Target Acquired

Ambion's David Dorris and Qiagen's Walter Tian on how to exploit the benefits of RNAi.

NGP. Sharp’s was a bold statement – do you agree with him and does it still ring true today?

WT. The statement stands as true today. More and more researchers are applying RNAi in basic research as well as in drug discovery, and we now know more about its mechanism and associated molecular machinery. More researchers are applying RNAi in a high throughput screening, and significant advances are being made to apply RNAi for in vivo application, and therapeutics development.

DD. Sharp is certainly right. As a research tool, RNAi has developed at an even faster pace than anyone might have predicted. It is now being used extensively in drug target identification and validation projects, as well as to answer basic biological questions about gene function and for pathway analysis. First used to study one to a handful of genes at a time, the technology is now being applied on a much larger scale. Several genomewide RNAi screens in Drosophila cells and C. elegans have been reported, and the first genomewide RNAi screens in human cells are nearing completion. The purpose of these screens is to ascertain a particular gene’s role in a pathway or biological process under study, for basic functional genomics studies or for target identification or validation projects. Of course, that is only part of the story. Several groups are investigating RNAi’ s use a therapeutic tool, and the first siRNA drugs are now in clinical trials, and show promise for treating age related macular degenerate.

NGP. So why’s RNAi so revolutionary and what are the critical factors for its success?

DD. RNAi is a revolutionary technology for determining gene function by selectively inhibiting the expression of any gene. It is ‘revolutionary’ because it works with essentially any gene, is fast and easy to do, is remarkably specific and, perhaps most importantly, can be used for genome-scale reverse genetics studies in human, mouse, C. elegans, Drosophila, and other higher eukaryotic systems.

The required reagents for RNAi experiments are really quite simple. You need a dsRNA that is completely complementary to the gene transcript(s) you wish to target, a means to deliver that dsRNA to cells, and a way to detect the biological effect of reducing target gene expression (an assay).

In mammalian systems, RNAi is typically induced using short interfering RNAs (siRNAs) complementary to a desired target mRNA. siRNAs are generally 21bp double-stranded RNA molecules with dinucleotide 3’ overhangs and are most often synthesized chemically by oligo manufacturers such as Ambion. They can also be generated intracellulary from short hairpin RNA expressed from a DNA construct. When using siRNAs to trigger RNAi, the most critical factors for success are the design of the siRNAs, which is now done by extensively tested algorithms, the quality of the molecule itself, and the efficiency of siRNA delivery into the desired cell type.

NGP. What is the potential impact on the drug development market of the technologies with which you are involved?

WT. Specific to RNAi technology, it provides another tool for system biology, in drug target discovery and compound screening. Unlike microarray technology, RNAi provides true insight into cellular functions associated with various genes. By combining RNAi with gene expression profiling, biomarker analysis and high throughput compound screening, more valid drug targets can discovered early with confidence.

NGP. More specifically, what is an siRNA library? How can they be fully utilized and why are they a big discussion point at present?

WT. An siRNA library is a pre-built siRNA set, delivered in either 96 well or 384 well plate formats, ready for high throughput analysis. Typically, these library sets target gene families of significance to basic biology and drug development, such as kinase and phosphatase genes, GPCR and druggable genome. There is an increasing trend toward whole genome screening. Advances in siRNA design and synthesis, high throughput transfection protocol, and high content analysis, have made it possible for such genome-wide RNAi screening, even at academic centers.

There are four critical factors that should be considered for successful high throughout RNAi: design and selection of siRNA library, development of high throughput transfection protocol, appropriate screening assays, and related expertise in automation and data analysis.

DD. When combined with plate-based and cell-based assays, including higher content, multiparameter assays, libraries of effective siRNAs allow researchers to quickly evaluate dozens to 10s of thousands of genes for their role in cellular processes.

