CiteULike: jyuh's rnai

Integrating experimental and analytic approaches to improve data quality in genome-wide RNAi screens.

XD Zhang — 2008-07-15T04:34:52-00:00

Journal of biomolecular screening : the official journal of the Society for Biomolecular Screening, Vol. 13, No. 5. (June 2008), pp. 378-389.

RNA interference (RNAi) not only plays an important role in drug discovery but can also be developed directly into drugs. RNAi high-throughput screening (HTS) biotechnology allows us to conduct genome-wide RNAi research. A central challenge in genome-wide RNAi research is to integrate both experimental and computational approaches to obtain high quality RNAi HTS assays. Based on our daily practice in RNAi HTS experiments, we propose the implementation of 3 experimental and analytic processes to improve the quality of data from RNAi HTS biotechnology: (1) select effective biological controls; (2) adopt appropriate plate designs to display and/or adjust for systematic errors of measurement; and (3) use effective analytic metrics to assess data quality. The applications in 5 real RNAi HTS experiments demonstrate the effectiveness of integrating these processes to improve data quality. Due to the effectiveness in improving data quality in RNAi HTS experiments, the methods and guidelines contained in the 3 experimental and analytic processes are likely to have broad utility in genome-wide RNAi research.

A method with flexible and balanced control of false negatives and false positives for hit selection in RNA interference high-throughput screening assays: a statistical terminology.

E Schechtman — 2008-07-15T04:35:14-00:00

Journal of biomolecular screening : the official journal of the Society for Biomolecular Screening, Vol. 13, No. 4. (April 2008), pp. 309-311.

Zhang suggests a new method that is flexible and controls the balance between false negatives and false positives for hit selection in RNA high-throughput screening assays. The author shows that the same decision rules and balances can be expressed by familiar statistical terms such as type I error and power and hence connects the new method to known statistical tools. (Journal of Biomolecular Screening 2008:309-311).

Median absolute deviation to improve hit selection for genome-scale RNAi screens.

N Chung — 2008-07-15T04:36:05-00:00

Journal of biomolecular screening : the official journal of the Society for Biomolecular Screening, Vol. 13, No. 2. (February 2008), pp. 149-158.

High-throughput screening (HTS) of large-scale RNA interference (RNAi) libraries has become an increasingly popular method of functional genomics in recent years. Cell-based assays used for RNAi screening often produce small dynamic ranges and significant variability because of the combination of cellular heterogeneity, transfection efficiency, and the intrinsic nature of the genes being targeted. These properties make reliable hit selection in the RNAi screen a difficult task. The use of robust methods based on median and median absolute deviation (MAD) has been suggested to improve hit selection in such cases, but mean and standard deviation (SD)-based methods are still predominantly used in many RNAi HTS. In an experimental approach to compare these 2 methods, a genome-scale small interfering RNA (siRNA) screen was performed, in which the identification of novel targets increasing the therapeutic index of the chemotherapeutic agent mitomycin C (MMC) was sought. MAD values were resistant to the presence of outliers, and the hits selected by the MAD-based method included all the hits that would be selected by SD-based method as well as a significant number of additional hits. When retested in triplicate, a similar percentage of these siRNAs were shown to genuinely sensitize cells to MMC compared with the hits shared between SD- and MAD-based methods. Confirmed hits were enriched with the genes involved in the DNA damage response and cell cycle regulation, validating the overall hit selection strategy. Finally, computer simulations showed the superiority and generality of the MAD-based method in various RNAi HTS data models. In conclusion, the authors demonstrate that the MAD-based hit selection method rescued physiologically relevant false negatives that would have been missed in the SD-based method, and they believe it to be the desirable 1st-choice hit selection method for RNAi screen results.

Antisense, RNAi, and gene silencing strategies for therapy: Mission possible or impossible?

Elizabeth Rayburn — 2008-06-29T05:43:43-00:00

Drug Discovery Today, Vol. 13, No. 11-12. (June 2008), pp. 513-521.

Antisense oligonucleotides can regulate gene expression in living cells. As such, they regulate cell function and division, and can modulate cellular responses to internal and external stresses and stimuli. Although encouraging results from preclinical and clinical studies have been obtained and significant progress has been made in developing these agents as drugs, they are not yet recognized as effective therapeutics. Several major hurdles remain to be overcome, including problems with efficacy, off-target effects, delivery and side effects. The lessons learned from antisense drug development can help in the development of other oligonucleotide-based therapeutics such as CpG oligonucleotides, RNAi and miRNA.

