Reduced Target Gene Expression Research Articles

RNA silencing in Chlamydomonas: mechanisms and tools

The generation of a comprehensive EST library and the sequencing of its genome set the stage for reverse genetics approaches in Chlamydomonas reinhardtii. However, these also require tools for the specific downregulation of target gene expression. Consequently, a large number of diverse constructs were developed aimed at reducing target gene expression in Chlamydomonas via the stable expression of antisense or inverted repeat-containing RNA. Double-stranded RNA (dsRNA) generated by the annealing of antisense and sense RNAs or by hairpin formation of an inverted repeat, feeds into the RNA silencing pathway. In this pathway, dsRNA is cleaved into approximately 25-bp small interfering RNAs (siRNAs) by the endonuclease Dicer. One of the two complementary strands of a siRNA is then loaded onto an Argonaute-like protein present as core component within larger complexes. Guided by this single-stranded RNA, the Argonaute-like protein either detects homologous transcripts and cleaves these endonucleolytically, or initiates transcriptional gene silencing. This article summarizes current information derived mainly from the Chlamydomonas genome project on components that are assumed to be involved in RNA silencing mechanisms in Chlamydomonas. Furthermore, all approaches employed in Chlamydomonas to date to downregulate target gene expression by antisense or inverted repeat constructs are reviewed and discussed critically.

71. Intratumoral Delivery of Small Interfering RNA for Inhibition of Tumor Progression in Mice

RNA interference (RNAi) is a post-transcriptional gene silencing event in which short double-stranded RNA (siRNA) degrades target mRNA in a sequence-specific manner. Silencing oncogenes or other genes contributing to tumor cell malignancy or progression by siRNA or siRNA-expressing vector offers a therapeutic treatment for cancer patients. For treating cancer patients with RNAi effector, its delivery to tumor cells is one of the key factors, because the gene silencing event is limited in the cells reached by the RNAi effector. To examine whether siRNA or siRNA-expressing vector is effective in reducing target gene expression in tumor cells in vivo, we developed tumor cell lines that stably express reporter genes for a convenient, sensitive and quantitative evaluation of the RNAi effect. Namely, mouse melanoma B16-BL6 cells were stably transfected with firefly (model target gene of RNAi) and sea pansy (indicator of tumor cell number) luciferases to obtain B16-BL6/dual Luc cells. We found that the ratio of the luciferase activities in the tumor cells can be used as an indicator of RNAi. When the siRNA-expressing plasmid DNA (pDNA) was added to the cells, the luciferase activity decreased with time and reached a trough level at 2 days. Then, the activity returned to the initial level at 12 days. siRNA also showed a similar inhibitory profile, but the decrease and recovery in the luciferase activity were faster than those of siRNA-expressing pDNA. Then, the B16-BL6/dual Luc cells were inoculated into the footpad of mice to examine whether the luciferase expression is suppressed by the delivery of RNAi effectors in vivo. A single injection of either siRNA or siRNA-expressing pDNA into the tumor tissue followed by electroporation (1000 V/cm, 5 ms, 4 Hz, 12 pulses) reduced the luciferase activity to about 30% of the control value. Next, we investigated whether silencing the expression of genes related to tumor cell progression by RNAi can inhibit the tumor cell growth. To this end, |[beta]|-catenin and hypoxia-inducible factor 1|[alpha]|(HIF1|[alpha]|) were selected as the target. Transfection of siRNA-expressing pDNA targeting to b-catenin or HIF1a reduced each target gene expression in cultured B16 cells to about 20% (|[beta]|-catenin) and 25% (HIF1|[alpha]|) at mRNA level compared with those in B16 cells transfected with pDNA expressing no effective siRNA. In addition, the treatment resulted in the decrease in the number of B16 cells to about 50% (|[beta]|-catenin) and 70% (HIF1|[alpha]|) of a control value at 3 days. Then we investigated whether the intratumoral delivery of siRNA-expressing pDNA targeting to |[beta]|-catenin or HIF1|[alpha]| can be a therapeutic treatment against the proliferation of the tumor cells in mice. To investigate this possibility, we administered the siRNA-expressing pDNA targeting to |[beta]|-catenin or HIF1|[alpha]| to the tumor-bearing mice by the same procedure as above. As a result, we found that the intratumoral delivery of siRNA-expressing pDNA targeting |[beta]|-catenin or HIF1|[alpha]| inhibited the tumor growth.

Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors

BackgroundRNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death.ResultsAs expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing.ConclusionsOur data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems.

