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Results for “Small interfering RNA”

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PubMed2004

Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing.

In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing.

Arabidopsis↗

PubMed2005

DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal.

In RNA interference, the RNase-III enzyme Dicer processes exogenous double-stranded RNA into small interfering RNAs (siRNAs). siRNAs guide RNA-induced silencing complexes to cleave homologous transcripts, enabling gene-specific knock-down. In plants, double-stranded RNA is processed into siRNA species of 21 nucleotides (nt) and 24 nt (ref. 5), but, unlike in nematodes, the Dicer enzymes involved in this processing have not been identified. Additionally, in both plants and nematodes, systemic signals with RNA components convey the sequence-specific effects of RNA interference between cells. Here, we describe Arabidopsis thaliana mutants with altered silencing cell-to-cell movement beyond the vasculature. At least three SILENCING MOVEMENT DEFICIENT genes (SMD1, SMD2 and SMD3) are required for trafficking, the extent of which correlates with siRNA levels in the veins. Five alleles defective in synthesis of 21-nt, but not 24-nt, siRNAs carry mutations in Dicer-like 4 (DCL4) that are involved in biogenesis of trans-acting siRNAs. We show that the biogenesis and function of trans-acting siRNA can be genetically uncoupled from a bona fide DCL4-dependent pathway that accounts for RNA interference and for production of the 21-nt siRNA component of the plant cell-to-cell silencing signal.

Alleles↗

PubMed2004

New paradigms for gene transfer: RNA trans-splicing and small interfering RNA as therapeutic strategies.

If successful, the sustained and regulated expression of therapeutic proteins secreted from a variety of tissues would revolutionize the medical treatment of hematologic diseases. The current paradigm that has dominated the gene therapy field since its inception has been the transfer of complementary DNAs (cDNAs) that encode for therapeutic proteins. The transfer of cDNAs can only correct autosomal recessive and sex-linked disorders. In most cases, cDNAs are constructed that lack their endogenous regulatory elements and therefore lose their intrinsic regulation of gene expression. In this article we will describe the use of RNA species to either suppress unwanted gene activity or to repair defective genes. Examples of RNA inhibition and repair will be discussed.

Gene Transfer Techniques↗

PubMed2004

Small interfering RNA molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism.

RNA silencing or interference (RNAi) is a sequence-specific, post-transcriptional process of mRNA degradation. The degradation of target gene mRNA can be induced by short dsRNA molecules (21-25-nt) corresponding to the sequence of the target gene to be silenced. Short dsRNA molecules have been shown to be very effective in inducing RNA silencing in several human cell lines. In this study, we have shown that short dsRNA molecules corresponding to the human rhinovirus-16 (HRV-16) genome induce effective inhibition of the viral replication in cell culture. This inhibition is sequence-specific and dose-dependent. A single or double nucleotide sequence change in an effective dsRNA molecule can significantly reduce the ability of the molecule to induce RNA silencing. Reducing the length of siRNA molecules to 19-nt or shorter abolishes their activity. Therefore, the results of this study demonstrate certain siRNA molecules are inhibitory for the replication of HRV-16 when transfected into human cells; further studies are warranted to explore the potential clinical value of these siRNA molecules as anti-human rhinovirus agents.

Antiviral Agents↗

PubMed2004

Prophylactic and therapeutic effects of small interfering RNA targeting SARS-coronavirus.

OBJECTIVES: To identify and characterize the siRNA duplexes that are effective for inhibition of SARS-CoV infection and replication in the non-human primate cells. This in vitro study will serve as the foundation for development of novel anti-SARS therapeutics. METHODS: 48 siRNA sequences were designed for targeting regions throughout entire SARS-CoV genome RNA including open-reading frames for several key proteins. Chemically synthesized siRNA duplexes were transfected into foetal rhesus kidney (FRhK-4) cells prior to or after SARS-CoV infection. The inhibitory effects of the siRNAs were evaluated for reductions of intracellular viral genome copy number and viral titres in the cell culture medium measured by Q-RT-PCR and CPE-based titration, respectively. Four siRNA duplexes were found to achieve potent inhibition of SARS-CoV infection and replication. A prolonged prophylactic effect of siRNA duplexes with up to 90% inhibition that lasted for at least 72 h was observed. Combination of active siRNA duplexes targeting different regions of the viral genome resulted in therapeutic activity of up to 80% inhibition. CONCLUSION: Chemically synthesized siRNA duplexes targeting SARS-CoV genomic RNA are potent agents for inhibition of the viral infection and replication. The location effects of siRNAs were revealed at both genome sequence and open-reading frame levels. The rapid development of siRNA-based SARS-CoV inhibitors marked a novel approach for combating newly emergent infectious diseases.