An image score inference system for RNAi genome-wide screening based on fuzzy mixture regression modeling.

Jun Wang — 2008-06-25T06:07:02-00:00

Journal of biomedical informatics (29 April 2008)

With recent advances in fluorescence microscopy imaging techniques and methods of gene knock down by RNA interference (RNAi), genome-scale high-content screening (HCS) has emerged as a powerful approach to systematically identify all parts of complex biological processes. However, a critical barrier preventing fulfillment of the success is the lack of efficient and robust methods for automating RNAi image analysis and quantitative evaluation of the gene knock down effects on huge volume of HCS data. Facing such opportunities and challenges, we have started investigation of automatic methods towards the development of a fully automatic RNAi-HCS system. Particularly important are reliable approaches to cellular phenotype classification and image-based gene function estimation. We have developed a HCS analysis platform that consists of two main components: fluorescence image analysis and image scoring. For image analysis, we used a two-step enhanced watershed method to extract cellular boundaries from HCS images. Segmented cells were classified into several predefined phenotypes based on morphological and appearance features. Using statistical characteristics of the identified phenotypes as a quantitative description of the image, a score is generated that reflects gene function. Our scoring model integrates fuzzy gene class estimation and single regression models. The final functional score of an image was derived using the weighted combination of the inference from several support vector-based regression models. We validated our phenotype classification method and scoring system on our cellular phenotype and gene database with expert ground truth labeling. We built a database of high-content, 3-channel, fluorescence microscopy images of Drosophila Kc(167) cultured cells that were treated with RNAi to perturb gene function. The proposed informatics system for microscopy image analysis is tested on this database. Both of the two main components, automated phenotype classification and image scoring system, were evaluated. The robustness and efficiency of our system were validated in quantitatively predicting the biological relevance of genes.

Using iterative cluster merging with improved gap statistics to perform online phenotype discovery in the context of high-throughput RNAi screens

Zheng Yin — 2008-06-06T07:33:06-00:00

BMC Bioinformatics, Vol. 9 (05 June 2008), 264.

Constraint factor graph cutbased active contour method for automated cellular image segmentation in RNAi screening

C Chen — 2008-04-29T09:12:54-00:00

Journal of Microscopy, Vol. 230, No. 2. (May 2008), pp. 177-191.

Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides

Rudy Juliano — 2008-06-18T07:34:29-00:00

Nucl. Acids Res. (16 June 2008), gkn342.

The potential use of antisense and siRNA oligonucleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effective intracellular delivery of the active molecules. In this Survey and Summary, we review recent reports on delivery strategies, including conjugates of oligonucleotides with various ligands, as well as use of nanocarrier approaches. These are discussed in the context of intracellular trafficking pathways and issues regarding in vivo biodistribution of molecules and nanoparticles. Molecular-sized chemical conjugates and supramolecular nanocarriers each display advantages and disadvantages in terms of effective and nontoxic delivery. Thus, choice of an optimal delivery modality will likely depend on the therapeutic context. 10.1093/nar/gkn342

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Mark Kleinman — 2008-03-27T04:35:18-00:00

Nature (26 March 2008)

Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference.

T Endoh — 2008-06-06T12:47:33-00:00

Bioconjugate chemistry, Vol. 19, No. 5. (May 2008), pp. 1017-1024.

HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

Efficient in vivo delivery of siRNA to the liver by conjugation of alpha-tocopherol.

K Nishina — 2008-06-06T12:46:56-00:00

Molecular therapy : the journal of the American Society of Gene Therapy, Vol. 16, No. 4. (April 2008), pp. 734-740.

RNA interference is a powerful tool for target-specific knockdown of gene expression. However, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ, which is essential for therapeutic applications, has not been established. In this study we used alpha-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The alpha-tocopherol was covalently bound to the antisense strand of 27/29-mer siRNA at the 5'-end (Toc-siRNA). The 27/29-mer Toc-siRNA was designed to be cleaved by Dicer, producing a mature form of 21/21-mer siRNA after releasing alpha-tocopherol. The C6 hydroxyl group of alpha-tocopherol, associated with antioxidant activity, was abolished. Using this new vector, intravenous injection of 2 mg/kg of Toc-siRNA, targeting apolipoprotein B (apoB), achieved efficient reduction of endogenous apoB messenger RNA (mRNA) in the liver. The downregulation of apoB mRNA was confirmed by the accumulation of lipid droplets in the liver as a phenotype. Neither induction of interferons (IFNs) nor other overt side effects were revealed by biochemical and pathological analyses. These findings indicate that Toc-siRNA is effective and safe for RNA interference-mediated gene silencing in vivo.

