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PubMed2004

RNA interference: a potent tool for gene-specific therapeutics.

RNA interference (RNAi) is a process through which double-stranded RNA induces the activation of cellular pathways, leading to potent and selective silencing of genes with homology to the double strand. Much excitement surrounding small interfering RNA (siRNA)-mediated therapeutics arises from the fact that this approach overcomes many of the shortcomings previously experienced with approaches such as antibodies, antisense oligonucleotides and pharmacological inhibitors. Induction of RNAi through administration of siRNA has been successfully used in treatment of hepatitis, viral infections, and cancer. In this review we will present a brief history of RNAi, methods of inducing RNAi, application of RNAi in the therapeutic setting, and the possibilities of using this highly promising approach in the context of transplantation.

Animals↗

PubMed2003

RNA interference targeting Fas protects mice from fulminant hepatitis.

RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. However, its potential to treat or prevent disease remains unproven. Fas-mediated apoptosis is implicated in a broad spectrum of liver diseases, where inhibiting hepatocyte death is life-saving. We investigated the in vivo silencing effect of small interfering RNA (siRNA) duplexes targeting the gene Fas (also known as Tnfrsf6), encoding the Fas receptor, to protect mice from liver failure and fibrosis in two models of autoimmune hepatitis. Intravenous injection of Fas siRNA specifically reduced Fas mRNA levels and expression of Fas protein in mouse hepatocytes, and the effects persisted without diminution for 10 days. Hepatocytes isolated from mice treated with Fas siRNA were resistant to apoptosis when exposed to Fas-specific antibody or co-cultured with concanavalin A (ConA)-stimulated hepatic mononuclear cells. Treatment with Fas siRNA 2 days before ConA challenge abrogated hepatocyte necrosis and inflammatory infiltration and markedly reduced serum concentrations of transaminases. Administering Fas siRNA beginning one week after initiating weekly ConA injections protected mice from liver fibrosis. In a more fulminant hepatitis induced by injecting agonistic Fas-specific antibody, 82% of mice treated with siRNA that effectively silenced Fas survived for 10 days of observation, whereas all control mice died within 3 days. Silencing Fas expression with RNAi holds therapeutic promise to prevent liver injury by protecting hepatocytes from cytotoxicity.

Animals↗

PubMed2003

[RNA interference is developmentally regulated: analysis of expression of suffix, a short Drosophila retroelement].

Sense and antisense transcripts of suffix and F element were detected at different stages of Drosophila development. A short RNA, similar in size to the full-length suffix transcript, was also found. It was suggested that it could originate from the master copy of the element. Though transcription pattern of these elements changed during the development, transcription remained symmetrical. These data indicate that formation of dsRNA in the cells and triggering of the RNA interference mechanisms are possible at all stages of Drosphila development. Analysis of total RNA samples from all stages of Drosophila development showed that 21- to 25-nt long suffix--specific small interfering RNAs (siRNA), the obligatory products of the RNA interference, were detected only in pupae. Thereafter, RNA interference mechanisms are developmentally regulated and the formation of dsRNA is necessary but not sufficient for launching this potent and specific machinery of post-transcription silencing.

Animals↗

PubMed2002

Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi).

Short 21-mer double-stranded RNA (dsRNA) molecules have recently been employed for the sequence-specific silencing of endogenous human genes. This mechanism, called RNA interference (RNAi), is extremely potent and requires only a few dsRNA molecules per cell to silence homologous gene mRNA expression. We used dsRNA targeting the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement. Transfection of dsRNA specific for the M-BCR/ABL fusion mRNA into K562 cells depleted the corresponding mRNA and the M-BCR/ABL oncoprotein. This was demonstrated by real-time quantitative PCR and Western blots. The BCR/ABL knockdown was accompanied by strong induction of apoptotic cell death. Leukemic cells without BCR/ABL rearrangement were not killed by M-BCR/ABL-dsRNA. In addition, to corroborate the extraordinary sequence specificity of RNAi, we designed another RNA oligo matching the M-BCR/ABL fusion site but having two point mutations within its central region. We show that these two point mutations abolished both p210 reduction and induction of apoptosis in K562 cells. Finally, we compared leukemic cell killing by RNAi to that caused by the ABL kinase tyrosine inhibitor, STI 571, Imatinib. For full induction of apoptosis, dsRNA targeting M-BCR/ABL required 24 h more than Imatinib. This may be caused by the relatively long half-life of the BCR/ABL oncoprotein, which is not targeted by the RNAi mechanism, but is affected by STI 571. When we applied ds M-BCR/ABL RNA and STI 571 in combination, we did not observe a further increase in the induction of apoptosis. Nevertheless, these data may open a field for further studies towards gene-therapeutic approaches using RNA interference to kill tumor cells with specific genetic abnormalities.

