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PubMed2005

Dissecting cancer pathways and vulnerabilities with RNAi.

The latest generation of molecular-targeted cancer therapeutics has bolstered the notion that a better understanding of the networks governing cancer pathogenesis can be translated into substantial clinical benefits. However, functional annotation exists for only a small proportion of genes in the human genome, raising the likelihood that many cancer-relevant genes and potential drug targets await identification. Unbiased genetic screens in invertebrate organisms have provided substantial insights into signaling networks underlying many cellular and organismal processes. However, such approaches in mammalian cells have been limited by the lack of genetic tools. The emergence of RNA interference (RNAi) as a mechanism to suppress gene expression has revolutionized genetics in mammalian cells and has begun to facilitate decoding of gene functions on a genome scale. Here, we discuss the application of such RNAi-based genetic approaches to elucidating cancer-signaling networks and uncovering cancer vulnerabilities.

Animals↗
PubMed2005

The therapeutic potential of RNA interference.

In recent years, we have witnessed the discovery of a new mechanism of gene regulation called RNA interference (RNAi), which has revitalized interest in the development of nucleic acid-based technologies for therapeutic gene suppression. This review focuses on the potential therapeutic use of RNAi, discussing the theoretical advantages of RNAi-based therapeutics over previous technologies as well as the challenges involved in developing RNAi for clinical use. Also reviewed, are the in vivo proof-of principle experiments that provide the preclinical justification for the continued development of RNAi-based therapeutics.

Animals↗
PubMed2006

Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant.

During vitellogenesis, one of the most tightly regulated processes in oviparous reproduction, vitellogenins are incorporated into the oocyte through vitellogenin receptor (VgR)-mediated endocytosis. In this paper, we report the cloning of the VgR cDNA from Blattella germanica, as well as the first functional analysis of VgR following an RNA interference (RNAi) approach. We characterized the VgR, VgR mRNA and protein expression patterns in pre-adult and adult stages of this cockroach, as well as VgR immunolocalization in ovarioles, belonging to the panoistic type. We then specifically disrupted VgR gene function using RNAi techniques. Knockdown of VgR expression led to a phenotype characterized by low yolk content in the ovary and high vitellogenin concentration in the haemolymph. This phenotype is equivalent to that of the yolkless mutant of Drosophila melanogaster, which have the yl (VgR) gene disrupted. The results additionally open the perspective that development genes can be functionally analyzed via systemic RNAi in this basal species.

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↗
PubMed2006

In vivo and in vitro knockdown of FREP2 gene expression in the snail Biomphalaria glabrata using RNA interference.

RNA interference (RNAi) is reported here for the first time for Biomphalaria glabrata, the snail intermediate host for the human parasite Schistosoma mansoni. The fibrinogen-related protein 2 (FREP2) gene, normally expressed at increased levels following exposure to digenetic trematode parasites, such as S. mansoni or Echinostoma paraensei, was targeted for knockdown. Double-stranded RNA (dsRNA) corresponding to specific regions of the FREP2 gene was introduced into snails by direct injection into the hemolymph, 2 days prior to exposure to trematodes, or added to co-cultures of B. glabrata embryonic (Bge) cells and E. paraensei sporocysts. After introduction of FREP2 dsRNA, expression levels of FREP2 were significantly reduced, to 20-30% of control values. In addition, we were able to disrupt expression of the house-keeping myoglobin gene, further confirming the feasibility of RNAi for B. glabrata. Cross-reactivity in RNAi has not been observed either among four FREP gene subfamilies or between FREP2 and myoglobin. Establishment of RNAi techniques in B. glabrata provides an important tool for clarifying the function of genes believed to play a role in host-parasite interactions, specifically between B. glabrata and its trematode parasites, including S. mansoni.

Animals↗
PubMed2002

Stable suppression of gene expression by RNAi in mammalian cells.

In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, approximately 22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (approximately 500 nt). This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of approximately 22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable "knock-down" cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.

Animals↗
PubMed2005

A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference.

The application of RNA interference (RNAi) to stem cell-based therapies will require highly specific and lineage-restricted gene silencing. Here we show the feasibility and therapeutic potential of coregulating transgene expression and RNAi in hematopoietic stem cells. We encoded promoterless small-hairpin RNA (shRNA) within the intron of a recombinant gamma-globin gene. Expression of both gamma-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage-specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34(+) cells from patients with sickle cell anemia led to erythroid-specific expression of the gamma-globin transgene and concomitant reduction of endogenous beta(S) transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny.

Anemia, Sickle Cell↗
PubMed2006

Design of expression vectors for RNA interference based on miRNAs and RNA splicing.