Efficient construction of an inverted minimal H1 promoter driven siRNA expression cassette: facilitation of promoter and siRNA sequence exchange.

H Nassanian — 2008-06-06T12:45:08-00:00

PLoS ONE, Vol. 2, No. 1. (2007)

BACKGROUND: RNA interference (RNAi), mediated by small interfering RNA (siRNA), is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC). The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template. PRINCIPAL FINDINGS: We show here the use of a ligase chain reaction (LCR) to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each). Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells. CONCLUSIONS: The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette.

Gene expression silencing with specific small interfering RNA goes beyond specificity a study of key parameters to take into account in the onset of small interfering RNA off-target effects

Vankoningsloo — 2008-05-15T18:39:15-00:00

FEBS Journal, Vol. 275, No. 11. (June 2008), pp. 2738-2753.

A PCR based method to construct small interference RNA expression vectors.

Zhiyong Zhang — 2008-06-04T16:05:20-00:00

Molecular biology reports (12 April 2008)

Small interference RNAs (siRNA) have been shown to be useful in the field of gene therapy and gene function studies. As a siRNA expression vector, pSilencer employ RNA polymerase III promoters and could stably produce siRNA for weeks. But once one siRNA sequence was inserted into the pSilencer vector, the other siRNA sequence will hardly be reconstructed, because the site of siRNA production has been occupied and difficult to be changed, so it is not suitable for screen of effective siRNA sequence. To solve this problem, we constructed the subclone pSilcencer329, which generated from pSilencer3.1, then developed a PCR based method of constructing siRNA expression vectors, and generated pSilencerBCL2L2 recombinants efficiently. This method was proven to be effective, reliable, and less expensive, and thus will be of great help in regular gene silencing studies, and will be especially suitable for large scale gene function analysis.

OligoWalk: an online siRNA design tool utilizing hybridization thermodynamics.

Zhi John Lu — 2008-06-04T15:50:49-00:00

Nucleic acids research (19 May 2008)

Given an mRNA sequence as input, the OligoWalk web server generates a list of small interfering RNA (siRNA) candidate sequences, ranked by the probability of being efficient siRNA (silencing efficacy greater than 70%). To accomplish this, the server predicts the free energy changes of the hybridization of an siRNA to a target mRNA, considering both siRNA and mRNA self-structure. The free energy changes of the structures are rigorously calculated using a partition function calculation. By changing advanced options, the free energy changes can also be calculated using less rigorous lowest free energy structure or suboptimal structure prediction methods for the purpose of comparison. Considering the predicted free energy changes and local siRNA sequence features, the server selects efficient siRNA with high accuracy using a support vector machine. On average, the fraction of efficient siRNAs selected by the server that will be efficient at silencing is 78.6%. The OligoWalk web server is freely accessible through internet at http://rna.urmc.rochester.edu/servers/oligowalk.

Fundamental differences in the equilibrium considerations for siRNA and antisense oligodeoxynucleotide design.

Zhi John Lu — 2008-06-04T15:50:55-00:00

Nucleic acids research (15 May 2008)

Both siRNA and antisense oligodeoxynucleotides (ODNs) inhibit the expression of a complementary gene. In this study, fundamental differences in the considerations for RNA interference and antisense ODNs are reported. In siRNA and antisense ODN databases, positive correlations are observed between the cost to open the mRNA target self-structure and the stability of the duplex to be formed, meaning the sites along the mRNA target with highest potential to form strong duplexes with antisense strands also have the greatest tendency to be involved in pre-existing structure. Efficient siRNA have less stable siRNA-target duplex stability than inefficient siRNA, but the opposite is true for antisense ODNs. It is, therefore, more difficult to avoid target self-structure in antisense ODN design. Self-structure stabilities of oligonucleotide and target correlate to the silencing efficacy of siRNA. Oligonucleotide self-structure correlations to efficacy of antisense ODNs, conversely, are insignificant. Furthermore, self-structure in the target appears to correlate with antisense ODN efficacy, but such that more effective antisense ODNs appear to target mRNA regions with greater self-structure. Therefore, different criteria are suggested for the design of efficient siRNA and antisense ODNs and the design of antisense ODNs is more challenging.

