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At least 73 records · Page 4Linked to original sources
PubMed2006

Antiviral RNAi therapy: emerging approaches for hitting a moving target.

The field of directed RNA interference (RNAi) has rapidly developed into a highly promising approach for specifically down regulating genes to alleviate disease pathology. This technology is especially well-suited to treating viral infections, and numerous examples now illustrate that a wide range of viruses can be inhibited with RNAi, both in vitro and in vivo. One principle that has arisen from this work is that antiviral RNAi therapies must be tailored to the unique life cycle of each pathogen, including the choice of delivery vehicle, route of administration, gene(s) targeted and regulation and duration of RNAi induction. Although effective strategies will be customized to each virus, all such therapies must overcome similar challenges. Importantly, treatment strategies must compensate for the inevitable fact that viral genome sequences evolve extremely rapidly, and computational and bioinformatics approaches may aid in the development of therapies that resist viral escape. Furthermore, all RNAi strategies involve the delivery of nucleic acids to target cells, and all will therefore benefit from the development of enhanced gene design and delivery technologies. Here, we review the substantial progress that has been made towards identifying effective antiviral RNAi targets and discuss strategies for translating these findings into effective clinical therapies.

Genetic Engineering↗
PubMed2004

RNAi-mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion, angiogenesis and tumor growth in gliomas.

RNA interference (RNAi) provides a powerful method for gene silencing in eukaryotic cells, including proliferating mammalian cells. Here, we determined whether RNAi could be utilized to inhibit the expression of proteases implicated in the extracellular matrix degradation, which is characteristic of tumor progression. We have previously shown that antisense stable clones of uPAR and cathepsin B were less invasive and did not form tumors when injected intracranially ex vivo. Since antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high molar concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPAR and cathepsin B in this study. We found that the expression of double-stranded RNA leads to the efficient and specific inhibition of endogenous uPAR and cathepsin B protein expression in glioma cell lines as determined by Western blotting. We also found the RNAi of uPAR and cathepsin B reduces glioma cell invasion and angiogenesis in in vitro and in vivo models. Intratumoral injections of plasmid vectors expressing hpRNA for uPAR and cathepsin B resulted in the regression of pre-established intracranial tumors. Further, RNAi for uPAR and cathepsin B inhibited cell proliferation and reduced the levels of pERK and pFAK compared to controls. Taken together, our findings indicate for the first time that RNAi operates in human glioma cells with potential application for cancer gene therapy.

Cathepsin B↗
PubMed1986

Cloning and characterization of rat 4.5S RNAI genes.

Genomic clones containing genes for 4.5S RNAI, which is an abundant small nuclear RNA found in rodent cells, were obtained from a rat genomic library. Thirty-four clones that formed RNase A resistant hybrids with 3'-end-labeled 4.5S RNAI were isolated, and seven of them (clones lambda I39, lambda I41, lambda I42, lambda I51, lambda I106, lambda I123 and lambda I154) were characterized by sequencing and in vitro transcription. Clones lambda I41 and lambda I123 carry one and two genes, respectively, with identical sequences to that of 4.5S RNAI and are actively transcribed in vitro. However, the other five clones contain sequences that seem to be pseudogenes for 4.5S RNAI, since they have nucleotide substitutions or deletions in the sequence corresponding to 4.5S RNAI or are not transcribed. Four clones (lambda I39, lambda I42, lambda I106 and lambda I154) were found to have 13-18 nucleotide-long direct repeats flanking the 4.5S RNAI sequences. The genomic organization of the genes and their related sequences is discussed.

Animals↗
PubMed2002

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins.

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.

Amino Acid Sequence↗
PubMed2004

Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library.

The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available, "phenoclustering" strategies can be used to begin understanding the complex molecular networks involved in development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing the possibilities for phenome mapping in C. elegans.

Animals↗
PubMed2006

Validation of RNAi silencing specificity using synthetic genes: salicylic acid-binding protein 2 is required for innate immunity in plants.

RNA interference (RNAi) is widely used to specifically silence the expression of any gene to study its function and to identify and validate therapeutic targets. Despite the popularity of this technology, recent studies have shown that RNAi may also silence non-targeted genes. Here we demonstrate the utility of a quick, efficient and robust approach to directly validate the specificity of RNAi as an alternative to indirect validation of RNAi through gene expression profiling. Our approach involves reversing (complementing) the RNAi-induced phenotype by introducing a synthetic version of the target gene that is designed to escape silencing. This synthetic gene complementation approach can also be used for mutational analysis of the target gene, or to provide a functional version of a defective protein after silencing the defective gene by RNAi. Using this approach we demonstrate that the loss of systemic acquired resistance, a form of innate immunity in plants, is indeed due to the silencing of salicylic acid-binding protein 2 rather than to off-target effects.

