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PubMed2005

The RNA interference revolution.

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

Animals↗
PubMed2004

siRNAs: mechanism of RNA interference, in vivo and potential clinical applications.

Small interfering RNAs are currently the most widely used nucleic acid-based sequence-specific gene silencing molecules. These molecules mediate RNA interference--a natural post-transcriptional gene-silencing pathway. Given the high reliability and higher efficiency of small interfering RNA-mediated RNA interference, compared to earlier reverse genetic technologies, this is now the preferred technique in functional genomics. Furthermore, the exquisite specificity and exceptional gene-silencing potency of small interfering RNAs has resulted in intense research related to potential target-specific therapeutic applications of these molecules. This review will discuss the mechanism of RNA interference and applications of the pathway in molecular biology including functional genomics will be overviewed. The article will outline in vivo and potential clinical applications of small interfering RNA molecules.

Gene Targeting↗
PubMed2005

RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays.

RNA interference (RNAi) is a process in which double-strand RNA (dsRNA) directs the specific degradation of a corresponding target mRNA. The mediators of this process are small dsRNAs, of approximately 21 bp in length, called small interfering RNAs (siRNAs). siRNAs, which can be prepared in vitro in a number of ways and then transfected into cells, can direct the degradation of corresponding mRNAs inside these cells. Hence, siRNAs represent a powerful tool for studying gene functions, as well as having the potential of being highly specific pharmaceutical agents. Some limitations in using this technology exist because the preparation of siRNA in vitro and screening for siRNAs efficient in RNAi can be expensive and time-consuming processes. Here, we demonstrate that custom oligonucleotide arrays can be efficiently used for the preparation of defined mixtures of siRNAs for the silencing of exogenous and endogenous genes. The method is fast, inexpensive, does not require siRNA optimization and has a number of advantages over methods utilizing enzymatic preparation of siRNAs by digestion of longer dsRNAs, as well as methods based on chemical synthesis of individual siRNAs or their DNA templates.

Green Fluorescent Proteins↗
PubMed2003

Vector-based in vivo RNA interference: dose- and time-dependent suppression of transgene expression.

RNA interference (RNAi) induced by delivery of a small-interfering RNA (siRNA)-expressing vector was characterized in mice. siRNA-expressing plasmid DNA (pDNA) was injected by a hydrodynamics-based procedure along with pDNA encoding an exogenous target luciferase gene. A comparative study showed that stem-loop-type siRNA-expressing pDNA was superior, in terms of the transgene suppressive efficacy, to the tandem-type in the liver following systemic delivery of these pDNAs. Transgene suppression occurred in the liver, kidney, and lung as well as muscle. The degree of suppression was dependent on the dose of siRNA-expressing pDNA and the time at which transgene expression was determined following simultaneous injection of siRNA-expressing and target pDNAs. A reduction in transgene expression became apparent at 1 day after injection, whereas a lower degree of inhibition was obtained before this, as early as 6 h even in mice treated with an excess of siRNA-expressing pDNA. These results suggest that delivery of siRNA-expressing pDNA requires a period of time for induction of RNAi. A study of sequential injections revealed that prior injection of siRNA-expressing pDNA produced a significant suppression for at least 1 day, which disappeared within 4 days. Confocal microscopic studies indicated that the localization of the cells with successful delivery of transgene was different between primary and secondary hydrodynamics-based injections, accounting for the less effective inhibition following the sequential injections. Taken together, these results demonstrate that vector-based in vivo RNAi is a dose- and time-dependent process and offers the possibility of suppressing endogenous targets in a variety of somatic cells.

Animals↗
PubMed2003

Human immunodeficiency virus type 1 escape from RNA interference.

Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.

Cell Line↗
PubMed2006

RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding.

RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene (EposCXE1) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene (EposPBP1) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of EposPBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed.

Animals↗
PubMed2005

Fast screening target sites for RNA interference using a cell-free system.

A fast and simple procedure to screen target sites for RNA interference by using RNA in a cell-free system of Hela cells, and then evaluating the efficiency by Northern blotting, is described. This procedure produces results with an identical reliability compared to those described previously but which are more time-consuming than this present method.