RNA interference (RNAi) mediates sequence-specific post-transcriptional gene silencing in many eukaryotes and is used for reverse genetic studies and therapeutics. RNAi is triggered by double-stranded small interfering RNAs (siRNAs), which can be processed from small hairpin RNAs generated from an expression vector. In some recently described vectors, the siRNAs are expressed from synthetic stem-loop precursors of microRNAs (miRNAs) driven by polymerase II promoters. We have designed new RNAi vectors, designated pSM155 and pSM30, that take into consideration miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. Like the original miRNA vectors, we show that the pSM155 and pSM30 constructs efficiently down-regulate expression of firefly luciferase and an endogenous gene, phospholipase D2. Moreover, the expression of a coexpressed fluorescent marker is substantially improved by this new design. Another improvement of these new vectors is incorporation of two inverted BsmBI sites placed internal to the arms of the new miRNA-based vectors, so oligos used for cloning are shorter and the cost is reduced. These RNAi vectors thus provide new tools for gene suppression.

Base Sequence↗
PubMed2006

Silencing of human alpha-synuclein in vitro and in rat brain using lentiviral-mediated RNAi.

Human alpha-synuclein overexpression and its toxic accumulation in neurons or glia are known to play key roles in the pathogenesis of Parkinson's disease and other related neurodegenerative synucleinopathies. Several single point mutations in the alpha-synuclein gene, as well as gene duplication and triplication, have been linked to familial Parkinson's disease. Moreover, genetic variability of the alpha-synuclein gene promoter is associated with idiopathic Parkinson's disease. Silencing of the human alpha-synuclein gene by vector-based RNA interference (RNAi) is a promising therapeutic approach for synucleinopathies. Here, we report identification of a 21-nucleotide sequence in the coding region of human alpha-synuclein that constitutes an effective target for robust silencing by RNAi and demonstrate allele-specific silencing of the A53T mutant of human alpha-synuclein. Furthermore, we have developed a plasmid vector-based RNAi for silencing of human alpha-synuclein in vitro. Lastly, using a dual cassette lentivirus that co-expresses an alpha-synuclein-targeting small hairpin RNA (shRNA) and enhanced green fluorescent protein (EGFP) as a marker gene, we demonstrate effective silencing of endogenous human alpha-synuclein in vitro in the human dopaminergic cell line SH-SY5Y and also of experimentally expressed human alpha-synuclein in vivo in rat brain. Our results demonstrate potent silencing of human alpha-synuclein expression in vitro and in vivo by viral vector-based RNAi and provide the tools for developing effective gene silencing therapeutics for synucleinopathies, including Parkinson's disease.

Alleles↗
PubMed2006

RNA interference: a potential revolution in disease therapy.

Gene therapy is the most recent face of modern biotechnology, which has shown promise of disease diagnosis and management at a newer and the most sophisticated level. The reason of enormous interest in gene therapy lies in the fact that most of lethal pathologies including hepatitis B and C, AIDS and carcinogenesis are refractory to contemporary treatments. These techniques, at least in controlled environment, allow turning on and off various genomic sequences. Relevant to this subject, RNA interference (RNAi) is the most recent, highly precise method to silence almost any gene of choice. Currently, RNAi for various disorders is being tried in humans; if this therapeutic strategy works at clinical level as well, it would be possible to design therapies against certain intractable pathologies including hepatitis C and AIDS.

Genetic Engineering↗
PubMed2006

Assessing RNAi frequency and efficiency in Ophiostoma floccosum and O. piceae.

Ophiostoma species are an economically important group of saprophytic and pathogenic fungi that grow in trees or wood. Ophiostoma like O. piceae and O. floccosum produce melanin, a pigment that stains lumber and logs. We used such species as model organisms for characterizing the molecular mechanisms in fungal melanin production. Because homologous recombination is rare in the Ophiostoma, identifying gene function in this group is challenging. We addressed this by assessing RNA interference (RNAi) as an alternative to gene replacement. For this, we built different inverted repeat transgene (IRT) constructs to down-regulate the polyketide synthase (PKS1) gene of the melanin pathway in O. piceae and O. floccosum. Transformation with IRT-PKS reduced mRNA levels for the PKS1 gene, and consequently decreased pigmentation in transformants. We showed that the PKS1 RNAi efficiency was proportional to the length of the dsRNA expressed from IRT constructs. These results indicated that RNAi is an appropriate tool for functional analysis of genes in Ophiostoma.

Ascomycota↗
PubMed2005

A simple strategy for generation of gene knockdown constructs with convergent H1 and U6 promoters.

RNA interference (RNAi) is a powerful tool for functional genetic studies in model organisms and mammalian cells. To facilitate rapid construction of gene knockdown constructs and RNAi libraries for known genes of mammalian cells, a new and simple strategy to produce small interfering RNA (siRNA) expression vectors with two opposing polymerase III promoters was developed. The design involved a one-step PCR amplification and single cloning procedure to construct a dual promoter siRNA expression vector. The forward primer is identical for all PCR reactions, only a single reverse primer that contains the siRNA targeting sequence has to be synthesized in the construction of each individual vector. This single primer design is cost-effective and it reduces the risk of sequence errors during synthesis of long oligos. Sense and antisense strands of siRNA duplexes were transcribed from the same template and this eliminated the need to synthesize long hairpin-forming oligonucleotides. Our study demonstrated that this vector design could mediate potent inhibition of expression of both exogenous and endogenous genes in mammalian cells.