Esterases↗
PubMed2006

RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster.

Drosophila melanogaster has a robust and efficient innate immune system, which reacts to infections ranging from bacteria to fungi and, as discovered recently, viruses as well. The known Drosophila immune responses rely on humoral and cellular activities, similar to those found in the innate immune system of other animals. Recently, RNAi or 'RNA silencing' has arisen as a possible means by which Drosophila can react to a specific pathogens, transposons and retroviral elements, in a fashion similar to that of a traditional mammalian adaptive immune system instead of in a more generalized and genome encoded innate immune-based response. RNAi is a highly conserved regulation and defence mechanism, which suppresses gene expression via targeted RNA degradation directed by either exogenous dsRNA (cleaved into siRNAs) or endogenous miRNAs. In plants, RNAi has been found to act as an antiviral immune response system. Here we show that RNAi is an antiviral response used by Drosophila to combat infection by Drosophila X Virus, a birnavirus, as well. Additionally, we identify multiple core RNAi pathway genes, including piwi, vasa intronic gene (vig), aubergine (aub), armitage (armi), Rm62, r2d2 and Argonaute2 (AGO2) as having vital roles in this response in whole organisms. Our findings establish Drosophila as an ideal model for the study of antiviral RNAi responses in animals.

Animals↗
PubMed2002

Confirming specificity of RNAi in mammalian cells.

RNA interference (RNAi) is a process of sequence-specific gene silencing. Recent advances in the understanding of RNAi have provided practical tools to silence gene expression in mammalian cells, opening new possibilities for studying the functions of genes and proteins. It is important to ensure that an observed effect of RNAi is due to silencing of the intended target. Indeed, it is possible that an siRNA may silence more than one messenger RNA that is homologous in the region complementary to the siRNA. Considering that we know little about how RNAi works in mammalian cells, other artifacts may be yet to be recognized. Thus, we suggest approaches to rescue the effect of RNAi by ectopically expressing the protein of interest. These approaches involve introducing silent mutations into the complementary DNA of the protein and targeting RNAi to the untranslated regions of the gene.

3' Untranslated Regions↗
PubMed1994

Molecular analysis of RNAI control of repB translation in IncB plasmids.

The translation of RepA, the replication initiation protein of the IncB plasmid pMU720, requires that its mRNA (RNAII) folds to form a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. The formation of this pseudoknot is dependent in turn on the translation and correct termination of a leader peptide, RepB. A small countertranscript RNA, RNAI, controls the replication of pMU720 by interacting with RNAII to negatively regulate the expression of repA both directly, by sequestering the proximal bases required for pseudoknot formation, and indirectly, by inhibiting the translation of repB. Inhibition of the translation of repB by RNAI was found to depend on the close proximity of the RNAI-RNAII complex to the translational initiation region of repB, indicating that the primary mechanism of RNAI control involves steric hindrance. Disruption of RNAI control of repB had only a small effect on the copy number of the IncB plasmid, indicating that inhibition of the expression of repA by RNAI is achieved predominantly by inhibition of pseudoknot formation rather than by inhibition of repB translation.

Bacterial Proteins↗
PubMed2006

Generation of RNAi libraries for high-throughput screens.

The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi) has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA) with homology to endogenous genes in Caenorhabditis elegans (C elegans), RNAi technology has been adapted to various high-throughput screens (HTS) for genome-wide loss-of-function (LOF) analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.

Journal Article↗
PubMed2005

Silencing the epidermal growth factor receptor gene with RNAi may be developed as a potential therapy for non small cell lung cancer.