Biotechnology↗
PubMed2006

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf.

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitf-specific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

Animals↗
PubMed2003

Silencing of Sm proteins in Trypanosoma brucei by RNA interference captured a novel cytoplasmic intermediate in spliced leader RNA biogenesis.

In Trypanosoma brucei the small nuclear (sn) RNAs U1, U2, U4, and U5, as well as the spliced leader (SL) RNA, bind the seven Sm canonical proteins carrying the consensus Sm motif. To determine the function of these proteins in snRNA and SL RNA biogenesis, two of the Sm core proteins, SmE and SmD1, were silenced by RNAi. Surprisingly, whereas the level of all snRNAs, including U1, U2, U4, and U5 was reduced during silencing, the level of SL RNA was dramatically elevated, but the levels of U6 and spliced leader-associated RNA (SLA1) remained unchanged. The SL RNA that had accumulated in silenced cells lacked modification at the cap4 nucleotide but harbored modifications at the cap1 and cap2 nucleotides and carried the characteristic psi. This SL RNA possessed a longer tail and had accumulated in the cytoplasm in 10 and 50 S particles that were found by in situ hybridization to be present in "speckles." We propose a model for SL RNA biogenesis involving a cytoplasmic phase and suggest that the trypanosome-specific "cap4" nucleotides function as a signal for export and import of SL RNA out and into the nucleus. The SL RNA biogenesis pathway differs from that of U sn ribonucleoproteins (RNPs) in that it is the only RNA that binds Sm proteins that were stabilized under Sm depletion in a novel RNP, which we termed SL RNP-C.

Active Transport, Cell Nucleus↗
PubMed2005

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase.

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 microg/microL. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Animals↗
PubMed2000

Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target.

A sensitive cellular assay system for RNA interference was developed using the firefly luciferase gene as target. RNA interference was noted not only in Drosophila cultured cells but Chinese hamster cells (CHO-K1) as well, although double-stranded RNA required for the latter was 2500 times more than for the former. Cognate double-stranded RNA as short as 38 bp was found to be still capable of inducing RNA interference in Drosophila cultured cells.

Animals↗
PubMed2005

About the nature of RNA interference.

In the context of yet unclarified issues of RNA interference (RNAi), it is discussed that RNAi-induced histone modification may not only have the purpose of inactivating native genes by blocking their transcription in the sense direction but may also simultaneously trigger transcription of the corresponding antisense strand to form double-stranded RNA for posttranscriptional gene-silencing in cells lacking RNA replicase activities. Invading foreign genetic traits may be posttranscriptionally silenced through complementary transcripts from specific, highly variable genomic regions, which are able to finally match any given sequence by the appropriate recombination and processing of their transcripts. The information to fight these traits may additionally become anchored in the genome, to provide at least a temporary "immunity" and may be inherited at least for a few generations. It is further proposed that: (1) RNA viruses evolved from constituents of the RNAi machinery through the capture of functions essential for their maintenance and replication and (2) viruses and RNAi are mutually interacting components of a universal and predominant genetic steering system that is involved in the modulation of gene expression on the cellular level and simultaneously constitutes a driving force for evolution, particularly in imperfect organisms. Such a model would deliver explanations for yet unresolved issues of RNAi, the clarification of which will have a significant impact on its future medical and biotechnological application.

Animals↗
PubMed2006

Therapeutic potential of RNA interference for neurological disorders.

During the past decade, numerous molecular mediators of neurodegenerative diseases and neurological disorders have been identified and validated, yet few novel therapies have emerged and the unmet medical needs remain high. These molecular mediators belong to target classes such as ion channels, neurotransmitters and neurotransmitter receptors, cytokines, growth factors, enzymes and other proteins. In some cases, substantial pre-clinical validation exists, but the molecular target has not been readily druggable with small molecules, proteins or antibodies. RNA interference represents a therapeutic approach applicable to such non-druggable targets. Both non-viral and viral delivery strategies are being undertaken for in vivo silencing of molecular targets by RNA interference, which has resulted in robust efficacy in animal models of Alzheimer's disease, ALS, Huntington's disease, spinocerebellar ataxia, anxiety, depression, neuropathic pain, encephalitis and glioblastoma. These proof-of-concept data in animal models, together with the commencement of clinical trials using RNA interference for macular degeneration and respiratory syncytial virus infection, point to the potential of direct RNA interference for neurological disorders and neurodegenerative diseases.

