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PubMed2006

Silencing neurodegenerative disease: bringing RNA interference to the clinic.

RNA interference (RNAi) is a recently described conserved biological pathway where non-coding RNAs suppress the expression of specific genes. Research efforts in the RNAi field aim to gain a better understanding of how its underlying machinery is orchestrated, to define the biological role of this conserved pathway, determine how to effectively manipulate RNAi in the laboratory and to integrate all this knowledge to develop novel therapies for human disease. This review summarizes the advances in the design of therapeutic RNAi for neurodegenerative diseases and discusses some of the experimental steps required to bring this therapy to human clinical trials.

Animals↗
PubMed2006

Intronic microRNA (miRNA).

Nearly 97% of the human genome is composed of noncoding DNA, which varies from one species to another. Changes in these sequences often manifest themselves in clinical and circumstantial malfunction. Numerous genes in these non-protein-coding regions encode microRNAs, which are responsible for RNA-mediated gene silencing through RNA interference (RNAi)-like pathways. MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) with complete or partial complementarity, are useful for the design of new therapies against cancer polymorphisms and viral mutations. Currently, many varieties of miRNA are widely reported in plants, animals, and even microbes. Intron-derived microRNA (Id-miRNA) is a new class of miRNA derived from the processing of gene introns. The intronic miRNA requires type-II RNA polymerases (Pol-II) and spliceosomal components for their biogenesis. Several kinds of Id-miRNA have been identified in C elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNAs are able to induce RNA interference in not only human and mouse cells, but in also zebrafish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of intron-mediated gene silencing via functional miRNA in cell and in vivo. These findings suggest an intracellular miRNA-mediated gene regulatory system, fine-tuning the degradation of protein-coding messenger RNAs.

Journal Article↗
PubMed2003

Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain.

RNA interference (RNAi) is an evolutionarily conserved mechanism of posttranscriptional gene-specific silencing. For in vivo applications, RNAi has been hampered until recently by inefficient delivery methods and by the transient nature of the gene suppression. Lentiviral vectors (LVs) hold great promise for gene therapeutic applications, pharmaceutical target validation, and functional genomics because stable gene transfer is mediated both in dividing and nondividing cells. We have used a lentiviral vector-based system for RNAi. We produced human immunodeficiency virus type 1-derived LVs encoding a short hairpin RNA specific for enhanced green fluorescent protein (EGFP) mRNA that were capable of inhibiting EGFP expression in mammalian cells. EGFP knockdown persisted after multiple passages of the cells. Of particular interest, our RNAi LVs were equally effective in suppression and prevention of EGFP expression after stereotactic injection in adult mouse brain. Therefore, we believe that the use of LVs for stable RNAi in brain will become a powerful aid to probe gene function in vivo and for gene therapy of diseases of the central nervous system.

Animals↗
PubMed2005

Sampling from natural populations with RNAI reveals high outcrossing and population structure in Caenorhabditis elegans.

Despite a nearly worldwide distribution in nature, Caenorhabditis elegans exhibits low levels of genetic polymorphism, possibly as an indirect consequence of low levels of outcrossing. In the laboratory, Caenorhabditis elegans males are produced at low rates and are steadily eliminated from cultures, so that reproduction happens largely through self-fertilization in hermaphrodites. C. elegans is increasingly the focus of evolutionary research; however, natural outcrossing rates are difficult to measure because mating tests with laboratory strains are usually required to identify C. elegans. We sampled natural populations of C. elegans with an RNA interference (RNAi) assay. Heterozygosities and polymorphism patterns revealed surprisingly high levels of population structure and outcrossing (approximately 22% of individuals are estimated to be the result of outcrossing and not self-fertilization). The finding of strong local population structure, together with low levels of diversity on local and global scales, suggests a metapopulation model of frequent extinction and recolonization of local populations. The occurrence of substantial outcrossing suggests that the extinction of local populations is probably not driven by the accumulation of harmful mutations.

Adenosine Triphosphatases↗
PubMed2005

Sequence-specific suppression of mdr1a/1b expression in mice via RNA interference.

