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Commentary
Harnessing the Power of RNA Interference to Advance Anticancer Drug Development
Cancer Drug Development Laboratory, Translational Genomics Research Institute, Gaithersburg, Maryland 20878,2
1 To whom requests for reprints should be addressed, at Cancer Drug Development Laboratory, Translational Genomics Research Institute, 20 Firstfield Road, Suite 110, Gaithersburg, MD 20878. Phone: (240) 631-1607, extension 228; Fax: (240) 631-1918; E-mail: smousses{at}tgen.org
The creation of specific inhibitors against molecular targets that play a role in the cancer phenotype is the basis for a rational strategy for anticancer drug development. Telomerase activity has been observed in almost all of the human tumors, yet it is completely lacking in normal somatic cells (1). Telomerase activity leads to the addition of TTAGGG repeat units at the end of telomeres, protecting the cell from senescence. This led to the hypothesis that telomerase activity results in a selective survival advantage for neoplastic cells. Therefore, molecules involved in telomere maintenance, such as the catalytic subunit of human telomerase, and template RNA component of telomerase are enticing for targets for therapeutic intervention (1). Better inhibitors against genes involved in this process would be very useful tools for investigating this mechanism in experimental systems. Furthermore, specific inhibitors may also increase the clinical success of strategies aimed at decreasing telomerase activity. Kosciolek et al. (2) report of a novel solution to this problem, by developing RNAi-based3 inhibitors to these targets resulting in the successful inhibition of telomerase activity in various cancer cell lines. This type of effort to develop telomerase activity-based therapeutic strategies reveals a more general solution to at least two critical challenges for current drug development. The first challenge relates to the ever-increasing need for specific reagents to conduct functional validation of drug targets, and the second is the difficult problem of developing therapeutic agents, with sufficient specificity and potency.
Genomic technologies have created a massive increase in the number of uncharacterized gene targets. Traditional approaches to functional analysis are dependent on time-consuming screens to first identify inhibitory reagents to use in target validation. This has created a major bottleneck in drug discovery processes. Synthetic RNAi-based inhibitors against specific targets can be generated much more readily. Such reagents, coupled with parallel screening platforms, can facilitate a much higher throughput to address the challenge of drug target validation and lead to the rapid discovery of the most appropriate targets for drug development.
Traditional screening for small chemical compound inhibitors against promising drug targets in cancer have proven to be very costly, labor intensive, and result in very limited clinical success. The binding profiles for most of the current anticancer drugs include numerous significant nonspecific interactions that may contribute to the toxic side effects and narrow therapeutic windows. New approaches to drug discovery to address the issues of specificity and efficacy include a trend toward increasing the complexity, diversity, and size of chemical compound libraries used for target screening, which are then analyzed by ultra high-throughput screening capabilities. The application of rational drug design, based on protein structures, promises to help this situation. Unfortunately, these efforts are also slow and have greatly increased the cost of developing new drugs, but have not significantly increased the clinical success rate for new drug compounds. Biologically derived agents such as antibodies and nucleic acid-based agents such as antisense DNA oligonucleotides can potentially achieve much higher specificity, and have shown improved efficacy against gene products compared with chemical compound inhibitors but are often difficult to develop and manufacture. Therefore, the second challenge in cancer drug development will be to develop inhibitors against these targets that are not only potent at inhibiting the activity of a gene product but to also achieve sufficient biochemical specificity against that target.
RNAi is a powerful new technology with the potential to address both the target validation and the inhibitor specificity challenges in drug development. It was observed early on that introduction of dsRNA into plant cells lead to a bizarre post-transcriptional gene silencing phenomenon. This mechanism turned out to be common to many species of plants and animals, and is suspected to have evolved as an intracellular viral defense mechanism in eukaryotes against foreign RNA and for transposon silencing. Introducing a dsRNA, of which the sequence is homologous to a particular gene, into cells of various plant and animal species resulted in silencing of the expression of the targeted gene in a very potent and sequence-specific manner.
However, in mammalian cells, the introduction of long dsRNA also triggered a nonspecific IFN response, which precluded the use of long, dsRNA for specific gene silencing. It was then discovered that once the dsRNA enters a cell, it is initially cleaved by an enzyme called dicer, and that it is the products of this cleavage, which are 21–23 nucleotide dsRNAs called siRNAs, that mediate the gene silencing through an interaction with a protein complex called RNA-induced silencing complex. Two seminal papers (3–5) then independently reported that one could use 21–23 nucleotide dsRNA as synthetic versions of siRNA, and they demonstrated that transfection of these synthetic siRNA into cells triggers gene silencing in a potent and sequence-specific manner, but without triggering the nonspecific IFN response associated with dsRNA longer than 30 nucleotides. This discovery made it possible and practical to chemically synthesize siRNA, which can be applied as a molecular biology tool for gene silencing in mammalian cells, and made it possible to consider siRNA as a viable therapeutic agent for humans (5).
In this latest application of siRNA to target disease genes, Kosciolek et al. (2) created siRNA-based agents, which inhibited telomerase activity. These kinds of inhibitors could be extremely useful tools for investigating the functional role of telomerase activity in various experimental systems. Additional development of siRNA inhibitors that inhibit telomerase activity also hold promise as therapeutic agents to treat cancer. Their work provides sufficient evidence that such an approach warrants additional investigation into the utility of these agents, but it also highlights some of the limitations of our current mastery of this technology. There are still many challenges in determining the ideal design of siRNA sequence and structure design to achieve the most effective silencing. New rules for more accurately predicting optimal target sites are now emerging (5).4 Despite these limitations, it appears that RNAi-based inhibitors have a higher rate of success, are generally more potent, and are consequently enjoying much wider and faster dissemination than other nucleic acid-based strategies for inhibiting gene expression.
Although it is still early in the development of RNAi technologies, and many challenges lay ahead, RNAi-based agents represent an exciting therapeutic strategy for inhibiting gene targets, with many advantages over traditional drug compounds. One main advantage is the higher level of specificity that can be achieved with nucleic acid-based agents compared with chemical compounds that bind to protein targets. Furthermore, factors such as protein folding, cellular localization of the target, and general accessibility of the proteins and their drug binding sites limit the efficacy of traditional drug compounds for many targets. Consequently, there are many gene products that are very difficult, if not impossible, to inhibit. However, RNAi-inducing reagents can potentially silence any gene in the transcriptome, in a sequence-specific manner, making them the ideal research tool for studying gene function. It is only a matter of time before siRNA agents will face the ultimate test in clinical trials of RNAi-based therapeutics.
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3 The abbreviations used are: RNAi, RNA interference; dsRNA, double-stranded RNA; siRNA, short interfering RNA.
4 N. J. Caplen, personal communication.
Received 1/31/03; accepted 1/31/03.
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