AsiDesigner: exon-based siRNA design server considering alternative splicing.

Young-Kyu Park — 2008-06-04T15:51:01-00:00

Nucleic acids research (14 May 2008)

RNA interference (RNAi) with small interfering RNA (siRNA) has become a powerful tool in functional and medical genomic research through directed post-transcriptional gene silencing. In order to apply RNAi technique for eukaryotic organisms, where frequent alternative splicing results in diversification of mRNAs and finally of proteins, we need spliced mRNA isoform silencing to study the function of individual proteins. AsiDesigner is a web-based siRNA design software system, which provides siRNA design capability to account for alternative splicing for mRNA level gene silencing. It provides numerous novel functions including the designing of common siRNAs for the silencing of more than two mRNAs simultaneously, a scoring scheme to evaluate the performance of designed siRNAs by adopting currently known key design factors, a stepwise off-target searching with BLAST and FASTA algorithms and checking the folding secondary structure energy of siRNAs. To do this, we developed a novel algorithm to evaluate the common target region, where siRNAs can be designed to knockdown a specific mRNA isoform or more than two mRNA isoforms from a target gene simultaneously. The developed algorithm and the AsiDesigner were tested and validated as very effective throughout widely performed gene silencing experiments. It is expected that AsiDesigner will play an important role in functional genomics, drug discovery and other molecular biological research. AsiDesigner is freely accessible at http://sysbio.kribb.re.kr/AsiDesigner/.

The art and design of genetic screens: RNA interference.

Michael Boutros — 2008-06-04T15:06:12-00:00

Nature reviews. Genetics (3 June 2008)

The remarkable gene knockdown technique of RNAi has opened exciting new avenues for genetic screens in model organisms and human cells. Here we describe the current state of the art for RNAi screening, and stress the importance of well-designed assays and of analytical approaches for large-scale screening experiments, from high-throughput screens using simplified homogenous assays to microscopy and whole-animal experiments. Like classical genetic screens in the past, the success of large-scale RNAi surveys depends on a careful development of phenotypic assays and their interpretation in a relevant biological context.

Merging molecular imaging and RNA interference: Early experience in live animals.

Alexei A Bogdanov — 2008-06-01T16:36:07-00:00

Journal of cellular biochemistry (4 February 2008)

The rapid development of non-invasive imaging techniques and imaging reporters coincided with the enthusiastic response that the introduction of RNA interference (RNAi) techniques created in the research community. Imaging in experimental animals provides quantitative or semi-quantitative information regarding the biodistribution of small interfering RNAs and the levels of gene interference (i.e., knockdown of the target mRNA) in living animals. In this review we give a brief summary of the first imaging findings that have potential for accelerating the development and testing of new approaches that explore RNAi as a method for achieving loss-of-function effects in vivo and as a promising therapeutic tool. J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.

Chemical modification: the key to clinical application of RNA interference?

DR Corey — 2008-05-14T06:41:41-00:00

The Journal of clinical investigation, Vol. 117, No. 12. (December 2007), pp. 3615-3622.

RNA interference provides a potent and specific method for controlling gene expression in human cells. To translate this potential into a broad new family of therapeutics, it is necessary to optimize the efficacy of the RNA-based drugs. As discussed in this Review, it might be possible to achieve this optimization using chemical modifications that improve their in vivo stability, cellular delivery, biodistribution, pharmacokinetics, potency, and specificity.

The impact of target site accessibility on the design of effective siRNAs

Hakim Tafer — 2008-05-09T21:16:16-00:00

Nature Biotechnology, Vol. 26, No. 5. (27 April 2008), pp. 578-583.

Analysis of siRNA specificity on targets with double-nucleotide mismatches.

Cecilia Dahlgren — 2008-05-13T08:15:01-00:00

Nucleic acids research (17 April 2008)

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that approximately 35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.

Systemic Leukocyte-Directed siRNA Delivery Revealing Cyclin D1 as an Anti-Inflammatory Target

Dan Peer — 2008-02-01T12:44:38-00:00

Science, Vol. 319, No. 5863. (1 February 2008), pp. 627-630.