Animals↗

PubMed2006

Use of herpes simplex virus type 1-based amplicon vector for delivery of small interfering RNA.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. The use of DNA-based plasmid vectors to achieve transient and stable expression of siRNA has been developed to avoid the problems of double-stranded oligonucleotides transfection. These vectors direct the transcription of small hairpin RNAs (shRNAs) from a polymerase-III (H1 or U6)-RNA gene promoter. However, numerous disadvantages remain, including low transfection efficiency and difficulty in transfecting primary cells. To overcome some of these problems, the use of viral vectors for siRNA delivery has been described. Retroviral, adenoviral, adeno-associated and herpes viral shRNAs delivery systems have been successfully used to silence genes, in vitro and in vivo. The use of a herpes simplex virus type 1 (HSV-1)-based amplicon vector for siRNA delivery into mammalian cells, using human polyomavirus BK (BKV)-transformed cells as a model system is described. The results demonstrate the ability of amplicon vectors to inhibit the expression of BKV T-Ag and tumorigenicity of BKV-transformed cells. We show that the use of the amplicon vector is highly efficient for the delivery of siRNA molecules. The unique ability of these vectors to deliver multiple copies of siRNA may provide a useful tool in the development of novel anticancer therapy.

Animals↗

PubMed2003

Lack of efficacy of 'naked' small interfering RNA applied directly to rat brain.

UNLABELLED: The intrastriatal infusions of 'naked' small interfering RNA (siRNA) targeted to dopamine D1 receptors (1.0-10.0 nmol over 3 days) did not reduce dopamine D1 receptor messenger RNA levels or receptor protein, assessed by [125I] SCH 23982 binding in intact rats. This was in contrast to results in vitro where a 76% reduction in dopamine D1 receptor ligand binding could be observed. CONCLUSION: The results suggest that synthetic siRNA, when applied directly to rat brain, is not capable of inducing RNA interference.

Animals↗

PubMed2005

[Inhibition of mouse hepatocyte apoptosis by anti-caspase-12 small interfering RNA].

OBJECTIVES: To study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs) against caspase-12. METHODS: The Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type IV, and apoptosis were induced with 4 micromol/L thapsigargin (TG). The three kingds of siRNAs targeting different gene sites (130, 214, 521) were synthetized chemically. The single-stranded RNAs were annealed to produce double-stranded siRNAs, then the mouse primary hepatocytes were transfected by oligofectamine package. The inhibition of caspase-12 was analyzed with RT-PCR and Western-blot. The viable hepatocytes following the induction of apoptosis were evaluated with MTT. RESULTS: All the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA. The siRNA (214) were more effective than the other two when the concentration was 100 nmol/L. The caspase-12 mRNA expression was inhibited by 52.08%, while that of siRNA (521) was 30.73% (t=4.30, P <0.05). However when the concentration was 200 nmol/L, the inhibitions were similar (88.07%, 86.22% and 89.41% respectively). siRNA (214) could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43% ( t=4.30, P <0.01). Contrasted with apoptotic hepatocytes, the cell activity, which was analyzed with MTT, increased by 48.76% (t=2.23, P <0.01). CONCLUSION: siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.

Animals↗

PubMed2005

Small interfering RNA inhibits SARS-CoV nucleocapsid gene expression in cultured cells and mouse muscles.

SARS-CoV is a newly identified coronavirus that causes severe acute respiratory syndrome (SARS). Currently, there is no effective method available for prophylaxis and treatment of SARS-CoV infections. In the present study, the influence of small interfering RNA (siRNA) on SARS-CoV nucleocapsid (N) protein expression was detected in cultured cells and mouse muscles. Four siRNA expression cassettes driven by mouse U6 promoter targeting SARS-CoV N gene were prepared, and their inhibitory effects on expression of N and enhanced green fluorescence protein (EGFP) fusion protein were observed. A candidate siRNA was proved to down-regulate N and EGFP expression actively in a sequence-specific manner. The expression vector of this siRNA was constructed and confirmed to reduce N and EGFP expression efficiently in both cultured cells and adult mouse muscles. Our findings suggest that the siRNA should provide the basis for prophylaxis and therapy of SARS-CoV infection in human.

