RNAi Goes Genomic: Target Identification and Validation with siRNAs

Recent genomics and proteomics initiatives, including microarray-based transcriptional profiling and analyses of protein interaction networks, have led to the identification of large numbers of potential drug targets. The downside is that the functions of most of these potential targets are poorly understood. Because substantial amounts of basic biological research are required to understand each target’s value, many of these candidate targets are stalled in industry pipelines.

Artificial induction of the natural RNA interference (RNAi) pathway by delivery of short interfering RNAs (siRNAs; 21 bp double-stranded RNA molecules complementary to a particular messenger RNA) is now routinely used to silence specific target genes in cultured cells, and more recently, animal models. Because of its ease of use, low cost, and adaptability to high throughput applications, RNAi has revolutionized the study of gene function. In the drug discovery industry, RNAi screens are now popular for drug target identification and validation. In this capacity, RNAi can be first used to screen for target genes that are important for a disease state or function; then, RNAi can be used to validate that target’s involvement with the disease or function.

RNAi experiments in mammalian cells for target identification purposes require high quality libraries of siRNAs corresponding to either a subset of genes or the entire set of genes in the genome. Because of the large percentage of drugs targeting kinases, siRNA libraries targeting each gene within the human kinome (for example, Ambion's Silencer® Kinase siRNA Library) are of particular interest, as are siRNA libraries targeting the "druggable genome." Additional requirements for RNAi screening in cultured mammalian cells include robust siRNA delivery methods and adequate experimental controls. When these components are combined with cell-based and other high quality assays, a wealth of information can be gleaned about potential drug targets in a short amount of time.

Intelligent siRNA Design
siRNA design is a crucial factor for determining the efficacy of the siRNA as a silencing reagent. Therefore, siRNA library choice should be made cautiously to ensure use of effective, specific, and potent siRNAs. Design rules have led to the development of siRNA design algorithms by a number of academic groups and biotechnology siRNA suppliers. Most siRNA design algorithms, including the one used by Ambion, incorporate sequence criteria such as differential stabilities of the duplex termini, melting temperature of the duplex, nucleotide content of the 3' overhangs, base composition at certain locations, and position within the mRNA, as well as strict specificity requirements to minimize off-target effects. However, the most important attribute of any algorithm is ultimately its performance as tested experimentally. Therefore an algorithm that has been tested on many siRNAs targeting as many different endogenous genes should produce more effective libraries leading to more useful data (Figure 1).

As more is learned about siRNAs, it is becoming increasingly clear that using intelligently designed, highly potent siRNAs that efficiently silence their target gene at low siRNA concentrations (e.g., Ambion's Silencer® siRNAs) is advantageous. Transfection of low siRNA concentrations (typically <30 nMthe optimal siRNA concentration will depend upon the method and efficiency of siRNA delivery, the cell line used, and the effectiveness of the siRNA sequence) reduces the potential for off-target effects and induction of the antiviral response. Low siRNA concentrations also allow the use of less siRNA per experiment, saving reagent costs and permitting the use of multiple siRNAs targeting different genes in a single experiment.

Individual vs. Pooled siRNAs
With randomly designed siRNAs, it can be beneficial to use several siRNAs to a specific target (an siRNA "pool") to increase the chances of reducing target gene expression. However, with the advent of more sophisticated design algorithms that identify very specific and effective siRNAs, this advantage is lost. Screens using siRNA pools show that combinations of siRNAs do not appear to function synergistically to affect target gene expression, and on occasion, less active siRNAs can interfere with the activities of the higher efficacy siRNAs. Screening with three individual siRNAs per gene significantly decreases both false positive and false negative rates, which can be as high as 50% with siRNA pools, resulting in enhanced confidence in RNAi screening data, reduction in the chance that important genes will be missed, and reduction in the amount of time spent following up on false positive "hits" from the screen.