Lung cancer has emerged as a leading cause of cancer death in the world. Non-small cell lung cancer (NSCLC) accounts for 75-80% of all lung cancers. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. Double stranded RNA (dsRNA)-mediated RNA interference (RNAi) has shown promise in gene silencing, the potential of which in developing new methods for the therapy of NSCLC needs to be tested. We report here RNAi induced effective silencing of the epidermal growth factor receptor (EGFR) gene, which is over expressed in NSCLC. NSCLC cell lines A549 and SPC-A1 were transfected with sequence- specific dsRNA as well as various controls. Immune fluorescent labeling and flow cytometry were used to monitor the reduction in the production of EGFR protein. Quantitative reverse-transcriptase PCR was used to detect the level of EGFR mRNA. Cell count, colony assay, scratch assay, MTT assay in vitro and tumor growth assay in athymic nude mice in vivo were used to assess the functional effects of EGFR silencing on tumor cell growth and proliferation. Our data showed transfection of NSCLC cells with dsRNA resulted in sequence specific silencing of EGFR with 71.31% and 71.78 % decreases in EGFR protein production and 37.04% and 54.92% in mRNA transcription in A549 and SPC-A1 cells respectively. The decrease in EGFR protein production caused significant growth inhibition, i.e.: reducing the total cell numbers by 85.0% and 78.3%, and colony forming numbers by 63.3% and 66.8%. These effects greatly retarded the migration of NSCLC cells by more than 80% both at 24 h and at 48 h, and enhanced chemo-sensitivity to cisplatin by four-fold in A549 cells and seven-fold in SPC-A1. Furthermore, dsRNA specific for EGFR inhibited tumor growth in vivo both in size by 75.06% and in weight by 73.08%. Our data demonstrate a new therapeutic effect of sequence specific suppression of EGFR gene expression by RNAi, enabling inhibition of tumor proliferation and growth. However, in vivo use of dsRNA for gene transfer to tumor cells would be limited because dsRNA would be quickly degraded once delivered in vivo. We thus tested a new bovine lentiviral vector and showed lentivector-mediated RNAi effects were efficient and specific. Combining RNAi with this gene delivery system may enable us to develop RNAi for silencing EGFR into an effective therapy for NSCLC.

Journal Article↗
PubMed2006

Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans.

C. elegans contains numerous small RNAs of ~21-24 nt in length. The microRNAs (miRNAs) are small noncoding RNAs produced by DCR-1- and ALG-dependent processing of self-complementary hairpin transcripts. Endogenous small interfering RNAs (endo-siRNAs), associated with ongoing silencing of protein-coding genes in normal worms, are produced by mechanisms that involve DCR-1 but, unlike miRNAs, also involve RDE-2, RDE-3, RDE-4, RRF-1, and RRF-3. The tiny noncoding (tncRNAs) are similar to endo-siRNAs in their biogenesis except that they are derived from noncoding sequences. These endo-siRNA- and tncRNA-based endogenous RNAi pathways involve some components, including DCR-1 and RDE-4, that are shared with exogenous RNAi, and some components, including RRF-3 and ERI-1, that are specific to endogenous RNAi. rrf-3 and eri-1 mutants are enhanced for some silencing processes and defective for others, suggesting cross-regulatory interactions between RNAi pathways in C. elegans. Microarray expression profiling of RNAi-defective mutant worms further suggests diverse endogenous RNAi pathways for silencing different sets of genes.

Animals↗
PubMed2003

RNAi as a gene therapy approach.

RNA duplexes of 21 - 23 nucleotides (nts), with approximately 2 nt 3' overhangs (called small interfering RNAs or siRNAs), have recently been shown to mediate sequence-specific inhibition of gene expression in mammalian cells via a post-transcriptional gene silencing (PTGS) mechanism termed RNA interference (RNAi). RNAi has been rapidly adopted as a functional genomics tool in a wide range of species, has been adapted to allow for the transient or stable knockdown of gene expression generation in cell lines and animals, and has been developed for high-throughput analysis of gene function in Caenorhabditis elegans. With an increasing list of genes successfully knocked-down by RNAi in mammalian cells and improvements in the delivery of siRNAs to cells, including in vivo delivery to mice, attention is now turning to assessing the potential RNAi has as a gene therapy approach. RNAi is likely to have the greatest impact as a therapeutic tool in two key clinical areas, cancer and infectious disease, but it also has the potential as a therapy for other disorders including some dominant genetic diseases. This review will describe the status of the science behind this novel mechanism and will illustrate the therapeutic potential of RNAi-based technologies, using examples from these critical clinical research areas.

Animals↗
PubMed2006

Therapeutic potential of RNAi in metabolic diseases.

Over the past years RNA interference (RNAi) has exploded as a new approach to manipulate gene expression in mammalian systems. More recently, RNAi has acquired interest as a potential therapeutic strategy. This review focuses on the potential therapeutic use of RNAi for metabolic diseases, the current understanding of RNAi biology, and how RNAi has been utilized to study the role of different genes in the pathogenesis of diabetes and obesity. Also reviewed are the in vivo proof-of-principle experiments that provide the preclinical justification for the development of RNAi-based therapeutics for diabetes and the key challenges that currently limit its application in the clinical setting.