PURPOSE: RNA interference (RNAi) is a powerful tool for silencing gene expression posttranscriptionally. The purpose of this study was to examine whether in vivo RNAi can be induced against endogenous mdr1a/1b in adult mice and to assess the feasibility of generating P-glycoprotein (P-gp) knockdown mice based on RNAi by a very simple intravenous injection of synthetic small interfering RNA (siRNAs) or siRNA-expressing plasmid DNAs. METHODS: The targeted sequences for silencing mdr1a specifically or mdr1a/1b simultaneously were examined in an in vitro study using a mouse colon carcinoma cell line, colon26 cells, in culture. Mice were repeatedly treated with intravenous synthetic siRNAs or siRNA-expressing plasmid DNAs in naked form administered via a large-volume and high-speed injection, i.e., the hydrodynamics-based procedure. The amount of targeted mRNA and P-gp in the liver were determined by real-time polymerase chain reaction and Western blot analysis, respectively. RESULTS: Among several targeted sequences, two and one optimized sequences were selected for mdr1a and mdr1a/1b, respectively, in the in vitro study. Following administration of synthetic siRNAs or siRNA-expressing plasmid DNAs directed against mdr1a, the mRNA level in the liver was significantly reduced to approximately 50-60% of that in control mice. Furthermore, a slight reduction was observed at the protein level. Similar results were obtained in the experiments using siRNA-expressing pDNA directed against mdr1a/1b. CONCLUSIONS: Our results demonstrate that sequence-specific suppression of mdr1 gene expression is possible at the mRNA level as well as the protein level in mice following intravenous delivery of siRNA effectors.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

[Effect of silencing epidermal growth factor receptor expression by RNA interference on the growth of nasopharyngeal carcinoma cell 5-8F].

OBJECTIVE: To observe the growth and cell cycle changes of nasopharyngeal carcinoma cell 5-8F in response to silencing of the expression of epidermal growth factor receptor (EGFR) with RNA interference (RNAi), and explore the possible relationships between EGFR and the occurrence, differentiation and progression of nasopharyngeal carcinoma. METHOD: Three EGFR-specific small interfering RNAs (siRNAs) were obtained by in vitro transcription and synthesis and were transiently transfected into 5-8F cells. The EGFR expression levels in the transfected cells were detected by reverse transcription (RT)-PCR and Western blotting, respectively. After EGFR expression silencing, the growth and cell cycle changes of the cells were observed. RESULTS: EGFR mRNA contents and protein levels decreased by approximately 67.5% and 77%, respectively, after RNAi of 5-8F cells, and the cell proliferation decreased and cell cycle arrest at G1 phase occurred in association with EGFR silencing. CONCLUSION: EGFR silencing by RNAi can reduce the proliferation of nasopharyngeal carcinoma cells and induce cell cycle arrest at G1 phase, which sheds light on the possible use of RNAi for further investigation of the pathogenesis and gene therapy of nasopharyngeal carcinoma.

Cell Line, Tumor↗
PubMed2003

Expression of siRNA from a single transcript that includes multiple ribozymes in mammalian cells.

RNA interference (RNAi) has been developed recently as a powerful tool for silencing of mRNAs in various organisms. In mammalian cells, the introduction of small interfering RNAs (siRNAs) can inhibit gene expression in a sequence-specific manner without induction of the nonspecific degradation that is activated by long double-stranded RNA (dsRNA) (>30 nt). Here, we report a method for generating siRNAs in mammalian cells using a self-cleaving ribozyme-expressing vector. Four ribozymes within transcripts that were expressed under the control of a cytomegalovirus (CMV) or tRNAVal promoter excised, in cis, specific sense and antisense sequences from primary transcripts and generated siRNAs in HeLa cells. The siRNAs generated by the ribozymes were able to decrease the expression of a firefly gene for luciferase. These results suggest that polymerase II (pol II) systems, particularly in view of the availability of many potential tissue-specific promoters, and pol III systems, in which siRNAs are generated by a trimming-ribozyme (TRz) system as described here, should be useful in efforts to suppress the expression of specific genes.

5' Untranslated Regions↗
PubMed2007

Use of RNA interference to dissect defense-signaling pathways in rice.

The RNA interference (RNAi) technique is a powerful tool to suppress gene expression and has been widely used for functional discovery of eukaryotic genes. To dissect defense-signaling pathways in rice, it is important to generate a series of rice mutant lines deficient in or insensitive to major signal molecules such as jasmonic acid and ethylene. Here we describe an RNAi protocol for generating and characterizing transgenic gene-silencing lines defective in rice jasmonic acid signaling. The RNAi technique should be useful for effective suppression of host genes encoding signaling components and facilitating the dissection of defense signal pathways in rice.