Animals↗

PubMedDate not supplied

Use of lentiviral vectors for delivery of small interfering RNA.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. Although in the original studies expression of siRNA in mammalian cells was achieved via the transfection of double stranded oligonucleotides, subsequent studies described the use of plasmids to achieve long-term and stable expression of siRNA. Recently, several groups have described the use of retroviral vectors for siRNA delivery. However, retroviral vectors require active cell division for gene transfer and also suffer from the problem of gene-silencing. In this report we have modified a commercially available self-inactivating lentiviral vector for the delivery of siRNA into mammalian cells. We demonstrate the ability of this modified vector to efficiently transfer siRNA into HeLa S3 cells and downregulate p53 expression. Our results suggest that lentiviruses are efficient vectors for delivery of siRNA into mammalian cells. Based on the known ability of these vectors to infect both dividing and non-dividing cells and achieve long-term multilineage gene expression, their use as a therapeutic tool for the delivery of siRNA deserves further study.

DNA Primers↗

PubMed2005

Development of new EBV-based vectors for stable expression of small interfering RNA to mimick human syndromes: application to NER gene silencing.

We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPA(KD) and XPC(KD) (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced (HSA)KIN17. Several clones displaying undetectable protein levels of XPA, XPC, or (HSA)kin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, (HSA)KIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPA(KD) and XPC(KD) HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPC(KD) HeLa clones were more sensitive to UVC than their XPA(KD) or KIN17(KD) counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPA(KD) cells recovered a normal UVC sensitivity, XPC(KD) cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, (HSA)kin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles.

Blotting, Southern↗

PubMed2004

Enhancement of hypoxia-induced tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1alpha.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is an important transcriptional factor that is activated when mammalian cells experience hypoxia, a tumor microenvironmental condition that plays pivotal roles in tumor progression and treatment. In this study, we examined the idea of down-regulating HIF-1alpha in tumor cells for therapeutic gain. We show that the expression levels of HIF-1alpha can be significantly attenuated by use of the recently established small interfering RNA technology in combination with adenovirus-mediated gene transfer. Down-regulation of the HIF-1alpha protein enhanced hypoxia-mediated tumor cell apoptosis in vitro. Subcutaneous tumor growth was also prevented from cells with attenuated HIF-1alpha expression. In addition, intratumoral injection of adenovirus encoding the HIF-1alpha-targeted small interfering RNA had a small but significant effect on tumor growth when combined with ionizing radiation. Therefore, our results provide proof of HIF-1alpha as an effective target for anticancer therapy. They also suggest that an adenovirus-based small interfering RNA gene transfer approach may be a potentially effective adjuvant strategy for cancer treatment.

Apoptosis↗

PubMed2006

Local knockdown of genes in the brain using small interfering RNA: a phenotypic comparison with knockout animals.

BACKGROUND: Recent reports have suggested effectiveness of RNA interference (RNAi) for the analysis of gene functions in the brain. This study sought to determine the efficiency of local small interfering RNA (siRNA) injections, comparing this approach with animals generated through classical gene targeting. METHODS: Small interfering RNA against dopamine transporter (DAT) (35 microg/14 days) or tyrosine hydroxylase (TH) (15 microg/3 days) was injected into the ventral tegmental/substantia nigra areas of the brain of adult wildtype or DAT-knockout mice, respectively. RESULTS: Local injections of siRNA resulted in a 35% to 40% reduction of DAT and TH protein levels in the striatum, respectively. Despite negligible effect of DAT knockdown on novelty-induced locomotion, the locomotor response of DAT siRNA treated animals to amphetamine was blunted similar to what is observed in the DAT heterozygote animals. Since incomplete reduction of TH levels in normal mice does not produce behavioral effects, TH siRNA experiments were carried out in DAT-knockout animals that show increased dependence on newly synthesized dopamine. Knockdown of TH in these animals resulted in reduced basal locomotion. CONCLUSIONS: Local injection of siRNA in the brain reduced gene expression by 40% to 50%, suggesting that siRNA-mediated knockdown of genes in the brain can be a complementary tool to classical transgenesis for the analysis of gene functions.

