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PubMed2006

Interfering antiviral immunity: application, subversion, hope?

RNA interference (RNAi), initially recognized as a natural antiviral mechanism in plants, has rapidly emerged as an invaluable tool to suppress gene expression in a sequence-specific manner in all organisms, including mammals. Its potential to inhibit the replication of a variety of viruses has been demonstrated in vitro and in vivo in mouse and monkey models. These results have generated profound interest in the use of this technology as a potential treatment strategy for viral infections for which vaccines and drugs are unavailable or inadequate. In this review, we discuss the progress made within the past 2-3 years towards harnessing the potential of RNAi for clinical application in viral infections and the hurdles that have yet to be overcome.

Animals↗
PubMed2006

Regulation of gene expression in mouse embryos and its embryonic cells through RNAi.

The ability to direct cell differentiation of embryo-derived cells should have considerable practical applications. A prerequisite for success is the availability of robust tools that allows efficient knockdown of specific genes to elucidate gene function and direct differentiation. Loss-of-function studies in the mouse have traditionally been carried out using gene knockout animals to obtain mutant embryonic cells. Despite improvements in this technology, it still remains a laborious method. In addition, the use of knockout for studying preimplantation embryonic cells is limited due to maternal contributions. RNA interference (RNAi) is a powerful genetic approach for specific gene silencing of target genes in which 21-23 nt small interfering RNAs work as sequence-specific RNAi mediators. Using RNAi approaches, gene function has been eliminated in mouse embryonic stem cells and carcinoma cells to direct cell differentiation. In mouse oocytes and embryos, RNAi eliminated both maternal and zygotic transcripts and disturbed normal development.

Animals↗
PubMed2004

Selection and characterization of RNA interference-deficient trypanosomes impaired in target mRNA degradation.

Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1, encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with alpha-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized. At the cellular level, only RiD-1 trypanosomes showed a significant increase in doubling time with the concomitant accumulation of cells defective in the completion of cytokinesis. At the RNA level, both clones accumulated wild-type amounts of small interfering RNAs and displayed elevated levels of retroposon transcripts, the hallmark of RNAi deficiency in T. brucei. Importantly, both RiD-1 and RiD-2 clones were defective in the degradation of target mRNA, suggesting an impairment of the activity of AGO1, the putative RNAi endonuclease. Since in RiD cells the AGO1 gene was not mutated and was expressed at wild-type levels, we propose that in trypanosomes the cleavage of mRNA by AGO1 is regulated by the interaction with another factor(s).

Alleles↗
PubMed2003

Coupling of RNAi-mediated target downregulation with gene replacement.

Short interfering RNA (siRNA)-mediated knockdown of deleterious endogenous transcripts has potential applications for the treatment of hereditary diseases. In situations where the mutant and wildtype transcripts cannot be discriminated from one another by siRNAs, it may be necessary to simultaneously carry out gene replacement with a modified form of the target RNA that is resistant to siRNA activity. To test this possibility, we have taken advantage of a potent siRNA that knocks down EGFP mRNA. In this system, wild-type EGFP expression is suppressed by the siRNA, whereas an EGFP construct with codon modifications in the target region that is otherwise fully functional is not downregulated. When expression of the wild-type message is inhibited, EGFP expression can be simultaneously restored by transfecting these cells with the codon-modified version of EGFP. These studies provide a detailed methodology and system for testing this strategy with RNA interference (RNAi).

Animals↗
PubMed2006

In praise of manual high-throughput screening.

RNA interference (RNAi) and automated high-throughput screening is a promising combination. But the first systematic large-scale mapping of genetic interactions in an animal shows that manual methods still have advantages over sophisticated automated screens.

Animals↗
PubMed2003

A new opaque variant of maize by a single dominant RNA-interference-inducing transgene.

In maize, alpha-zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD alpha-zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants.

Genes, Dominant↗
PubMed2004

Inhibition of EGFP expression by siRNA in EGFP-stably expressing Huh-7 cells.

The RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally, and causes the degradation of an mRNA containing the same sequence. In this present study, an alternative approach was used to in vitro synthesize enhanced green fluorescent protein (EGFP) specific short interfering RNA (siRNA) using T7 RNA polymerase, and a pEGFP-N1 transfected, human hepatoma cell line Huh-7 derived Huh-7-N cell clone was established. When introduced the siRNA into the EGFP expressing Huh-7-N cells, the EGFP specific siRNA was able to specifically inhibit the expression of EGFP in Huh-7-N. In comparison with that in wild-type Huh-7 or that in Huh-7 co-transfected with pEGFP-N1, the inhibition of EGFP specific siRNA in Huh-7-N cells is more significant and repeatable. It is concluded that a cell clone Huh-7-N, which stably expresses EGFP, has been established, and the in vitro synthesized EGFP siRNA can be used in silencing the EGFP gene expression. This Huh-7-N/EGFP specific siRNA system has been proved reliable and convenient, and can also be applied widely as control in other RNA interference studies.

Cell Line↗
PubMed2005

Computational design of antiviral RNA interference strategies that resist human immunodeficiency virus escape.

Recently developed antiviral strategies based upon RNA interference (RNAi), which harnesses an innate cellular system for the targeted down-regulation of gene expression, appear highly promising and offer alternative approaches to conventional highly active antiretroviral therapy or efforts to develop an AIDS vaccine. However, RNAi is faced with several challenges that must be overcome to fully realize its promise. Specifically, it degrades target RNA in a highly sequence-specific manner and is thus susceptible to viral mutational escape, and there are also challenges in delivery systems to induce RNAi. To aid in the development of anti-human immunodeficiency virus (anti-HIV) RNAi therapies, we have developed a novel stochastic computational model that simulates in molecular-level detail the propagation of an HIV infection in cells expressing RNAi. The model provides quantitative predictions on how targeting multiple locations in the HIV genome, while keeping the overall RNAi strength constant, significantly improves efficacy. Furthermore, it demonstrates that delivery systems must be highly efficient to preclude leaving reservoirs of unprotected cells where the virus can propagate, mutate, and eventually overwhelm the entire system. It also predicts how therapeutic success depends upon a relationship between RNAi strength and delivery efficiency and uniformity. Finally, targeting an essential viral element, in this case the HIV TAR region, can be highly successful if the RNAi target sequence is correctly selected. In addition to providing specific predictions for how to optimize a clinical therapy, this system may also serve as a future tool for investigating more fundamental questions of viral evolution.

Computational Biology↗
PubMed2004

Control of HIV-1 replication by RNA interference.

Small interfering RNAs (siRNAs) have been shown to direct sequence-specific inhibition of gene expression in mammalian cells. siRNAs are RNA duplexes of 21-23 nucleotides (nts) with approximately 2nt 3' overhangs that can induce degradation of their homologous target mRNAs without interferon responses in mammalian cells. The degradation of the target occurs at the post-transcriptional level, meaning a post-transcriptional gene silencing (PTGS) mechanism called as RNA interference (RNAi). RNAi has emerged as an efficient method to inhibit gene expression in mammalian cells with increasingly successful cases of knockdown of many specific genes. Recent works have shown that the use of RNAi could inhibit HIV-1 replication by targeting viral or cellular genes. RNAi can be considered as a gene-specific therapeutic option for controlling HIV-1 replication. However, the control of HIV-1 replication has become complex because of the limited effectiveness of existing anti-HIV-1 agents and the high speed mutation rate of the HIV-1 genome. Careful assessments are required for the potential of RNAi as a gene therapy approach for controlling HIV-1 replication. This review will discuss the status of the science using RNAi for controlling HIV-1 replication and will describe possible problems for therapeutic applications of RNAi-mediated technologies for HIV-1 behind this novel mechanism.

Gene Expression Regulation, Viral↗
PubMed2006

Influenza A virus NS1 protein does not suppress RNA interference in mammalian cells.

