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Results for “RNA Interference”

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At least 145 records · Page 8Linked to original sources
PubMed2006

Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.

RNA-interference (RNAi) is a potent mechanism, conserved from plants to humans for specific silencing of genes, which holds promise for functional genomics and gene-targeted therapies. Here we show that bacteria engineered to produce a short hairpin RNA (shRNA) targeting a mammalian gene induce trans-kingdom RNAi in vitro and in vivo. Nonpathogenic Escherichia coli were engineered to transcribe shRNAs from a plasmid containing the invasin gene Inv and the listeriolysin O gene HlyA, which encode two bacterial factors needed for successful transfer of the shRNAs into mammalian cells. Upon oral or intravenous administration, E. coli encoding shRNA against CTNNB1 (catenin beta-1) induce significant gene silencing in the intestinal epithelium and in human colon cancer xenografts in mice. These results provide an example of trans-kingdom RNAi in higher organisms and suggest the potential of bacteria-mediated RNAi for functional genomics, therapeutic target validation and development of clinically compatible RNAi-based therapies.

Animals↗
PubMed2005

Functional genomic analysis of RNA interference in C. elegans.

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.

Amino Acid Motifs↗
PubMed2005

RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) was first discovered in the nematode Caenorhabditis elegans (Fire et al., 1998; Guo and Kemphues, 1995). The completion of the C. elegans genome in 1998 coupled with the advent of RNAi techniques to knock down gene function ushered in a new age in the field of functional genomics. There are four methods for double-stranded RNA (dsRNA) delivery in C. elegans: (1) injection of dsRNA into any region of the animal (Fire et al., 1998), (2) feeding with bacteria producing dsRNA (Timmons et al., 2001), (3) soaking in dsRNA (Tabara et al., 1998), and (4) in vivo production of dsRNA from transgenic promoters (Tavernarakis et al., 2000). In this chapter, we discuss the molecular genetic mechanisms, techniques, and applications of RNAi in C. elegans.

Animals↗
PubMed2005

RNA interference and potential therapeutic applications of short interfering RNAs.

RNA interference is an endogenous gene-silencing mechanism that involves double-stranded RNA-mediated sequence-specific mRNA degradation. The discovery of this pathway together with the elucidation of the structure and function of short interfering RNAs--the effector molecules of RNA interference--has had an enormous impact on experimental biology. RNA interference technologies are currently the most widely utilized techniques in functional genomic studies. Furthermore, there is an intense research effort aimed at developing short interfering RNAs for therapeutic purposes. A number of proof-of-principle experiments have demonstrated the clinical potential of appropriately designed short interfering RNAs in various diseases including viral infections, cancer and neurodegenerative disorders. Already, in such a short time from their discovery, Acuity Pharmaceuticals (August 2004) and Sirna Therapeutics (September 2004) have filed Investigational New Drug applications with the US FDA to begin clinical trials with modified siRNA molecules in patients with age-related macular degeneration. This review will give a brief overview of the mechanism of RNA interference and applications of the pathway in experimental biology will be discussed. The article will focus on recent developments related to the use of RNA interference technologies in mammalian systems and on potential clinical applications of short interfering RNA-mediated RNA interference.

Genetic Therapy↗
PubMed2005

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor.

Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80-90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.

Animals↗
PubMed2004

Unlocking the potential of the human genome with RNA interference.

The discovery of RNA interference (RNAi) may well be one of the transforming events in biology in the past decade. RNAi can result in gene silencing or even in the expulsion of sequences from the genome. Harnessed as an experimental tool, RNAi has revolutionized approaches to decoding gene function. It also has the potential to be exploited therapeutically, and clinical trials to test this possibility are already being planned.

Animals↗
PubMed2006

Silencing neurodegenerative disease: bringing RNA interference to the clinic.

RNA interference (RNAi) is a recently described conserved biological pathway where non-coding RNAs suppress the expression of specific genes. Research efforts in the RNAi field aim to gain a better understanding of how its underlying machinery is orchestrated, to define the biological role of this conserved pathway, determine how to effectively manipulate RNAi in the laboratory and to integrate all this knowledge to develop novel therapies for human disease. This review summarizes the advances in the design of therapeutic RNAi for neurodegenerative diseases and discusses some of the experimental steps required to bring this therapy to human clinical trials.

Animals↗
PubMed2004

RNA interference and the use of small interfering RNA to study gene function in mammalian systems.