The future of antisense therapy: combination with anticancer treatments

The current direction in cancer research is rational drug design, which is based on the evidence that transformed cells are characterized by alterations of genes devoted to the regulation of both cell proliferation and apoptosis. A variety of approaches have been carried out to develop new agents selective for cancer cells. Among these, antisense oligonucleotides (ASOs) are one of such class of new agents able to inhibit specifically the synthesis of a particular cancer-associated protein by binding to protein-encoding RNA, thereby preventing RNA function. In the past decade, several ASOs have been developed and tested in preclinical and clinical studies. Many have shown convincing in vitro reduction in target gene expression and promising activity against a wide variety of tumors. However, because of the multigenic alterations of tumors, the use of ASOs as single agents does not seem to be effective in the treatment of malignancies. Antisense therapy that interferes with signaling pathways involved in cell proliferation and apoptosis are particularly promising in combination with conventional anticancer treatment. An overview of the progress of ASOs used in combination therapy is provided.

RNA interference may be more potent than antisense RNA in human cancer cell lines.

1. RNA interference (RNAi) is a newly discovered cellular pathway for the silencing of sequence-specific genes at the mRNA level by the introduction of the cognate double-stranded (ds) RNA. Because antisense (AS) mechanisms have similar effects, we compared these two effects in human cancer cell lines, considering a possible application of RNAi for cancer therapy. 2. We tested RNAi effects by transfecting human hepatoma and pancreatic cancer cell lines with AS and sense (S) RNA expression plasmids corresponding to the exogenous luciferase gene or the endogenous c-raf gene in the form of complexes with a cationic lipopolyamine or a tumour-targeting peptide vector we developed. In addition, we compared the effects of small interfering RNA and AS oligoDNA complexed with the peptide vector. 3. From the viewpoint of AS actions, the effect of the AS RNA may be cancelled by the S RNA, although, interestingly, we found that the combination of the AS and S RNA expression plasmids was more effective than the AS RNA expression plasmids alone in reducing target gene expression, whereas the S RNA expression plasmids had no effects. The combination of the luciferase AS and S RNA had no effects on the expression of either the beta-galactosidase gene or the c-raf gene. In the presence of 2-aminopurine (an inhibitor of dsRNA-activated protein kinase), the inhibitory effect of the combination of AS and S RNA on gene expression did not change in the case of the endogenous c-raf gene, but was reduced in the case of the exogenous luciferase gene. The effect of 22 nucleotide RNA duplexes corresponding to the luciferase gene was by one order stronger than that of the phosphorothioate AS DNA. 4. Thus, it is suggested that RNAi may be more potent than AS RNA in reducing target gene expression in human cancer cell lines, regardless of the length of dsRNA. With further studies on the RNAi phenomenon in cancer cells, RNAi could provide a novel approach for cancer gene therapy.

Combination Antigene Therapy Targeting c-myc and c-erbB2 in the Ovarian Cancer COC1 Cell Line

Objective. Antigene therapy targeting only one oncogene in ovarian cancer has made much progress, although it still has some limitations. To explore the potential for combination antigene therapy in ovarian cancer, we examined the in vitro effects of liposmal antisense phosphorothioate oligodeoxynucleotides targeting c-erbB2 and c-myc (LF–c-erbB2/c-myc AS-ODNs) in the human ovarian cancer COC1 cell line.Methods. COC1 cells were treated differently as follows: group A with single LF–c-erbB2 AS-ODNs; group B with single LF–c-myc AS-ODNs; group C with combination LF–c-erbB2/c-myc AS-ODNs; and group D as untreated control. Cell proliferation was studied by MTT assay and clonal cultures. RT-PCR was used to measure gene expression of c-erbB2 and c-myc before and after transfection. Morphologic changes in the COC1 cells were observed with the electron microscope.Results. Single antigene therapy targeting c-erbB2 or c-myc could reduce target gene expression and inhibit COC1 cell growth by 61.9 ± 9.3 and 64.5 ± 11.2%, respectively. However, combination antigene therapy could not only suppress expression of c-erbB2 and c-myc simultaneously, but also inhibit COC1 cell proliferation with a higher inhibitory rate of 82.6 ± 12.1%. Apart from that, the combination agents could induce COC1 cell apoptosis.Conclusions. Our study suggests that combination antigene therapy targeting c-erbB2 and c-myc can inhibit COC1 cell proliferation and gene expression of c-erbB2 and c-myc. Furthermore, its effectiveness is much higher than that of individual antigene therapy.

Reduction of target gene expression by a modified U1 snRNA.

Although the primary function of U1 snRNA is to define the 5' donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5' donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], beta-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3' cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.

Expression of a reporter gene is reduced by a ribozyme in transgenic plants.

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduced npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant expressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achieving a significant reduction in target gene expression.

Spontaneous reactivation of thymidine kinase deficient (TK −) herpes simplex virus (HSV) infection without acyclovir (ACV) pressure in a patient with the acquired immunodeficiency syndrome (AIDS) : J.J. Sasadeusz, B.A. Rennie, S.L. Sacks. University of British Columbia, Vancouver, BC Canada

Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70–98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70–90%, while an equivalent dose of ACV produced only 40–50% inhibition when the treatment was applied between 1 and 3 hpi. Seven daily topical treatments of 100 μg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10 μg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5–50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.