Apoptosis↗

PubMed2005

Inhibition of xenogeneic response in porcine endothelium using RNA interference.

BACKGROUND: Rejection mediated by antibody recognition of the alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) is a major barrier in porcine-to-human xenotransplantation. Because the synthesis of alpha-Gal is dependent on alpha1,3 galactosyltransferase (alpha1,3GT), methods of blocking this enzyme are needed. RNA interference induced by small interfering RNA (siRNA) is a powerful technique for allowing the silencing of mammalian genes with great specificity and potency. In this study, we use siRNA for silencing of alpha1,3GT with the purpose of reducing expression of the alpha-Gal epitope and subsequently decreasing immunogenicity of porcine endothelial cells. METHODS: alpha1,3GT-specific and control siRNAs were transfected into the porcine aortic endothelial cell line, PED. alpha-Gal expression was assessed by Western blotting, flow cytometry, and immunofluorescence. Protection from human-complement and natural killer (NK)-cell-mediated cytotoxicity was evaluated by Cr-release assays after incubation of PED with normal human serum (NHS) and NK92 cell, respectively. RESULTS: RNA interference was successfully achieved in PED as witnessed by the specific knock-down of alpha1,3GT mRNA levels. Flow cytometric analysis using the Griffonia simplicifolia isolectin B4 lectin confirmed the suppression of alpha1,3GT activity as evidenced by decreased alpha-Gal. Functional relevance of the knock-down phenotype was illustrated by the finding that silenced PED were protected from cytotoxicity of NHS. Protection from NK-mediated cytotoxicity was not observed. CONCLUSIONS: Our data are the first to demonstrate that RNA interference is a potent tool to down modulate alpha-Gal expression and to protect endothelial cells from complement-mediated cytotoxicity. Gene silencing by siRNA may represent a new approach for overcoming hyperacute and acute vascular rejection.

Animals↗

PubMed2007

RNA interference in mice.

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for functional genomics in mammalian cells. Furthermore, RNAi holds promise as a simple, fast and cost-effective approach to studying mammalian gene function in vivo and as a novel therapeutic approach. This review provides an overview of the progress of RNAi in vivo, with emphasis on systemic/local siRNA delivery, viral shRNA vectors, shRNA vector transgenic mice and conditional systems to control shRNA vectors. Taken together, the data from 80 in vivo studies show that RNAi is a useful tool that offers new opportunities for functional genomics in mice.

Animals↗

PubMed2003

Characterization of RNA interference in an Anopheles gambiae cell line.

Introduction of double stranded RNA into invertebrate cells often results in posttranscriptional silencing of target genes through a mechanism termed RNA interference (RNAi). Double-stranded RNA is cleaved by an RNAse III-like enzyme, termed dicer, to small interfering RNAs (siRNAs). In Drosophila, these siRNAs are incorporated in the RNA induced silencing complex (RISC) and mediate degradation of target mRNA. The RISC complex contains members of Argonaute (Ago) family of proteins. We show here that RNAi in a hemocyte cell line of Anopheles gambiae, the principal malaria vector in Africa, requires expression of dicer-2, Ago2 and Ago3 proteins. Furthermore, we demonstrate that RNAi in the mosquito does not spread outside of the target region, suggesting that RNA dependent RNA polymerase mediated transitive amplification is absent in the mosquito.

Animals↗

PubMed2004

A resource for large-scale RNA-interference-based screens in mammals.

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.

Animals↗

PubMed2006

Silencing of HIV-1 with RNA interference: a multiple shRNA approach.

Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). Previously, we have shown that stable expression of a single shRNA targeting the HIV-1 Nef gene strongly inhibits HIV-1 replication. However, this was not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presented a way out for the virus, as single nucleotide substitutions in the target region abolished inhibition. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against conserved HIV-1 regions. Therefore, we screened 86 different shRNAs targeting highly conserved regions. We identified multiple shRNAs that act as potent inhibitors of virus replication. We show, for the first time, that expression of three different shRNAs from a single lentiviral vector results in similar levels of inhibition per shRNA compared to single shRNA vectors. Thus, their combined expression results in a much stronger inhibition of virus production. Moreover, when we infected cells transduced with a double shRNA viral vector, virus escape was delayed. These results confirm that RNAi has great potential as an antiviral gene therapy approach and support our efforts to develop this strategy for treatment of HIV-1-infected individuals.

Base Sequence↗

PubMedDate not supplied

RNA interference: on the road to an alternate therapeutic strategy!

RNA interference (RNAi) is a newly described natural biological phenomenon mediated by small interfering RNA (siRNA) molecules which target viral mRNA for degradation by cellular enzymes. RNAi has become a method of choice for studying gene function, especially in mammalian systems. With proof-of-concept studies already presented against a wide variety of human pathogens and several innovative methods of delivering the siRNA to a wide variety of primary cells available, the role for siRNA as a potential therapeutic strategy is becoming increasingly clear. This review presents recent advances in this direction.

Acquired Immunodeficiency Syndrome↗

PubMed2003

RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription.

Influenza A virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. Here we show that short interfering RNAs (siRNAs) specific for conserved regions of the viral genome can potently inhibit influenza virus production in both cell lines and embryonated chicken eggs. The inhibition depends on the presence of a functional antisense strand in the siRNA duplex, suggesting that viral mRNA is the target of RNA interference. However, siRNA specific for nucleocapsid (NP) or a component of the RNA transcriptase (PA) abolished the accumulation of not only the corresponding mRNA but also virion RNA and its complementary RNA. These siRNAs also broadly inhibited the accumulation of other viral, but not cellular, RNAs. The findings reveal that newly synthesized NP and PA proteins are required for influenza virus transcription and replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection in humans.

Animals↗

PubMed2005

RNA interference toward UMP1 induces proteasome inhibition in Saccharomyces cerevisiae: evidence for protein oxidation and autophagic cell death.

The proteasome is a large intracellular protease that is responsible for a large portion of intracellular proteolysis, in particular the degradation of a majority of short-lived and oxidized proteins. Inhibition of proteasome function occurs in response to multiple stressors, with proteasome inhibition sufficient for the induction of a wide range of cytotoxic processes. Although considerable advances have been made in the understanding of the proteasome, and the effects of proteasome inhibition, our understanding of these topics in Saccharomyces cerevisiae has been slowed by the inability of proteasome inhibitors to penetrate and/or be retained in S. cerevisiae. Expression of UMP1 is necessary for proteasome assembly in S. cerevisiae, and in the present study we examined the effectiveness of RNA interference for UMP1 as a means of achieving proteasome inhibition in S. cerevisiae. Induction of RNA interference for UMP1 resulted in a dramatic decrease in UMP1 at the protein level, which was not observed in cells transformed with control vector. RNA interference caused an impairment in proteasome function, and increase in protein oxidation, with proteins involved in both stress response and energy metabolism showing increased oxidation. Interestingly, RNA interference induced cell death that seemed to be autophagic in nature, suggesting possible cross talk between the proteasome and the autophagic proteolytic pathways. Taken together, these data indicate that RNA interference may be a useful model with which to study the effects of proteasome inhibition in S. cerevisiae and demonstrate the ability of proteasome inhibition to induce cytotoxic alterations in S. cerevisiae.

Autophagy↗

PubMed2005

Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo.