Cyclopentanes↗
PubMed2003

In vivo gene silencing in Plasmodium berghei--a mouse malaria model.

RNA interference (RNAi) has emerged as a specific and efficient tool to silence gene expression in a variety of organisms and cell lines. An important prospect for RNAi technology is its possible application in the treatment of diseases using short interfering RNAs (siRNAs). However, the effect of siRNAs in adult animals and their potential to treat or prevent diseases are yet to be fully investigated. The main goal of the present study is to find out whether it was possible to carry out RNAi on circulating malaria parasite in vivo. To trigger RNAi in mouse malaria parasite, we used siRNAs corresponding to cysteine protease genes of Plasmodium berghei (berghepain-1 & 2). Intravenous injections of berghepains' siRNAs in infected animal resulted in characteristic enlargement of food vacuole in circulating parasites. Protein analysis of these treated parasites showed substantial accumulation of hemoglobin, which is reminiscent of the effect observed upon treating Plasmodium falciparum with different cysteine protease inhibitors. Parasites treated with berghepain 1 & 2 siRNAs showed marked reduction in the levels of their cognate mRNAs, thereby suggesting specific inhibition of berghepains' gene expression in vivo. We also observed the generation of approximately 25 nt RNA species from berghepains' mRNAs in the treated parasites, which is a characteristic of an RNAi phenomenon. These results thus provide evidence that beyond its value for validation of gene functions, RNAi may provide a new approach for disease therapy.

Animals↗
PubMed2003

Induction of G0/G1 cell cycle arrest in ovarian carcinoma cells by the anti-inflammatory drug NS-398, but not by COX-2-specific RNA interference.

Cyclooxygenases, particularly COX-2, play an important role in tumor development and progression. We have previously shown that COX-2 expression is an independent prognostic factor in human ovarian carcinoma. In this study, we investigated the effects of the inhibition of COX isoforms by the NSAID NS-398 as well as by COX-isoform-specific RNA interference (RNAi) in the human ovarian carcinoma cell lines OVCAR-3 and SKOV-3. OVCAR-3 cells showed a constitutive expression of COX-1 and an induction of high levels of COX-2 and PGE(2) after stimulation with interleukin-1beta. In contrast, SKOV-3 cells were negative for both COX isoforms. In OVCAR-3 cells, PGE(2) production was inhibited by NS-398 in concentrations of 1 microM and by a COX-2-specific silencing RNA (siRNA), while a COX-1-specific siRNA did not have an effect. This suggests that COX-2 is the major source of PGE(2) in this cell line. To dissociate COX-2-specific and non-COX-2-specific effects on cell proliferation, a proliferation assay was performed after incubation of cells with NS-398 and COX siRNAs. NS-398 induced an inhibition of cell proliferation at concentrations of 50-500 microM, which are above the concentrations needed for the inhibition of PGE(2) production. This inhibitory effect was present in the COX-positive cell line OVCAR-3 as well as in the COX-negative cell line SKOV-3 and could not be reverted by addition of exogenous PGE(2). Neither COX-1- nor COX-2-specific siRNAs had an effect on cell proliferation of OVCAR-3 cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with NS-398, but not with COX siRNAs. These experiments suggest that NS-398 reduced cell proliferation in ovarian carcinoma cells by induction of G0/G1 cell cycle arrest independent of COX-2 inhibition. Our study shows that specific inhibition of COX isoforms by RNAi could be used to dissociate effects of NSAIDs. Furthermore, our results suggest that cell cycle arrest is one of the primary mechanisms responsible for the antiproliferative effects of NS-398 on ovarian carcinoma cells.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2003

Caenorhabditis elegans EVL-14/PDS-5 and SCC-3 are essential for sister chromatid cohesion in meiosis and mitosis.