Animals↗
PubMed2004

RNA interference for the identification of disease-associated genes.

RNA interference (RNAi) has emerged as a novel cellular mechanism regulating gene expression at the post-transcriptional level and as a powerful tool to control gene function experimentally. Recent advances in the biology and application of RNAi include the definition of improved criteria for selecting effective small interfering RNA (siRNA) sequences, and the generation of vectors for the delivery of siRNAs and stable silencing of genes in mammalian cells, tissues and animals. High-throughput screening projects based on RNAi have been initiated to search for genes involved in basic biological processes and in complex pathological conditions such as cancer, autoimmunity and degenerative disorders. This research is helping to identify novel therapeutic targets for a range of diseases and may translate into novel clinical applications for RNAi.

Animals↗
PubMed2006

Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction.

BACKGROUND: Hepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro. METHODS: RNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively. RESULTS: Here we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1 knockdown significantly augmented apoptosis sensitivity of HCC cells towards chemotherapy combined with PI3K inhibition. CONCLUSION: Our data suggest that specific downregulation of Mcl-1 by RNA interference is a promising approach to sensitize HCC cells towards chemotherapy and molecularly targeted therapies.

Antineoplastic Agents↗
PubMed2006

Lentivirus-mediated RNA interference therapy for human immunodeficiency virus type 1 infection.

RNA interference (RNAi) is a natural mechanism by which small interfering RNAs (siRNAs) operate to specifically and potently downregulate the expression of a target gene. This downregulation has been demonstrated by targeting siRNAs to the mRNA (posttranscriptional gene silencing) as well as to the gene promoter, regulating gene expression epigenetically by transcriptional gene silencing. These observations significantly broaden the role RNA plays in the cell and suggest that siRNAs could prove to be a potent future therapeutic for the treatment of diseases such as human immunodeficiency virus type 1 (HIV-1) infection. The specificity and simplicity of design and the ability to express siRNAs from mammalian promoters make the use of siRNAs to target and suppress virtually any gene or gene promoter of interest a soon-to-be-realized technology. However, the delivery and stable expression of siRNAs to target cells remain an enigma that could be surmounted, at least regarding the treatment of HIV-1 infection, by the application of lentiviral vectors to deliver and express anti-HIV-1 siRNAs in target cells. This review focuses on the development, delivery, and potential therapeutic use of antiviral siRNAs in treating HIV-1.

Genetic Therapy↗
PubMed2005

Targeting 2A protease by RNA interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice.

BACKGROUND: Enteroviridae such as coxsackievirus B3 (CVB3) are important infectious agents involved in viral heart disease, hepatitis, and pancreatitis, but no specific antiviral therapy is available. METHODS AND RESULTS: The aim of the present study was to evaluate the impact of RNA interference on viral replication, cytopathogenicity, and survival. Small interfering RNA (siRNA) molecules were designed against the viral 2A region (siRNA-2A), which is considered to be highly conserved and essential for both virus maturation and host cytopathogenicity. siRNA-2A exhibited a significant protective effect on cell viability mediated by marked inhibition of CVB3 gene expression and viral replication. In highly susceptible type I interferon receptor-knockout mice, siRNA-2A led to significant reduction of viral tissue titers, attenuated tissue damage, and prolonged survival. Repeated siRNA-2A transfection was associated with a further improvement of survival. Various control siRNA molecules had no protective effect in vitro or in vivo. CONCLUSIONS: RNA interference directed against the 2A protease encoding genomic region effectively confers intracellular immunity toward CVB3-mediated cell injury and improves survival, suggesting a potential role for RNA interference for future treatment options targeting enteroviral diseases.

Animals↗