Cyclin D1 (CyD1) is a pivotal cell cycleregulatory molecule and a well-studied therapeutic target for cancer. Although CyD1 is also strongly up-regulated at sites of inflammation, its exact roles in this context remain uncharacterized. To address this question, we developed a strategy for selectively silencing CyD1 in leukocytes in vivo. Targeted stabilized nanoparticles (tsNPs) were loaded with CyD1small interfering RNA (siRNA). Antibodies to 7 integrin (7 I) were then used to target specific leukocyte subsets involved in gut inflammation. Systemic application of 7 I-tsNPs silenced CyD1 in leukocytes and reversed experimentally induced colitis in mice by suppressing leukocyte proliferation and T helper cell 1 cytokine expression. This study reveals CyD1 to be a potential anti-inflammatory target, and suggests that the application of similar modes of targeting by siRNA may be feasible in other therapeutic settings. 10.1126/science.1149859

Cellular phenotype recognition for high-content RNA interference genome-wide screening.

J Wang — 2008-01-31T06:11:56-00:00

J Biomol Screen, Vol. 13, No. 1. (February 2008), pp. 29-39.

Genome-wide, cell-based screens using high-content screening (HCS) techniques and automated fluorescence microscopy generate thousands of high-content images that contain an enormous wealth of cell biological information. Such screens are key to the analysis of basic cell biological principles, such as control of cell cycle and cell morphology. However, these screens will ultimately only shed light on human disease mechanisms and potential cures if the analysis can keep up with the generation of data. A fundamental step toward automated analysis of high-content screening is to construct a robust platform for automatic cellular phenotype identification. The authors present a framework, consisting of microscopic image segmentation and analysis components, for automatic recognition of cellular phenotypes in the context of the Rho family of small GTPases. To implicate genes involved in Rac signaling, RNA interference (RNAi) was used to perturb gene functions, and the corresponding cellular phenotypes were analyzed for changes. The data used in the experiments are high-content, 3-channel, fluorescence microscopy images of Drosophila Kc167 cultured cells stained with markers that allow visualization of DNA, polymerized actin filaments, and the constitutively activated Rho protein Rac(V12). The performance of this approach was tested using a cellular database that contained more than 1000 samples of 3 predefined cellular phenotypes, and the generalization error was estimated using a cross-validation technique. Moreover, the authors applied this approach to analyze the whole high-content fluorescence images of Drosophila cells for further HCS-based gene function analysis.

Klotho RNAi induces premature senescence of human cells via a p53/p21 dependent pathway

Rita de Oliveira — 2008-01-28T04:07:02-00:00

FEBS Letters, Vol. 580, No. 24. (16 October 2006), pp. 5753-5758.

Klotho has recently emerged as a regulator of aging. To investigate the role of Klotho in the regulation of cellular senescence, we generated stable MRC-5 human primary fibroblast cells knockdown for Klotho expression by RNAi. Downregulation of Klotho dramatically induces premature senescence with a concomitant upregulation of p21. The upregulation of p21 is associated with cell cycle arrest at G1/S boundary. Knockdown of p53 in the Klotho attenuated MRC-5 cells restores normal growth and replicative potential. These results demonstrate that Klotho normally regulates cellular senescence by repressing the p53/p21 pathway. Our findings implicate Klotho as a regulator of aging in primary human fibroblasts.

www.rnaworkbench.com: A new program for analyzing RNA interference.

Radka Svobodová Vařeková — 2008-01-22T12:48:30-00:00

Comput Methods Programs Biomed (18 January 2008)

RNA interference (RNAi) has become an important tool to study and utilize gene silencing by introducing short interfering RNA (siRNA). In order to predict the most efficient siRNAs, a new software tool, RNA Workbench (RNAWB), has been designed and is freely available (after registration) on http://www.rnaworkbench.com. In addition to the standard selection rules, RNAWB includes the possibility of statistical analyses of the applied selection rules (criteria). The role of RNA secondary structures in the RNA interference process as well as the application of sequence rules are discussed to show the applicability of the software.

Nonviral delivery of synthetic siRNAs in vivo.

S Akhtar — 2008-01-18T02:14:51-00:00

J Clin Invest, Vol. 117, No. 12. (December 2007), pp. 3623-3632.