RNA interference technology is emerging as a very potent tool to obtain a cellular knockdown of a desired gene. In this work we used vector-based RNA interference to inhibit vascular endothelial growth factor (VEGF) expression in prostate cancer in vitro and in vivo. We demonstrated that transduction with a plasmid carrying a small interfering RNA targeting all isoforms of VEGF, dramatically impairs the expression of this growth factor in the human prostate cancer cell line PC3. As a consequence, PC3 cells loose their ability to induce one of the fundamental steps of angiogenesis, namely the formation of a tube-like network in vitro. Most importantly, our "therapeutic" vector is able to impair tumor growth rate and vascularization in vivo. We show that a single injection of naked plasmid in developing neoplastic mass significantly decreases microvessel density in an androgen-refractory prostate xenograft and is able to sustain a long-term slowing down of tumor growth. In conclusion, our results confirm the basic role of VEGF in the angiogenic development of prostate carcinoma, and suggest that the use of our vector-based RNA interference approach to inhibit angiogenesis could be an effective tool in view of future gene therapy applications for prostate cancer.

Animals↗

PubMed2001

A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.

The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression.

Animals↗

PubMed2006

[Selective knockdown of Angiotensin II receptor subtype 1a in rat vascular smooth muscle cells by RNA interference].

OBJECTIVE: To selectively knockdown the expression of Angiotensin II receptor subtype 1a (AT1aR) in rat vascular smooth muscle cells (VSMCs) by RNA interference and the sequential effects on cellular viability and proliferation. METHODS: The primary cultured rat aortic VSMCs were transfected by plasmids pAT1a-shRNA1 and pAT1a-shRNA2, each carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence, or by a control plasmid pGenesil-Control (pCon) carrying a nonspecific shRNA-coding sequence. The mRNA and protein expressions of AT1a, AT2 were analyzed by semi-quantified RT-PCR and Western blot, respectively and normalized to the internal control gene beta-actin. Cellular viability and proliferation were determined with methylthiazoletetrazolium (MTT) assay. RESULTS: AT1a mRNA and protein were reduced by 82% and 69% by pAT1a-shRNA1, 77% and 56% by pAT1a-shRNA2, respectively while no change was found in pCon treated VSMCs. AT2 receptor level in VSMCs remains unchanged after various treatments. The A(490nm) values obtained by MTT measurements were similar among groups in the absence of Ang II but decreased significantly in pAT1a-shRNA1 and pAT1a-shRNA2 treated VSMCs in the presence of Ang II. CONCLUSION: RNA interference can selectively knockdown AT1a expression in cultured VSMCs and attenuate the Ang II induced cell proliferation. Future studies are warranted to explore the potential role of RNA interference on AT1 function and as a new gene therapy tool for cardiovascular diseases.

Animals↗

PubMed2004

[RNA interference and its possible use in cancer therapy].

Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) is a powerful tool for silencing gene expression. This mechanism was initially considered as a strange phenomenon limited to few plant species. It has become clear that PTGS occurs in both plants and animals and has roles in viral defense and transposon silencing mechanisms. However, the use of RNA interference triggered by the introduction of small double-stranded RNA (dsRNA or siRNA) into mammalian cells as a tool to knock down expression of specific genes holds the promise to selectively inhibit expression of disease-associated genes in humans. On the other hand, there are about 40,000 protein-coding genes in the human genome, but the function of most of them remains unknown. RNAi technology has now been developed for systematically deciphering the functions and interactions of these thousands of genes.

Animals↗

PubMed2004

RNA interference for analysis of gene function in trypanosomatids.

Gene-specific silencing by RNA interference is a valuable tool for analysis of gene function in the protozoan parasite Trypanosoma brucei. The development of tetracycline-regulated vectors for production of double-stranded RNA has facilitated its widespread use. RNA interference provides a fast and efficient method for determining whether a gene is essential for growth and viability, reveals mechanistic information on gene function, and has greatly enhanced our understanding of complex biological processes. Finally, the creation of an RNA interference-based library has allowed, for the first time, an approach for conducting forward genetic experiments in this organism.

Animals↗

PubMed2005

RNA interference libraries prove their worth in hunt for tumor suppressor genes.

RNA interference has been promoted as an ideal tool for functional genomics, but to date the success stories have principally been in model organisms. Two papers in this issue of Cell change all that: use of RNA interference libraries targeting large proportions of the human genome to uncover two novel tumor suppressor genes. REST is a transcriptional repressor that silences neuron-specific gene expression, and PITX1 is a homeodomain transcription factor that promotes the expression of a negative regulator of Ras.

Gene Library↗