Gene Targeting↗
PubMed2005

[The application of RNAi to the medical genetics].

Experimental RNA interference (RNAi) leading to the selective knockdown of gene function is induced by introducing into cells either double stranded RNA (dsRNA), or short interfering RNA (siRNA) fragments into which dsRNA is cut. The siRNA triggers degradation of homologous messenger RNA (mRNA). Widely used as a research tool in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster and mouse to investigate the function of individual genes, RNAi has also been deployed in genome-wide, specific gene-knockdown screens. Recent rapid progress in the application of RNAi to mammalian cells, including neurons and muscle cells, offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAi-inducing molecules have raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Here, we review examples of the application of RNAi to worm, fly and mouse models of such diseases aimed at understanding their pathophysiology.

Gene Silencing↗
PubMed1988

Control of ColE1 replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII.

We have studied the interactions between the three molecules Rop, RNAI and RNAII that are involved in the regulatory mechanism controlling the replication of ColE1 plasmids. We show that it is possible to purify the two RNA molecules by passing an RNA mixture through an affinity column containing Rop immobilized to a solid support. The dissociation constants of the Rop-RNAI and Rop-RNAII complexes are of the order of 10(-4) M, several orders of magnitude higher than dissociation constants of stable protein-nucleic acid complexes (10(-10) M in the lambda repressor system). Although complete RNAI molecules have higher affinity, stem-and-loop I alone can also bind Rop, suggesting that this structure plays an important role in the interaction. Rop protects the stems of RNAI and RNAII from digestion by RNases while the sensitivity of the loops to digestion by RNase T1 is not affected by high concentrations of Rop. We propose a model for Rop-RNAI/RNAII interaction in which the dimeric protein acts as an adaptor between stem structures to position the two RNAs in the correct position for loop interaction.

Bacteriocin Plasmids↗
PubMed2003

In vivo knockdown of gene expression in brain cancer with intravenous RNAi in adult rats.

BACKGROUND: Gene silencing in mammalian cells is possible with RNA interference (RNAi) with expression plasmids that encode for short hairpin RNAs (shRNA) that hybridize to a specific sequence within a target mRNA. The limiting factor in developing RNAi therapeutics in mammals is the gene delivery system. METHODS: The present studies describe the production of anti-luciferase shRNA expression plasmids, which are encapsulated in the interior of 85 nm pegylated immunoliposomes (PILs). C6 rat glioma cells, permanently transfected with the luciferase gene, are implanted in the caudate-putamen nucleus of adult rats, which produces luciferase-expressing intracranial brain cancer. The PILs are targeted across the blood-brain barrier and across the tumor cell membrane in vivo with a monoclonal antibody (MAb) to the rat transferrin receptor (TfR). The TfRMAb is tethered to the tips of 1-2% of the poly(ethylene glycol) strands conjugated to the surface of the liposome. RESULTS: The TfRMAb-targeted PILs inhibit luciferase gene expression in the brain cancer by 90%, and this effect persists for at least 5 days after a single intravenous injection of 10 micro g/rat of plasmid DNA. RNAi therapy directed against the luciferase gene caused no change in expression of tumor gamma-glutamyl transpeptidase. Targeting the empty expression plasmid with the TfRMAb-PIL resulted in no change in luciferase activity in the brain cancer in vivo. CONCLUSIONS: In vivo RNAi is enabled with a new form of gene delivery system that encapsulates expression plasmids in PILs, which are targeted to distant sites based on the specificity of a receptor-specific monoclonal antibody. The combined application of the PIL gene delivery system and RNAi expression plasmids enables gene silencing in remote sites such as brain cancer in mammals after intravenous administration.

Animals↗
PubMed2001

RNAi As a tool for understanding germline development in Caenorhabditis elegans: uses and cautions.

RNA-mediated genetic interference (RNAi) has become a very useful tool for analyzing gene function in development and other processes. RNAi can be used as a complement to traditional genetic studies or as a primary means of determining biological function. However, the efficacy of RNAi depends on a variety of factors that the researcher must take into consideration. This review focuses on germline development in the nematode, Caenorhabditis elegans, and discusses the uses and limitations of RNAi in providing new information about gene function as well as the possible endogenous role RNAi plays in germline physiology.

Animals↗