Influenza A virus NS1 protein has been shown to suppress RNA interference (RNAi) in plants and Drosophila. Although it has not been demonstrated experimentally, NS1 has also been thought to inhibit RNAi in mammals as well as being an antagonist of interferon. In this study, the influence of NS1 protein from influenza virus strain A/WSN/33 on RNAi in mammalian cells was investigated. While transiently or stably expressed NS1 was fully competent to inhibit the interferon pathway in cultured cells, it did not suppress RNAi-mediated silencing of different reporter genes. These findings imply a significant difference in RNAi mechanism between mammals and plants.

Animals↗
PubMed2005

Specific interference of urokinase-type plasminogen activator receptor and matrix metalloproteinase-9 gene expression induced by double-stranded RNA results in decreased invasion, tumor growth, and angiogenesis in gliomas.

We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy.

Animals↗
PubMed2005

A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites.

The specificity of small interfering RNA (siRNA)-mediated gene silencing is a critical consideration for the application of RNA interference (RNAi). While the discovery of potential off-target effects by siRNAs is of concern, no systematic analysis has been conducted to explore the specificity of RNAi. Here, we present a study where a functionally validated siRNA (siCD46) was examined for silencing specificity on all possible 57 permutated target sites, each carrying a single-nucleotide mutation that would generate a mismatch when paired with siRNA antisense strand. We found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing. Surprisingly, mismatches formed between adenine (A) and cytosine (C), in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46. Northern blots showed that the silencing of fusion genes harboring the mutated target sites involved target mRNA degradation. This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is instrumental in the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.

Base Pair Mismatch↗
PubMed2005

RNA interference screening in ticks for identification of protective antigens.

Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important arthropod vector of pathogens in North America. Development of vaccines directed against tick proteins may effect reduction of tick infestations and transmission of tick-borne pathogens. The limiting step for the development of tick vaccines has been the identification of tick protective antigens. Reverse vaccinology approaches aimed at reducing animal experimentation while allowing for the rapid screening of pools of potential tick vaccine candidates would greatly facilitate progress towards the development of tick vaccines. Herein, we describe the screening of Ixodes scapularis cDNAs for identification of tick protective antigens using RNA interference (RNAi). The results of the RNAi screening were similar to those obtained previously using expression library immunization and demonstrated that RNAi could serve as a more rapid and cost-effective tool for vaccine antigen discovery in ticks and in other nonmodel organisms.

Animals↗
PubMed2006

Induction of accommodation model by combined RNA interference targeting 1,3-galactosyltransferase gene and low-dose GS-IB4 lectin in vitro.

OBJECTIVE: This study sought to mimic the interaction of xenograft endothelial cells and human serum in vitro after successfully silencing the expression of porcine alpha1,3-galactosyltransferase (alpha1,3GT) gene by RNA interference (RNAi), and to investigate the possibility of inducing accommodation in vitro by stimulation of alpha-Gal-specific binding lectin, Griffonia simplicifolia isolectin B4 (GS-IB4) and RNAi. MATERIALS AND METHODS: Various alpha-Gal expression patterns on a pig endothelial cell immortalized line (PED) was achieved by serial doses of small interfering RNA (siRNA) targeting porcinc alpha1,3GT gene. alpha1,3GT-siRNA transfected PEDs were exposed to increasing doses of GS-IB4 lectin (0.5, 2, and 8 microg/mL) for 4 hours before incubation with normal human serum (NHS). Accommodation phenomenon of PEDs in NHS was observed by 51Cr release and antibody/complement binding assays. RESULTS: With combined RNAi and low-dose GS-IB4 stimulation, PEDs remarkably inhibited complement-mediated cytotoxicity, which showed a better protective effect than using RNAi alone. At a concentration of 2 mug/mL, GS-IB4 exhibited the maximum protective effect. The expression of E-selectin on alpha1,3GT-siRNA transfected PEDs did not differ from that on parental PEDs with heat-inactivated NHS (HINHS) stimulation. Combined with GS-IB4 stimulation, however, it inhibited expression of E-selectin, which was GS-IB4 dose dependent, resulting in mean fluorescence intensity values of 98.5, 42.0, and 36.3 at 0.5, 2, and 8 microg/mL. The mRNA expression of the protective gene HO-1 was significantly up-regulated after treatment with RNAi and low-dose of GS-IB4. CONCLUSIONS: Combined RNAi and low-dose GS-IB4 induced pig endothelial cell accommodation in vitro. The level of alpha-Gal expression played an important role in the induction of accommodation.