The hot button issues around siRNA libraries revolve around the corresponding siRNAs’ potency and specificity. Effective siRNA design is key to avoid wasting both time and money. Ambion and few other companies use ‘intelligent’ siRNA design algorithms and validate a portion of their siRNAs to ensure that siRNAs within a library induce efficient gene silencing. Although current siRNA design algorithms yield a high percentage of effective siRNAs, the vast majority of siRNAs in available siRNA libraries have not been experimentally proven to knock down their intended target. In addition, siRNAs have been observed to exhibit sequence specific off-target effects. These effects can be reduced but not eliminated with both careful design and in some cases, chemical modifications. The use of multiple siRNAs per target – whether pooled or used individually – is generally accepted to be the best approach for dealing with this situation. Employing three or more distinct, individual siRNAs per target, and requiring two or more of these siRNAs to induce a given phenotype in the primary screen, significantly decrease both false positive and false negative rates as compared to screening with pools of siRNAs. Therefore most researchers have adopted this approach in their experiments.

NGP. Some researchers doubt that technologies such as RNAi will ever make it out of the lab, what are the kind of obstacles facing these relatively new processes and how can these problems be overcome?

WT. There are several challenges with RNAi. First of all, we must find a way to address off-target effects. These are non-specific knockdowns associated with siRNA sequences homology to unintended targets. In practice, there are several guidelines one could follow to minimize such effects.

Another significant hurdle is cell delivery. siRNA delivery into primary cells is still a big challenge, and existing reagents and technologies are not sufficient. This is a lesser issue for established cell lines.

Applying siRNA as a therapeutic agent presents its own challenges, in the form of siRNA stability, tissue specificity, etc. More progress is made in this area by chemically modifying siRNA, which add improved in vivo stability and tissue specificity.

DD. RNAi’s potential impact on the drug development market is enormous. The technology as it exists right now can be used to better characterize candidate drug targets, and the potential side effects of an antagonist to that target. However, to reach its potential as a therapeutic, hurdles such as efficient siRNA delivery in vivo and minimization of side effects, or off-target effects, need to be overcome. Targeted delivery is really the biggest issue limiting the usefulness of RNAi in vivo right now. Considerable progress has been made, both with conjugation and formulation approaches, but significant improvements are necessary to bring RNAi therapeutics into widespread use. And specificity is being addressed by Ambion and others through both siRNA design and chemical modifications.

NGP. Given this, what are your predictions over the next 10 years – will it meet its potential?

WT. Like many other technologies, RNAi holds great promise. However, it is never a magic bullet, and is not going to solve all the problems alone. By combining RNAi with other high throughput platforms, and by continuous improvement, it will no doubt significantly increase our effectiveness and efficiency in drug discovery and development. We will see an FDA-approved drug based on RNAi technology in 10 years.

DD. As a research tool, I believe that RNAi will continue as an integral part of most molecular and cellular biology laboratories. In fact, its use will undoubtedly expand until it is being employed by essentially every field of biological research. Screening, perhaps the biggest potential for RNAi, will be in its development as a therapeutic. Current successes indicate to me that we will likely have an approved siRNA based drug no later than 2010, with many others in various stages of the drug development pipeline. The hype around RNAi has been tremendous, but I do think that in this case, the hype is warranted.

Walter Tian
A scientist by training, Tian has 20 years of experience in biotech research and life science product marketing. In that time, Tian has successfully launched advanced technology platforms for basic research and drug discovery, such as cDNA microarrays and SNP genotyping and has two US patents in the field of human T lymphocyte immunology. At QIAGEN, Tian is responsible for the company strategy and product marketing in the field of RNAi.

David Dorris
Dorris joined Ambion in 2002 to lead the Custom RNA Services efforts at Ambion, with a focus on siRNA products and RNAi technology. He is responsible for the siRNA manufacturing operation, RNAi R&D and business development related to RNAi. Prior to joining Ambion, Dorris led an effort to develop the CodeLink DNA microarray platform at Motorola Life Sciences (now part of GE Healthcare).