In the past 2 years, extraordinary developments in RNA interference (RNAi)-based methodologies have seen small interfering RNAs (siRNA) become the method of choice for researchers wishing to target specific genes for silencing. In this review, an historic overview of the biochemistry of the RNAi pathway is described together with the latest advances in the RNAi field. Particular emphasis is given to strategies by which siRNAs are used to study mammalian gene function. In this regard, the use of plasmid-based and viral vector-based systems to mediate long-term RNAi in vitro and in vivo are described. However, recent work has shown that non-specific silencing effects and activation of the interferon response may occur following the use of some siRNA and delivery vector combinations. Future goals must therefore be to understand the mechanisms by which siRNA delivery leads to unwanted gene silencing effects in cells and, in this way, RNAi technology can reach its tremendous potential as a scientific tool and ultimately be used for therapeutic purposes.

Animals↗
PubMed2003

RNA interference (RNAi) in hematology.

RNA interference (RNAi), an evolutionary highly conserved process of post-transcriptional gene silencing, can be triggered by small interfering RNAs (siRNAs) that mediate sequence-specific mRNA degradation. Since the first reports in 1998, RNAi has rapidly been developed into an effective tool to specifically knock down gene expression in a wide variety of target cells. Accordingly, RNAi is currently used for both systematic functional genomics in several organisms and for specific therapeutic intervention in preclinical models of different diseases characterized by aberrant gene expression. However, since siRNAs are not replicated in mammalian cells during the process of RNAi, kinetic aspects of RNAi-induced gene silencing that eventually depend on the intracellular level of siRNA must be considered for each analytical or therapeutic application in these cells. We summarize here some aspects of siRNA design and delivery, of RNAi kinetics, potential side effects, and limitations of siRNA-mediated gene silencing, as well as putative RNAi targets for functional and/or therapeutic intervention in hematopoietic cells.

Animals↗
PubMed2004

Application of RNA interference to chicken embryos using small interfering RNA.

Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.

Animals↗
PubMed2006

Prospects of RNA interference therapy for cancer.

RNA interference (RNAi) is a powerful gene-silencing process that holds great promise in the field of cancer therapy. The discovery of RNAi has generated enthusiasm within the scientific community, not only because it has been used to rapidly identify key molecules involved in many disease processes including cancer, but also because RNAi has the potential to be translated into a technology with major therapeutic applications. Our evolving understanding of the molecular pathways important for carcinogenesis has created opportunities for cancer therapy employing RNAi technology to target the key molecules within these pathways. Many gene products involved in carcinogenesis have already been explored as targets for RNAi intervention, and RNAi targeting of molecules crucial for tumor-host interactions and tumor resistance to chemo- or radiotherapy has also been investigated. In most of these studies, the silencing of critical gene products by RNAi technology has generated significant antiproliferative and/or proapoptotic effects in cell-culture systems or in preclinical animal models. Nevertheless, significant obstacles, such as in vivo delivery, incomplete suppression of target genes, nonspecific immune responses and the so-called off-target effects, need to be overcome before this technology can be successfully translated into the clinical arena. Significant progress has already been made in addressing some of these issues, and it is foreseen that early phase clinical trials will be initiated in the very near future.

Drug Resistance, Neoplasm↗
PubMed2006

Testing the efficacy of RNA interference in Haemonchus contortus.

RNA interference (RNAi) is widely used in Caenorhabditis elegans to identify gene function and has been adapted as a high throughput screening method to identify genes involved in essential processes. We have been examining whether RNAi could also be used on the strongylid parasitic nematode Haemonchus contortus to study gene function. Eleven genes were targeted in L1 and exsheathed L3 H. contortus larvae with RNAi methodologies which have been shown to be effective in C. elegans and parasitic nematodes-feeding, soaking and electroporation. Reverse transcriptase-PCR and, where possible, protein assays were carried out to examine decreases in mRNA and protein levels. RNAi soaking in dsRNA to beta-tubulin and sec-23, a gene involved in vesicle transport, resulted in specific decreases in mRNA levels in exsheathed L3 larvae. No signs of specific decreases in expression levels were observed for the other nine genes tested. Following electroporation of dsRNA in L1 stage larvae, significant decreases were observed for two out of four genes tested. These findings suggest that the RNAi pathway is functional in H. contortus and that, under certain conditions, it is possible to suppress gene expression by RNAi. However, it only works on a limited number of genes and in some cases the effect is small and difficult to reproduce. This indicates that the RNAi approaches established for C. elegans and other nematodes have limited efficacy in H. contortus. This may reflect differences between nematode species in dsRNA uptake and transport into cells and between cells.

Animals↗
PubMed2004

Analysis of cytokine-induced NO-dependent apoptosis using RNA interference or inhibition by 1400W.