Animals↗
PubMed2004

Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting.

The imprinted regulation of H19 and Insulin-like growth factor 2 expression involves binding of the vertebrate insulator protein, CCCTC binding factor (CTCF), to the maternally hypomethylated differentially methylated domain (DMD). How this hypomethylated state is maintained during oogenesis and the role of CTCF, if any, in this process are not understood. With the use of a transgenic RNA interference (RNAi)-based approach to generate oocytes with reduced amounts of CTCF protein, we found increased methylation of the H19 DMD and decreased developmental competence of CTCF-deficient oocytes. Our results suggest that CTCF protects the H19 DMD from de novo methylation during oocyte growth and is required for normal preimplantation development.

Actins↗
PubMed2003

Stable and heritable gene silencing in the malaria vector Anopheles stephensi.

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles . This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.

Animals↗
PubMed2005

Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA.

BACKGROUND: West Nile virus is an emerging human pathogen for which specific antiviral therapy has not been developed. Recent studies have suggested that RNA interference (RNAi) has therapeutic potential as a sequence specific inhibitor of viral infection. Here, we examine the ability of exogenous small interfering RNAs (siRNAs) to block the replication of West Nile virus in human cells. RESULTS: WNV replication and infection was greatly reduced when siRNA were introduced by cytoplasmic-targeted transfection prior to but not after the establishment of viral replication. WNV appeared to evade rather than actively block the RNAi machinery, as sequence-specific reduction in protein expression of a heterologous transgene was still observed in WNV-infected cells. However, sequence-specific decreases in WNV RNA were observed in cells undergoing active viral replication when siRNA was transfected by an alternate method, electroporation. CONCLUSION: Our results suggest that actively replicating WNV RNA may not be exposed to the cytoplasmic RNAi machinery. Thus, conventional lipid-based siRNA delivery systems may not be adequate for therapy against enveloped RNA viruses that replicate in specialized membrane compartments.

Cell Line↗
PubMed2006

RNAi-based suppression and replacement of rds-peripherin in retinal organotypic culture.

Extensive mutational heterogeneity presents a significant barrier to the development of therapeutics for RDS-peripherin-linked autosomal-dominant retinitis pigmentosa (RP), for which more than 50 disease-related mutations have been identified to date. Mutation-independent suppression, using RNA interference (RNAi), together with simultaneous expression of a replacement rds gene (r-rds, which has been altered to escape suppression but nevertheless encodes wild-type protein) has been explored in COS-7 cells and mouse retinal explants. The efficacy of small interfering and short hairpin RNAs (si/shRNAs) silencing mouse rds, and the function of r-rds (containing degenerate substitutions in the RNAi target sequence) were analyzed at transcript (RT-PCR) and protein (ELISA) levels in COS-7 cells. "Dual-" and "triple-expression" constructs carrying the shRNA suppressor and the marker EGFP with or without the r-rds cassette were electroporated in vitro into retinal explants from 1-day-old pups. The retinae were dissociated at day 14, and transduced cells were FACS-sorted using the coexpressed EGFP marker and analyzed by RT-PCR. si/shRNAs decreased rds mRNA and protein expression by up to 82%, while r-rds was protected from suppression in COS-7 cells. Similarly, efficient RNAi-mediated suppression of endogenous rds was detected in retinal explants, while concomitant rescue of r-rds was also achieved. These data validate the concept of RNAi-based suppression coupled with replacement technology for the development of therapies targeting RDS-linked autosomal-dominant RP, and suggest that such approaches could potentially be used for other autosomal-dominant diseases with similarly extensive intragenic heterogeneity.

Animals↗