Necessary Controls
Proper negative and positive siRNA controls are critical for RNAi screens, The most obvious and therefore standard negative control is cells treated with an siRNA that does not target any specific gene. This negative control will serve to create the assay baseline and needs to be repeated on every microtiter plate. Caution must be taken when choosing negative control siRNAs, depending on the sensitivity of screening assay. Many investigators test multiple negative controls during the assay development phase of their screen. An additional wrinkle is to use controls that allow one to fully understand the impact of transfection on cellular behavior compared to untreated cells. This, too, is usually a standard step in assay development.

Robust positive controls for each microtiter plate are just as necessary as carefully chosen negative controls. The best positive controls can be used to insure that a minimal experimental threshold is met. The simplest example would be a positive control that induces apoptosis in a screen for genes that affect apoptosis. The most robust positive controls will be near their threshold of activity at the time when phenotypic effects are monitored. For example, if the positive control needs to reduce the target protein levels by at least 60% to induce apoptosis, then poor transfections can be easily identified (i.e., protein levels reduced by only 30%), and the experiment can be repeated.

High Throughput siRNA Delivery
siRNAs can be delivered into cultured mammalian cells by a number of methods, including most prominently, transfection and electroporation. Key to the effective use of siRNA libraries in screening studies are adapting these methods to deliver hundreds or thousands of active siRNAs to cells reproducibly, and with high efficiency, while minimizing cellular toxicity. Additionally, delivery methods that can be manually performed yet are adaptable to robotic liquid handling systems are needed for high throughput processing typically used for siRNA library screening.

Transfection with lipid-based reagents is used to deliver siRNAs to adherent cell lines. Reverse transfection, which involves simultaneous transfection and plating of cells, streamlines the siRNA delivery procedure, making it adaptable to robotic systems. This method of delivery also enhances delivery efficiency in some cell types, such as HepG2 cells, and results in high reproducibility. In the last year, reagents from Ambion and at least one other supplier have been developed specifically to enhance reverse transfection.

Many cell types, especially primary cells, cells grown in suspension, and cell lines of hematopoetic origin are difficult if not impossible to efficiently transfect using lipid-based reagents. For these recalcitrant cell lines, electroporation provides an alternative siRNA delivery method. One of the biggest technical challenges of electroporation is the lack of available instrumentation to perform electroporation in 96 and 384 well formats. Recently, electroporation instruments, such as Ambion's siPORTer™-96 Electroporation Chamber, have been developed that help overcome this obstacle. However, additional advancements in the technology are required to permit 384 well electroporation that can be used with today's automated liquid handling systems.

Recent Successes
The value of siRNA libraries for functional genomics has recently been demonstrated by several publications over the last year. In one report, Marino Zerial and colleagues performed high throughput RNAi screening with an Ambion siRNA library to identify kinases involved in two types of endocytosis: clathrin- and caveolae/raft-mediated endocytosis [Nature (2005) 436: 78-86]. They monitored this through infection of vesicular stomatitis virus, simian virus 40 and transferrin trafficking, as well as through cell proliferation and apoptosis assays. Specific sets of kinases were found to regulate each endocytic route, in some cases, by coordinate regulation. The large number of kinases involved suggests that signaling functions such as those controlling cell adhesion, growth, and proliferation are built into the endocytosis pathway.

In another ambitious screening project that is anticipated to be complete by the end of 2005, a consortium of researchers in Europe, known a the MitoCheck Consortium, is systematically silencing each gene in the human genome to better understand each gene's role in cell cycle progression. Using a genome-wide siRNA library supplied by Ambion, the group is trying to understand how phosphorylation regulates mitosis in human cells. These projects are just two examples of how the systematic silencing of a set of genes by RNAi is helping to elucidate the specific roles of genes in various biological pathways.

Looking Forward
The availability of genome-wide siRNA libraries from Ambion and others has made it possible to evaluate the effects of sequentially reducing the expression of individual genes on cellular processes, providing a powerful reverse genetics tool for use in human and rodent cells. RNAi experiments in different cell lines and even in primary cells dramatically enhance the current toolbox of expression profiling and other functional genomics experiments to discern whether identified drug targets are of high value. Although RNAi alone will not solve every aspect of target identification and validation, RNAi does provide real functional data for putative drug targets and not just a snapshot of a single datum point.