RNA interference has been used to silence gene expression and evaluate the contribution of a gene product to cell function. Here, we investigated conditions under which expression of an inducible protein, nitric oxide synthase 2 (NOS2), is decreased by RNA interference. Cytokine treatment of insulin-producing RINm5F cells results in NOS2 induction and cell death. Conditions used here favoured cytokine-induced apoptosis, for the first time--rather than necrosis, previously recorded. In RINm5F cells, transfected with NOS2-specific small interfering RNA followed by a 12 h exposure to cytokines, there was a significant reduction in NOS2 protein, nitrite, and apoptosis. There were no changes in these three parameters when experiments were carried out with unrelated vimentin siRNA. To interpret the NOS2-siRNA result further, we compared it with complete pharmacological inhibition of nitric oxide (NO) production by the NOS2 competitive inhibitor, 1400W, which lowered apoptosis by only 50% in the RINm5F cells. We conclude that the use of NOS2-specific siRNA has resulted in the subsequent lowering of expression of a cytokine-inducible protein whose function can be quantified. siRNA results have compared favourably with use of a pharmacological inhibitor of NOS2, in revealing the subtle, partial contribution of cytokine-induced NO to apoptosis induction in these insulin-producing cells.

Amidines↗
PubMed2005

RNA interference remarkably suppresses bcl-2 gene expression in cancer cells in vitro and in vivo.

Bcl-2 is an anti-apoptotic protein. If the level of Bcl-2 protein can be reduced sufficiently in tumors using RNA interference (RNAi) to target the gene message, the apoptosis of tumor cells may be promoted. In this study, we synthesized 19 nucleotides (nts) small interference RNA (siRNA) constructs suppressing bcl-2 gene expression in human tumor cells (HeLaB2 and BGC-823 cell lines) in vitro. The bcl-2 gene expression levels were significantly reduced when these siRNA were transfected into experimental two tumor cells for 72 hours. The apoptosis process was also examined in the tumor cells. Here we synthesized siRNA from a DNA template under the control of the RNA polymerase III promoter in transfected tumor cells. Using this DNA vector-based approach, we found that the siRNA efficiently and specifically inhibited the synthesis of protein encoded by the bcl-2 gene in HeLaB2 and BGC-823 tumor cells. Tumor growth was inhibited by 66.5% with 2mg/kg pSilencer 3.1H1-bcl-2 in mouse liver tumor-bearing BALB/c mice. This approach may prove to be a valuable clinical technique for the analysis of specific gene functions and gene therapy of malignant tumors that utilize the bcl-2 gene via RNA interference.

Animals↗
PubMed2004

[Advances in the molecular mechanism of RNA interference].

This paper reviews the latest development of RNA interference(RNAi). RNA interference is the process of sequence-specific degradation of homologous mRNA triggered by double-stranded RNA. As a technically simple and an effective genetic tool which can exert effect on the expression of gene and substitute for gene knock-out technique in some degree, RNAi phenomena have been broadly validated in diverse model organisms such as Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Neurospora crassa. Simultaneously, study on molecular mechanism of RNAi, which might be involved in the level of post-transcription, translation, genome methylation or conduction of silencing signals, is now making unceasing progress. Clear elucidation of the molecular mechanism could provide important theoretical references and powerful tools for the practical application in this field where RNAi may be put into the use of systematic gene screening, the discovery of new genes and the gene therapy for human tumor or other refractory human diseases.

Animals↗
PubMed2004

[RNA interference: mechanism and applications].

Gene expression in eukaryotes is controlled at transcriptional and post-transcriptional levels by RNA dependent mechanism called as "RNA Interference" or "RNA Inhibition". Small inhibitory RNAs (siRNAs) formed by the cleavage of double stranded RNAs activate the mechanisms which control cellular activities in organisms as diverse as protozoa, fungi, plants and primates. RNA interference participates in cellular functions such as heterochromatin formation, controlled DNA elimination, defence against foreign nucleic acids, maintenance of expression patterns via conserved proteins. Its effect is also generalized and amplified in some organisms. RNA interference is widely used as a biotechnological method for gene function studies and can also be potentially employed as a gene therapy approach for certain autoimmune, neoplastic and infectious diseases. In this review, the mechanism of RNA interference and its therapeutic applications have been discussed, focusing on viral diseases.

Animals↗
PubMed2003

RNA interference: antiviral weapon and beyond.

RNA interference (RNAi) is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA. The term encompasses related pathways found in a broad range of eukaryotic organisms, including fungi, plants, and animals. RNA interference is part of a sophisticated network of interconnected pathways for cellular defense, RNA surveillance, and development and it may become a powerful tool to manipulate gene expression experimentally. RNAi technology is currently being evaluated not only as an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop specific dsRNA based gene-silencing therapeutics. Several laboratories have been interested in using RNAi to control viral infection and many reports in Nature and in Cell show that short interfering (si) RNAs can inhibit infection by HIV-1, polio and hepatitis C viruses in a sequence-specific manner. RNA-based strategies for gene inhibition in mammalian cells have recently been described, which offer the promise of antiviral therapy.

Animals↗