Animals↗
PubMed2004

RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos.

Both murine endogenous retrovirus-L (MuERV-L) and intracisternal A particle (IAP), two autonomous long terminal repeat (LTR) retrotransposons, are activated during genome activation in the preimplantation mouse embryo, and both sense and antisense transcripts are detected in 2-cell and 8-cell stage embryos. Because RNA interference (RNAi) functions in the preimplantation mouse embryo, we analyzed the relationship between RNAi and MuERV-L and IAP expression by inhibiting RNAi and measuring relative changes of the levels of these transcripts. We inhibited the initial step in the RNAi pathway by injecting 1-cell embryos with mDicer siRNA or long mDicer dsRNA and analyzed MuERV-L and IAP expression at the 8-cell stage. This approach resulted in the targeted destruction of mDicer mRNA, but not Hdac1 mRNA, inhibited the RNAi pathway, and resulted in a 50% increase in IAP and MuERV-L transcript abundance. These results suggest that RNAi constrains expression of repetitive parasitic sequences in preimplantation embryos, and thereby contributes to preserving genomic integrity at a stage of development when the organism consists of only a few cells.

Animals↗
PubMed2005

Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi.

RNAi (RNA interference) and ASO (antisense oligonucleotide) technologies are the most commonly used approaches for silencing gene expression. However, the specificity of such powerful tools is an important factor to correctly interpret the biological consequences of gene silencing. In the present study, we examined the effects of acute loss of Ser/Thr kinase PDK1 (3-phosphoinositide-dependent kinase 1) expression using ASO and RNAi, and compared, for the first time, these two techniques using Affymetrix microarrays. We show that both ASO- and siRNA (small interfering RNA)-mediated knock-down of PDK1 expression strongly inhibited cell proliferation, although by different mechanisms, thereby questioning the specificity of these reagents. Using microarray analysis, we characterized the specificity of the ASO- and siRNA-mediated gene silencing of PDK1 by examining expression profiles 48 and 72 h following oligonucleotide transfection. At 48 h, a PDK1-dependent pattern of gene alterations was detectable, despite a large number of non-specific changes due to transfection of control nucleic acids. These non-specific alterations became more apparent at the 72 h time point, and obscured any PDK1-specific pattern. This study underscores the importance of defining appropriate control ASOs and siRNAs, using multiple oligonucleotides for each target and preferably short time points following transfection to avoid misinterpretation of the phenotype observed.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference.

RNA interference (RNAi) is a form of post-transcriptional gene silencing that has been described in a number of plant, nematode, protozoan, and invertebrate species. RNAi is characterized by a number of features: induction by double stranded RNA (dsRNA), a high degree of specificity, remarkable potency and spread across cell boundaries, and a sustained down-regulation of the target gene. Previous studies of RNAi have examined this effect in whole organisms or in extracts thereof; we have now examined the induction of RNAi in tissue culture. A screen of mammalian cells from three different species showed no evidence for the specific down-regulation of gene expression by dsRNA. By contrast, RNAi was observed in Drosophila Schneider 2 (S2) cells. Green fluorescent protein (GFP) expression in S2 cells was inhibited in a dose-dependent manner by transfection of dsRNA corresponding to gfp when GFP was expressed either transiently or stably. This effect was structure- and sequence-specific in that: (1) little or no effect was seen when antisense (or sense) RNA was transfected; (2) an unrelated dsRNA did not reduce GFP expression; and (3) dsRNA corresponding to gfp had no effect on the expression of an unrelated target transgene. This invertebrate tissue culture model should allow facile assays for loss of function in a well-defined cellular system and facilitate further understanding of the mechanism of RNAi and the genes involved in this process.

Animals↗
PubMed2004

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA.

RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.

Apoptosis↗
PubMed2005

Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels.

BACKGROUND: Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. METHODS: Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence. RESULTS: First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (<0.5 pmol). Second, various combinations of cationic lipids were tried and the best formulation was found to be a combination of JetSI with the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). Efficient inhibition of target, firefly luciferase was obtained with exceedingly low amounts of siRNA: 78 +/- 6% inhibition at 24 h post-transfection with 0.2 pmol siRNA. This inhibition was dose-dependent and specific. No effect was seen on the control gene, co-transfected Renilla luciferase, and the control mutated siRNA sequence had no effect on the targeted firefly luciferase. CONCLUSIONS: We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.

Analysis of Variance↗
PubMed2004

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Adenovirus E1A Proteins↗