Sister chromatid cohesion is fundamental for the faithful transmission of chromosomes during both meiosis and mitosis. Proteins involved in this process are highly conserved from yeasts to humans. In screenings for sterile animals with abnormal vulval morphology, mutations in the Caenorhabditis elegans evl-14 and scc-3 genes were isolated. Defects in cell divisions were observed in germ line as well as in vulval and somatic gonad lineages. Through positional cloning of these genes, we have shown that EVL-14 and SCC-3 are likely the only C. elegans homologs of the yeast sister chromatid cohesion proteins Pds5 and Scc3, respectively. Both evl-14 and scc-3 mutants displayed defects in the meiotic germ line. In evl-14 mutants, synaptonemal complexes (SCs) were detectable but more than the usual six DAPI (4',6'-diamidino-2-phenylindole)-positive structures were seen at diakinesis, suggesting that EVL-14/PDS-5 is important for the maintenance of sister chromatid cohesion in late prophase. In scc-3 mutant animals, normal SCs were not visible and approximately 24 DAPI-positive structures were seen at diakinesis, indicating that SCC-3 is necessary for sister chromatid cohesion. Immunostaining revealed that localization of REC-8, a homolog of the yeast meiotic cohesin subunit Rec8, to the chromosomes depends on the presence of SCC-3 but not that of EVL-14/PDS-5. scc-3 RNA interference (RNAi)-treated embryos were 100% lethal and displayed defects in cell divisions. evl-14 RNAi caused a range of phenotypes. These results indicate that EVL-14/PDS-5 and SCC-3 have functions in both mitosis and meiosis.

Animals↗
PubMed2006

RNA interference of the Arabidopsis putative transcription factor TCP16 gene results in abortion of early pollen development.

Pollen development is a fundamental and essential biological process in seed plants. Pollen mother cells generated in anthers undergo meiosis, which gives rise to haploid microspores. The haploid cells then develop into mature pollen grains through two mitotic cell divisions. Although several sporophytic and gametophytic mutations affecting male gametogenesis have been identified and analyzed, little is known about the underlying molecular mechanism. In this study, we investigated the function of the TCP16 gene, which encodes a putative transcription factor. Expression analysis of the promoter::GUS fusion gene revealed that TCP16 transcription occurred predominantly in developing microspores. GUS expression began at the tetrad stage and markedly increased in an early unicellular stage. Transgenic plants harboring a TCP16 RNA interference (RNAi) construct generated equal amounts of normal and abnormal pollen grains. The abnormal pollen grains exhibited morphological abnormality and degeneration of genomic DNA. The defective phenotype of the RNAi plants was first detectable at the middle of the unicellular stage. Our results therefore suggest that TCP16, a putative transcription factor, plays a crucial role in early processes in pollen development.

Arabidopsis↗
PubMed2005

Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA.

RNA interference (RNAi) has been shown to inhibit viral replication in some animals and plants. Whether the RNAi is functional in shrimp remains to be demonstrated. In vitro transcribed dsRNAs of YHV helicase, polymerase, protease, gp116, and gp64 were transfected into shrimp primary cell culture and found to inhibit YHV replication. dsRNA targeted to nonstructural genes (protease, polymerase, and helicase) effectively inhibited YHV replication. Those targeted structural genes (gp116 and gp64) were the least effective. These findings are the first evidence that RNAi-mediated gene silencing is operative in shrimp cells. This could be a powerful tool for studying gene function and to develop effective control of viral infection in shrimp.

Animals↗
PubMed2005

[Cloning of ACC oxidase gene and inhibition of endogenous gene expression with RNAi in cauliflower].

A fragment of 1202 bp of the candidate ACO gene was amplified from the cauliflower (brassica oleracea Var. botrytis) genome using the degenerated primers which were designed according to the consensus sequence of ACO amino acids among various plant species. The result of BLAST showed the sequence presented a very high match with the ACO genes from other plants; the homologue was from 83% to 99%. Three exons and two introns were identified in this sequence. The spliced length of mRNA was 756 nt and encoded 252 amino acids. The putative new gene was denominated BoACO, and submitted to GenBank (AY676466). Using the sequence, we constructed an RNA interference (RNAi) transformation vector through the way of BP cloning. The transformation into cauliflower was performed. Five regenerated plants with kanamycin resistance were obtained. And the transgene integrated into cauliflower genome was proved with PCR and Southern blotting. The expression of this ACO gene is down-regulated based on the Northern blotting in the transgenic plants. The activity of ACO enzyme was depressed significantly.