Animals↗

PubMed2003

Specific gene inhibition by adenovirus-mediated expression of small interfering RNA.

Specific gene inhibition in mammalian cells can be achieved by the use of small interfering RNA molecules (siRNA). These siRNA molecules can be chemically synthesized and transfected into cells, or directly expressed intracellularly from a plasmid DNA by the function of the cellular RNA polymerase III. We report here that the latter concept can be incorporated into an adenovirus vector to achieve specific gene inhibition in mammalian cells. As an examination of this approach, we have prepared an adenoviral vector capable of expressing siRNA molecules targeting p53 or VprBP/KIAA0800, a cellular protein that interacts with the HIV auxiliary protein Vpr. In both cases, specific reduction in the target protein level was observed after adenoviral infection. The reduction in the protein level was correlated with a specific reduction in the mRNA level. Since many cell types can be efficiently infected by adenoviruses, adenoviral vectors may serve as a useful alternative to other methods for siRNA delivery and gene inhibition, especially when the target cells are refractory to transfection by DNA or RNA.

Adenoviridae↗

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↗

PubMed2006

Small interfering RNA delivery to the liver by intravenous administration of galactosylated cationic liposomes in mice.

Although small interfering RNA (siRNA) is a potentially useful therapeutic approach to silence the targeted gene of a particular disease, its use is limited by its stability in vivo. For the liver parenchymal cell (PC)-selective delivery of siRNA, siRNA was complexed with galactosylated cationic liposomes. Galactosylated liposomes/siRNA complex exhibited a higher stability than naked siRNA in plasma. After intravenous administration of a galactosylated liposomes/siRNA complex, the siRNA did not undergo nuclease digestion and urinary excretion and was delivered efficiently to the liver and was detected in PC rather than liver non-parenchymal cells (NPC). Endogenous gene (Ubc13 gene) expression in the liver was inhibited by 80% when Ubc13-siRNA complexed with galactosylated liposomes was administered to mice at a dose of 0.29 nmol/g. In contrast, the bare cationic liposomes did not induce any silencing effect on Ubc13 gene expression. These results indicated that galactosylated liposomes/siRNA complex could induce gene silencing of endogenous hepatic gene expression. The interferon responses by galactosylated liposomes/siRNA complex were controlled by optimization of the sequence of siRNA. Also no liver toxicity due to galactosylated liposomes/siRNA complex was observed under any of the conditions tested. In conclusion, we demonstrated the hepatocyte-selective gene silencing by galactosylated liposomes following intravenous administration.

Animals↗

PubMedDate not supplied

The use of small interfering RNA to elucidate the activity and function of ion channel genes in an intact tissue.

Small interfering RNA (siRNA) directs the targeted destruction of mRNA encoding a specific protein, in a process known as RNA interference (RNAi). This stops translation of the targeted mRNA into protein, effectively silencing the gene. RNAi is a recent discovery, identified in mammalian cells in 2001, but it has rapidly advanced into a practical technique and is being used increasingly to investigate mammalian gene function. Tools are available to induce RNAi in cell lines, intact tissue preparations and even in vivo. Depending on the method used, loss of gene expression may be transient or sustained, enabling a wide range of functions to be investigated. RNAi therefore offers a powerful technique that can be used to produce targeted knockout of ion channel genes in mammalian cells. Its applications potentially include identification of ion channel function in health and disease, identification of novel channel genes and drug target validation. This paper outlines our current understanding of siRNA and the experimental requirements for producing efficient RNAi and gene silencing. Effective RNAi requires an appropriate siRNA sequence to be designed and an efficient method for delivering the siRNA to the cells of interest. Since not all potential siRNA sequences are effective, it is also important to verify the loss of gene expression by measuring the level of channel protein remaining. Limitations of the methods available for delivering siRNA are one of the main obstacles to producing efficient RNAi, especially in intact tissue preparations. Here we describe an in vitro method for targeted RNAi against the TASK-1 potassium channel gene in an isolated vascular preparation, using a DNA construct to direct the expression of siRNA, along with a non-viral method for transfecting cells within the vessel. Successful silencing of the TASK-1 gene is verified by immunostaining with an antibody directed against the TASK-1 protein.

Blood Vessels↗

PubMed2005

Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells.

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.

Antiviral Agents↗