitro or expressed by DNA-based vector. However, introduction of siRNA into mammalian cells by transfection limits the application of RNAi, especially when it is necessary to generate long-term gene silencing in vivo. Virus vector-mediated RNAi provides an alternative to transfection. In the present study, we investigated such transduction system and showed that retrovirus vector-mediated RNAi can substantially down-regulate expression of mouse adult beta-globin gene in MEL cells. The results suggest that retrovirus vector-delivered RNAi may find its use in functional genomics and in gene therapy.

Animals↗
PubMed2006

A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference.

RNA interference (RNAi) might be an efficient antiviral therapy for some obstinate illness. Herein, a retrovirus-based RNAi system was developed to drive expression and delivery of Hepatitis B virus (HBV)-specific short hairpin RNA (shRNA) in HepG2 cells. The levels of HBsAg and HBeAg and that of HBV mRNA were dramatically decreased by this RNAi system in HepG2 cells transfected with Topo-HBV plasmid. Retrovirus-based RNAi thus may be useful for therapy in HBV and other viral infections and provide new clues for prophylactic vaccine development.

Cell Line, Tumor↗
PubMed2006

Retraction of synapses and dendritic spines induced by off-target effects of RNA interference.

RNA interference (RNAi), which allows selective gene silencing, has been proposed for functional genomic analysis and for the treatment of human disease. However, induction of RNAi in mammalian cells by expression of double-stranded RNA can activate innate antiviral response pathways that perturb off-target gene expression. The activation and functional consequences of these effects in neurons are unknown. We find that expression of subsets of short hairpin RNAs (shRNAs) in rat hippocampal pyramidal neurons can have off-target effects that reduce the complexity of dendritic arbors and trigger the loss of dendritic spines. Morphological changes are accompanied by electrophysiological perturbations in passive membrane properties and a decrease in the number and strength of excitatory and inhibitory synapses. These perturbations depend on the shRNA sequence and are independent of the identity of the targeted protein. Our results indicate that off-target effects of RNAi severely perturb neuronal structure and function and may lead to the functional withdrawal of affected cells from the brain circuitry.

Adaptation, Physiological↗
PubMed2006

Optimization of feline immunodeficiency virus vectors for RNA interference.

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.

Cell Line, Tumor↗
PubMed2003

A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.

Animals↗
PubMed2006

Autoimmune targeting of key components of RNA interference.

RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production.

Animals↗
PubMed2002

Modulation of HIV-1 replication by RNA interference.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.

Cell Line↗
PubMed2003

Inhibition of hepatitis B virus expression and replication by RNA interference.

RNA interference (RNAi) is the process of sequence-specific gene silencing, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the target gene. Because it has been shown that RNAi can be accomplished in cultured mammalian cells by introducing small interfering RNAs (siRNAs), much effort has been invested in exploiting this phenomenon for experimental and therapeutic means. In this study, we present a series of experiments showing a significant reduction in hepatitis B virus (HBV) transcripts and proteins in cell culture, as well as in the viral replicative forms, induced by siRNA-producing vectors. The antiviral effect is sequence-specific and does not depend on active viral replication. In conclusion, our data suggest that RNAi may provide a powerful therapeutic tool, acting both on replication-competent and on replication-incompetent HBV.

Cells, Cultured↗
PubMed2002

Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference.

RNA interference (RNAi) is an ancient antiviral response that processes dsRNA and associates it into a nuclease complex that identifies RNA with sequence homology and specifically cleaves it. We demonstrate that RNAi mediated by 21-bp dsRNA specifically inhibits HIV-1 infection of permanent cell lines and primary CD4(+) T cells. Inhibition of HIV replication was measured by p24 Gag protein content in supernatant, Northern blot analysis, and DNA PCR for products of reverse transcription. The inhibition occurred at two points in the viral life cycle, after fusion and before reverse transcription and during transcription of viral RNA from integrated provirus. Treatment of HIV-infected activated CD4(+) T cells with a fluorine-derivatized siRNA that is resistant to RNase A yielded similar inhibition of HIV infection. In addition, the derivatized siRNA could be delivered without lipofectin complexing and in the presence of serum. The identification of RNAi activity against HIV-1 presents a new approach to study viral infections and a proof of concept of RNAi antiviral activity in mammalian cells.

Anti-HIV Agents↗