Sequence-specific gene silencing using small interfering RNA (siRNA) is a Nobel prize-winning technology that is now being evaluated in clinical trials as a potentially novel therapeutic strategy. This article provides an overview of the major pharmaceutical challenges facing siRNA therapeutics, focusing on the delivery strategies for synthetic siRNA duplexes in vivo, as this remains one of the most important issues to be resolved. This article also highlights the importance of understanding the genocompatibility/toxicogenomics of siRNA delivery reagents in terms of their impact on gene-silencing activity and specificity. Collectively, this information is essential for the selection of optimally acting siRNA delivery system combinations for the many proposed applications of RNA interference.

Therapeutic application of RNAi: is mRNA targeting finally ready for prime time?

Dirk Grimm — 2007-12-26T05:23:05-00:00

J. Clin. Invest., Vol. 117, No. 12. (3 December 2007), pp. 3633-3641.

With unprecedented speed, RNA interference (RNAi) has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool and perhaps our single most promising biotherapeutic for a wide array of diseases. Numerous studies document RNAi efficacy in laboratory animals, and the first clinical trials are underway and thus far suggest that RNAi is safe to use in humans. Yet substantial hurdles have also surfaced and must be surmounted before therapeutic RNAi applications can become a standard therapy. Here we review the most critical roadblocks and concerns for clinical RNAi transition, delivery, and safety. We highlight emerging solutions and concurrently discuss novel therapeutic RNAi-based concepts. The current rapid advances create realistic optimism that the establishment of RNAi as a new and potent clinical modality in humans is near. 10.1172/JCI34129

RNA interference against viruses: strike and counterstrike

Joost Haasnoot — 2007-12-08T05:44:10-00:00

Nature Biotechnology, Vol. 25, No. 12. (07 December 2007), pp. 1435-1443.

Inhibition of Integrin-Linked Kinase via a siRNA Expression Plasmid Attenuates Connective Tissue Growth Factor-Induced Human Proximal Tubular Epithelial Cells to Mesenchymal Transition.

Bi-Cheng Liu — 2007-11-20T02:09:29-00:00

Am J Nephrol, Vol. 28, No. 1. (2008), pp. 143-151.

Background: Increasing evidence suggests that connective tissue growth factor (CTGF) is involved in the epithelial-to-mesenchymal transition (EMT). The exact intracellular events that drive this process, however, are not fully understood. In this study, we investigated the role of integrin-linked kinase (ILK) in mediating CTGF-induced EMT. Methods: The expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin upon the stimulation by recombinant human CTGF (rhCTGF) in cultured human tubular epithelial cell line (HK-2) was detected by real-time RT-PCR and Western blot. Subsequently, the role of ILK was determined by using ILK siRNA. Results: rhCTGF increased the mRNA expression of alpha-SMA significantly in a dose- and time-dependent manner, while E-cadherin mRNA decreased in a dose- and time-dependent manner. alpha-SMA protein was up-regulated after stimulation by 5 ng/ml CTGF for 96 h, and increased further after stimulation by 50 ng/ml. An immunocytochemical study showed that alpha-SMA was initially detectable at 48 h, and increased further at 72 h, while there was almost no alpha-SMA immunostaining observed in the control group at the same time point. E-cadherin protein was also down-regulated in a dose-dependent manner. Transfection of HK-2 cells with ILK-siRNA significantly attenuated rhCTGF-induced alpha-SMA induction and E-cadherin repression. Conclusion: Our study suggested that ILK mediated the effect of EMT in proximal tubular epithelial cells stimulated by CTGF. Copyright (c) 2007 S. Karger AG, Basel.

Interfering with disease: a progress report on siRNA-based therapeutics

Antonin de Fougerolles — 2007-06-02T15:09:50-00:00

Nature Reviews Drug Discovery, Vol. 6, No. 6., pp. 443-453.

Enhancing and confirming the specificity of RNAi experiments

Bryan Cullen — 2006-08-24T06:57:37-00:00

Nature Methods, Vol. 3, No. 9. (23 August 2006), pp. 677-681.

Delivery of RNA interference.

CX Li — 2007-09-20T03:23:07-00:00

Cell Cycle, Vol. 5, No. 18. (September 2006), pp. 2103-2109.