Amino Acid Oxidoreductases↗
PubMed2006

A divergent transcription factor TFIIB in trypanosomes is required for RNA polymerase II-dependent spliced leader RNA transcription and cell viability.

Transcription by RNA polymerase II in trypanosomes deviates from the standard eukaryotic paradigm. Genes are transcribed polycistronically and subsequently cleaved into functional mRNAs, requiring trans splicing of a capped 39-nucleotide leader RNA derived from a short transcript, the spliced leader (SL) RNA. The only identified trypanosome RNA polymerase II promoter is that of the SL RNA gene. We have previously shown that transcription of SL RNA requires divergent trypanosome homologs of RNA polymerase II, TATA binding protein, and the small nuclear RNA (snRNA)-activating protein complex. In other eukaryotes, TFIIB is an additional key component of transcription for both mRNAs and polymerase II-dependent snRNAs. We have identified a divergent homolog of the usually highly conserved basal transcription factor, TFIIB, from the pathogenic parasite Trypanosoma brucei. T. brucei TFIIB (TbTFIIB) interacted directly with the trypanosome TATA binding protein and RNA polymerase II, confirming its identity. Functionally, in vitro transcription studies demonstrated that TbTFIIB is indispensable in SL RNA gene transcription. RNA interference (RNAi) studies corroborated the essential nature of TbTFIIB, as depletion of this protein led to growth arrest of parasites. Furthermore, nuclear extracts prepared from parasites depleted of TbTFIIB, after the induction of RNAi, required recombinant TbTFIIB to support spliced leader transcription. The information gleaned from TbTFIIB studies furthers our understanding of SL RNA gene transcription and the elusive overall transcriptional processes in trypanosomes.

Amino Acid Sequence↗
PubMed2005

Nucleic acid modulation of gene expression: approaches for nucleic acid therapeutics against cancer.

Most cancers are characterized by abnormal gene expression, which is thought to contribute to the pathogenesis and maintenance of the malignant phenotype; abnormal proliferation, maturation, and apoptosis. Silencing such genes would appear to be a rational approach to the therapy of cancer, and some preliminary clinical studies support this concept. Of the strategies available, the anti-mRNA gene silencing approach has attracted much attention and is the focus of this review. This strategy includes three types of agents: (1) single-stranded antisense oligonucleotides; (2) catalytically active oligonucleotides, such as ribozymes, and DNAzymes that possess inherent RNA cleaving activity; and (3) small interfering RNA (siRNA) molecules that induce RNA interference (RNAi). Among these agents, antisense oligonucleotides, especially phosphorothioate (PS) oligonucleotides, have been the most frequently used in clinical trials. In this article, we provide an overview of anti-mRNA gene silencing agents and their development for use as cancer therapeutics.

Animals↗
PubMed2005

Potential applications of RNA interference technology in the treatment of cancer.

Inhibition of growth and progression of cancer cells is a challenge with major potential impact. RNA interference (RNAi) technology has been rapidly developed as a laboratory tool for the downregulation of the expression of a gene of interest. Moreover, RNAi offers a new potential for gene therapy of particular neoplasms by the specific inhibition of a cancer-associated target. This article will briefly describe the mechanism and application possibilities of RNAi, and illustrate the therapeutic potential in cancer gene therapy. The utilization of RNAi technology as a potential therapeutic tool for the treatment of cancer will be discussed in detail for two specific targets; the Bcr-Abl tyrosine kinase and the multidrug transporter MDR1/P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Targeting primary human leukaemia cells with RNA interference: Bcr-Abl targeting inhibits myeloid progenitor self-renewal in chronic myeloid leukaemia cells.

We have investigated functional outcome of challenging primary chronic myeloid leukaemia (CML) cells with Bcr-Abl fusion sequence-directed RNA interference (RNAi). We targeted the Bcr-Abl b3a2 variant, by RNAi, in primary chronic phase CML cells, and detected strikingly reduced proliferation of myeloid precursor cells expressing this variant. Lack of an effect in cells expressing a distinct Bcr-Abl variant confirmed the specificity of the response. Through the functional targeting of an oncogene in primary human tumour cells, we have demonstrated that Bcr-Abl enhances CML progenitor cell amplification, and that RNAi may be suitable for development as a specific anti-leukaemia treatment.

Cell Proliferation↗