Early experiments in plants and lower animals showed that this post-transcriptional gene silencing could be effected by the introduction into cells of one of the intermediates in the RNAi pathway, small interfering RNAs (siRNAs), which are 21-23 nucleotide double-stranded RNA sequences that specifically cause homologous mRNA cleavage. When it was shown in 2001 that RNAi could be successfully applied to mammalian cell culture, RNAi took a giant step toward revolutionizing discovery biology and drug development strategies. More recent studies have expanded the use of RNAi beyond cell culture, showing that gene silencing can be demonstrated in vivo in mammals and that siRNAs can be targeted to specific tissues.

RNAi-induced gene silencing is now commonly used by scientists as a tool to characterize the individual biological roles of specific genes and to illuminate their participation in clinically important pathways or mechanisms (e.g., insulin metabolism, the cell cycle, or apoptosis. RNAi is the result of well-coordinated RNA-to-protein interactions; a key participant in this mechanism is the RNA-induced silencing complex (RISC), which plays a role in the binding of siRNA and its target mRNA to effect eventual mRNA cleavage and resulting gene suppression.

Numerous studies have shown that certain structural and sequence-specific characteristics of siRNA are highly significant in determining the success of its interaction with RISC. For example, thermodynamic stability profiles of siRNA duplexes led to the identification of key specific locations in the molecule where certain nucleotide base pairs provide a high degree of probability of ensuring siRNA functionality. To further refine the pool of functional siRNA candidates, bioinformatic screens can be used to identify unique siRNA sequences that will specifically target a gene without producing unintended silencing of other genes (i.e., off-target effects).


Promise of Therapeutics
The development and application of chemical modification patterns to the siRNA molecule that further enhance potency, mRNA target specificity, and in vivo stability provide additional promise for the use of RNAi in the development of therapeutics. Novel RNA synthesis technologies are well positioned for the rapid and reliable large-scale synthesis of siRNAs for use in high-throughput target validation strategies and for use as therapeutic agents themselves.

Current drug discovery and development programs are fed by fast-paced genome sequencing projects. These projects define the critical sets of genes that delineate normal biological function and lead to understanding of how genetic mutations or pathogens interfere with this normal function. To this end, cataloging whole genomes facilitates the identification of potential gene candidates against which small molecules or therapeutic agents may be developed to alleviate or abrogate disease-related syndromes or specific pathologies.

However, the drug-development process, and more specifically target validation, is often hampered by the plethora of sequence information, much of which remains to be fully characterized. While structure and function may be predicted from this genomic data, validation of candidate genes as suitable targets requires reliable, practical approaches to performing screens for functional analysis. Therefore, even with complete sequence information in hand, characterization of individual genes can be an involved and daunting task.

The serendipitous discovery of RNAi could not have been more opportune for the pharmaceutical industry, as the rapid output of functional information made possible by RNAi-based strategies alleviates the bottleneck of target validation. Some recent high-throughput analytical approaches include combining siRNA-mediated gene silencing with sophisticated microarray assays, complex cell-based assays, and comprehensive bioinformatics.

For example, several microarray studies of siRNA-treated cell populations revealed both the occurrence of unintended off-target effects and the actual genomewide expression profile of targeted gene silencing. These studies led to the development of modification strategies that enhance the specificity of siRNA-mediated silencing. In another study, siRNA-mediated silencing coupled with a sophisticated cell-based assay that employs a reliable, robust image analysis system permitted a complete phenotypic assessment of the effects of siRNA-induced knockdown of genes that are involved in the cell cycle.

Studies such as these illustrate the potential of new technologies to capture the widespread cellular impact of modulating gene function. The ultimate goal of integrating RNAi biochemistry and biology, high-throughput cell-based functional analyses, and bioinformatics is to provide a complete assessment of the biological impact of small-molecule therapies. The combination of these methodologies promises to accelerate the pace of drug discovery and enhance the reliability of early target identification and validation, maximizing the investment in successful therapeutic solutions.

William Marshall is vice president of research and development for Dharmacon, in Lafayette, Colo. E-mail: marshall.w@dharmacon.com.

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