Over the last few years, RNA Interference (RNAi), a naturally occurring mechanism of gene regulation conserved in plant and mammalian cells, has opened numerous novel opportunities for basic research across the field of biology. While RNAi has helped accelerate discovery and understanding of gene functions, it also has great potential as a therapeutic and potentially prophylactic modality. Challenging diseases failing conventional therapeutics could become treatable by specific silencing of key pathogenic genes. More specifically, therapeutic targets previously deemed "undruggable" by small molecules, are now coming within reach of RNAi based therapy. For RNAi to be effective and elicit gene silencing response, the double-stranded RNA molecules must be delivered to the target cell. Unfortunately, delivery of these RNA duplexes has been challenging, halting rapid development of RNAi-based therapies. In this review we present current advancements in the field of siRNA delivery methods, including the pros and cons of each method.

The best control for the specificity of RNAi.

M Sarov — 2007-09-20T02:48:42-00:00

Trends Biotechnol, Vol. 23, No. 9. (September 2005), pp. 446-448.

RNA interference (RNAi) is revolutionizing functional genomics. However, there are several reasons to be concerned about the specificity and off-target effects of this technique. A recent paper by Kittler et al. describes a straightforward way to validate RNAi specificity, which exploits the increasing availability of bacterial artificial chromosome (BAC) clone resources. Genetic rescue of the RNAi phenotype by BAC transgenesis is the best control yet described for specificity, and has further implications for reverse genetics.

Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer-small molecule interaction.

CI An — 2006-06-09T01:27:44-00:00

RNA, Vol. 12, No. 5. (May 2006), pp. 710-716.

Recent studies have uncovered extensive presence and functions of small noncoding RNAs in gene regulation in eukaryotes. In particular, RNA interference (RNAi) has been the subject of significant investigations for its unique role in post-transcriptional gene regulation and utility as a tool for artificial gene knockdown. Here, we describe a novel strategy for post-transcriptional gene regulation in mammalian cells in which RNAi is specifically modulated through RNA aptamer-small molecule interaction. Incorporation of an RNA aptamer for theophylline in the loop region of a short hairpin RNA (shRNA) designed to silence fluorescent reporter genes led to dose-dependent inhibition of RNAi by theophylline. shRNA cleavage experiments using recombinant Dicer demonstrated that theophylline inhibited cleavage of an aptamer-fused shRNA by Dicer in vitro. Inhibition of siRNA production by theophylline was also observed in vivo. The results presented here provide the first evidence of specific RNA-small molecule interaction affecting RNAi, and a novel strategy to regulate mammalian gene expression by small molecules without engineered proteins.

A protein alternative to RNAi.

D Evanko — 2007-09-20T02:49:41-00:00

Nat Methods, Vol. 3, No. 11. (November 2006)

A general method of targeting tagged proteins for rapid degradation in the cell promises to provide a powerful alternative to RNA interference (RNAi) for studying the functions of proteins in living cells.

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells

Ralf Kittler — 2007-04-22T04:51:32-00:00

PNAS, Vol. 102, No. 7. (15 February 2005), pp. 2396-2401.

RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function. 10.1073/pnas.0409861102

An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division

Ralf Kittler — 2004-12-23T14:17:35-00:00

Nature, Vol. 432, No. 7020. (23 December 2004), pp. 1036-1040.

A novel shRNA vector that enables rapid selection and identification of knockdown cells.

A Nagasaki — 2007-09-20T01:24:05-00:00

Plasmid, Vol. 58, No. 2. (September 2007), pp. 190-194.

Small interference RNA (siRNA) is a powerful tool for disrupting expression of specific genes in a variety of cells. We have developed a vector, piMARK, which mediates expression of both small hairpin RNA (shRNA) and the blasticidin resistance (Bsr) protein fused with enhanced green fluorescent protein (EGFP), enabling rapid selection and identification of knockdown cells. Using this vector, we targeted Ect2, a gene encoding a guanine nucleotide exchange factor for several small GTPases, in human cell lines. Incubation in the presence of 10mug/ml blasticidin S rapidly killed untransfected cells, so that after 24h >90% of surviving HeLa S3 cells emitted green fluorescence and >70% were binucleate as a result of the frequent failure of cell division. The GFP-Bsr fluorescence enabled easy identification of individual knockdown cells under a fluorescence microscope, which in turn enabled unambiguous assessment of the morphological consequences of silencing Ect2. Moreover, because untransfected cells rapidly died and detached from the substrate, they were easily removed by simply rinsing the culture dishes. It thus should be possible to analyze the biochemical consequences of gene silencing en masse in the absence of a background of untransfected cells.