RNAi screening is not limited to the early stages of the drug development process. For example, siRNAs can be used in toxicology experiments to test the metabolic rate of lead compounds. The lead discovery process can also include RNAi to knock down certain genes to differentiate between potential chemical leads (e.g., specificity of leads). Almost every scientist performing target validation experiments wants to perform siRNA experiments in animals to test for phenotypic effects after knockdown of their putative drug targets. Although still in its early stages, this approach already shows promise, and significant advances have been made. For instance, there are now more than 100 publications describing successful delivery of siRNAs to rodent lung, liver, brain, and solid tumors. Ambion and other siRNA suppliers are supporting these efforts by the manufacture of milligram and gram amounts of both modified and unmodified siRNAs ready for in vivo delivery. As these uses for RNAi become commonplace, RNAi technologies will provide a real decrease in the expenditures required to get a drug into clinical trials.

In addition to their application to the drug discovery process, siRNAs themselves are being evaluated as potential therapeutic agents. If realized, the impact on the pharmaceutical industry would be revolutionary. Synthetic siRNAs have been injected systemically and into defined tissues, and demonstrated to elicit target-specific responses. While these advances have spurred a flurry of investments and partnering in the biotech sector, significant hurdles remain.

The most significant hurdle for the therapeutic use of siRNA is delivery: how can an siRNA drug be targeted to a specific tissue? Delivery of nucleic acids to specific organs, tissues, and cells will require significant advances in nucleic acid chemistries, including possible novel conjugations and/or formulations to specifically target certain cells. However, significant progress has already been made. For instance, two siRNAs designed to treat age related macular degeneration are currently in clinical trials. In addition, several reports have described the successful targeting of siRNAs—using a variety of delivery routes and some using conjugation and encapsulation strategies—in rodent lung, liver, small intestine, and solid tumors.

Two other hurdles for siRNA therapeutics relate to challenges faced by all nucleic acid therapeutics: drug stability and manufacturability. The in vivo stability of siRNAs can be increased by a variety of modifications, including alterations to the sugar moiety, the backbone, and/or the base. These modifications are the result of years of research on antisense therapeutics, ribozyme therapeutics, and aptamer technologies, providing a head start for siRNA therapeutics. The current technologies for manufacturing nucleic acids on solid supports are sufficient for supplying kilogram quantities of materials for clinical trials, although new technologies will likely be required to produce the tons of RNA oligonucleotides that will be required to support a major pharmaceutical product.

Over the last four years, RNAi has revolutionized the study of gene function. Now routine for most labs trying to characterize the role of individual gene products, it has also enabled cost effective loss-of-function studies on a genome-wide scale. RNAi is now firmly entrenched as an invaluable tool in most drug discovery pipelines, and further advances in the technology will no doubt enhance the utility of the technique in identifying and validating potential drug targets.

Ambion, Inc., The RNA Company, is a leader in the development and supply of innovative, RNA-based life science research products to academic, biotech, and pharmaceutical researchers. Ambion has a long track record in developing products for handling, preserving, isolating, detecting, and measuring RNA. In addition to providing several highly effective siRNAs to each human, mouse, and rat gene (over 200,000 siRNAs), Ambion offers a wide selection of siRNA libraries, including genome-wide siRNA libraries and custom siRNA libraries. Active in RNAi research since 2001, Ambion has also been instrumental in adapting standard transfection and electroporation methods for high throughput siRNA delivery.

Effectiveness of Silencer siRNAs. This graph shows the distribution of gene silencing measured for 808 siRNAs, designed using an algorithm developed by Cenix BioScience (licensed exclusively to Ambion), targeting >300 endogenously-expressed human genes. Target mRNA levels were measured by qRT-PCR, determining dCt values normalized against 18S rRNA from samples harvested 48 hr after siRNA transfection into HeLa cells. These data demonstrate the effectiveness of the algorithm used to design Ambion's Silencer Pre-designed and Validated siRNAs and Silencer siRNA Libraries. Data courtesy of Cenix BioScience.