A more efficient RNAi inducible system for tight regulation of gene expression in mammalian cells and xenograft animals.

J Zhang — 2007-09-20T01:30:54-00:00

RNA, Vol. 13, No. 8. (August 2007), pp. 1375-1383.

Two types of tetracycline-controlled inducible RNAi expression systems have been developed that generally utilize multiple tetracycline operators (TetOs) or repressor fusion proteins to overcome the siRNA leakiness. Here, we report a novel system that overexpresses the tetracycline repressor (TetR) via a bicistronic construct to control siRNA expression. The high level of TetR expression ensures that the inducible promoter is tightly bound, with minimal basal transcription, allowing for regulation solely dependent on TetR rather than a TetR fusion protein via a more complicated mechanism. At the same time, this system contains only a single TetO, thus minimizing the promoter impairment occurring in existing systems due to the incorporation of multiple TetOs, and maximizing the siRNA expression upon induction. In addition, this system combines all the components required for regulation of siRNA expression into a single lentiviral vector, so that stable cell lines can be generated by a single transduction and selection, with significant reduction in time and cost. Taken together, this all-in-one lentiviral vector with the feature of TetR overexpression provides a unique and more efficient tool for conditional gene knockdown that has wide applications. We have demonstrated the high degree of robustness and versatility of this system as applied to several mammalian cells and xenograft animals.

Opportunities for treating chronic hepatitis B and C virus infection using RNA interference

Arbuthnot — 2007-06-20T04:39:49-00:00

Journal of Viral Hepatitis, Vol. 14, No. 7. (July 2007), pp. 447-459.

Applications of RNA interference: current state and prospects for siRNA-based strategies in vivo.

A Aigner — 2007-08-31T02:07:16-00:00

Appl Microbiol Biotechnol, Vol. 76, No. 1. (August 2007), pp. 9-21.

Within the recent years, RNA interference (RNAi) has become an almost-standard method for in vitro knockdown of any target gene of interest. Now, one major focus is to further explore its potential in vivo, including the development of novel therapeutic strategies. From the mechanism, it becomes clear that small interfering RNAs (siRNAs) play a pivotal role in triggering RNAi. Thus, the efficient delivery of target gene-specific siRNAs is one major challenge in the establishment of therapeutic RNAi. Numerous studies, based on different modes of administration and various siRNA formulations and/or modifications, have already accumulated promising results. This applies to various animal models covering viral infections, cancer and multiple other diseases. Continuing efforts will lead to the development of efficient and "double-specific" drugs, comprising of siRNAs with high target gene specificity and of nanoparticles enhancing siRNA delivery and target organ specificity.

Strategies for silencing human disease using RNA interference

Daniel Kim — 2007-02-17T21:01:12-00:00

Nature Reviews Genetics, Vol. 8, No. 3., pp. 173-184.

sIR: siRNA Information Resource, a web-based tool for siRNA sequence design and analysis and an open access siRNA database

Jyoti Shah — 2007-06-01T13:10:11-00:00

BMC Bioinformatics, Vol. 8 (31 May 2007), 178.

High-content siRNA screening.

E Krausz — 2007-03-22T18:14:55-00:00

Mol Biosyst, Vol. 3, No. 4. (April 2007), pp. 232-240.

Very recent developments in instrumentation and image analysis have made microscopy applicable to high-throughput screening (HTS). For 'High-Content Screening' modern automated microscopy systems provide a throughput of up to 100 000 (confocal) images, with amazingly high resolution, of cells fluorescently stained using multiple colours that are imaged simultaneously during the screen. Image analysis tools provide multi-parametric pattern extraction and quantification on-the-fly. Big pharmaceutical companies have presented image-based screens of more than 100 000 compounds, while academia has published data on large RNA interference screens for functional genomics.Numerous whole-genome sequencing projects have been completed and published. Gene annotation is still in flux. Nevertheless, about 23 000 human genes have been reliably annotated. Additionally, gene expression array technologies and proteomics have added further data on molecules present in cells and tissues. The major challenge of the present and future is to unravel the detailed function of all these gene products and their interaction. One way to gain insight, is to design oligonucleotides that induce lack-of-function phenotypes by specifically inhibiting protein production.

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression.

KJ Shin — 2006-11-01T15:15:08-00:00

Proc Natl Acad Sci U S A, Vol. 103, No. 37. (12 September 2006), pp. 13759-13764.

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.