New uses of RNAi might transform all of its possibilities into realities.
The discovery and application of RNA interference (RNAi) have already fundamentally changed our understanding of biology. The ability to “knock down” the expression of specific genes of interest has helped researchers decipher how genes or gene networks contribute to biological and pathological processes. Recently, the activity of synthetic small interfering RNA (siRNA) has created great interest in developing a new class of drugs based on RNA therapeutics.
Merck & Co., Inc., Whitehouse Station, N.J., recently established an RNA Therapeutics department to capitalize on the expertise obtained through the acquisition of Sirna Therapeutics, San Francisco, in 2006. Targeting RNA in humans also represents the next logical step in the research conducted over the past six years through the Rosetta Inpharmatics group, Seattle, Wash. In 2003, company researchers discovered siRNA off-target effects (inhibition of other genes besides the gene of interest) [Jackson et al. (2003) Nat. Biotechnol. 21:635-7]. Subsequently, the company established empirically-derived sequence selection rules and identified chemical modifications that allow for the creation of siRNA molecules with high potency and minimal off-target activity [Jackson et al. (2006) RNA 12:1179]. These advances have helped to optimize RNAi as a research tool.
The Rosetta Inpharmatics team, now the core of the Molecular Profiling department, has also pioneered integrated analyses of gene expression (mRNA), genotyping and other types of data to reconstruct the causal networks that drive common diseases [Schadt E. (2007) Nature Oct: S24 ]. This multidimensional approach has yielded numerous potential therapeutic targets. However, since most of the novel targets Merck researchers have identified—indeed, 80 percent of all therapeutic targets—are not “druggable” with small molecules or protein-based therapies, directly targeting RNA has become an increasingly attractive strategy for therapeutic intervention. This article provides insight into how Merck is integrating RNAi technology throughout the drug discovery and development process.
RNAi technology goes preclinical
siRNA has become a powerful screening tool for target identification because it can be used to show that inhibiting a particular target has a measurable effect on a cellular process. In 2002, Merck began using siRNA in cell-based assays to better understand disease and facilitate the identification of novel therapeutic targets. Optimization of siRNA through sequence selection and chemical modification led to broader use of siRNA-based screening, culminating in the development of automated, high-throughput protocols that allow researchers to perform genome-wide tests of genes that are relevant to nearly any cellular process. For example, Merck has used this approach to identify genes associated with beta-amyloid metabolism, a key component of Alzheimer’s disease [Majercak et al. (2006) PNAS 103;47:17967-72]. The ability to perturb pathways in this manner has the potential to lead to new therapeutic targets for Alzheimer’s disease and other common illnesses.
In addition to siRNA-based screening, researchers are studying how microRNAs (miRNAs)—naturally-occurring molecules that affect the activity of multiple genes simultaneously—regulate key disease-related gene networks. In collaboration with scientists from Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Stony Brook University, Stony Brook, N.Y., and Applied Biosystems, Foster City, Calif., Merck has identified miRNAs that appear to play an important regulatory role in the p53 tumor suppressor network, which is often dysregulated in cancer [He et al. (2007) Nature 447(7148):1130-4]. Additional research is pointing to potentially important roles of abnormal (increased or decreased) miRNA expression in numerous cancers.
Finally, the company is using RNAi technology as a tool to establish preclinical proof-of-concept. As in cells, siRNA can be used to produce a phenotype in model systems, which can help determine if a drug candidate has the expected biological effect. This information can facilitate decisions about whether or not to advance a candidate to clinical development.
In the clinic
Sirna Therapeutics identified various chemical modifications that optimize siRNA for clinical use by increasing siRNA potency; increasing the duration of the molecules’ activity; minimizing off-target effects; and avoiding cytokine activation, a natural consequence of introducing nucleic acids into the body. In collaboration with Allergan, Irvine, Calif., Sirna inititated the first human clinical trials with an optimized siRNA and was the first to demonstrate biological activity of an siRNA in humans.
Optimization of siRNA for clinical use has created both short-term and long-term opportunities. In the short-term, siRNA may provide a powerful means of establishing clinical proof of concept. By administering single doses of siRNA to humans and measuring robust biomarkers (e.g., blood glucose levels or LDL cholesterol), it should be possible to transiently “knock down” the expression of target genes and rapidly assess—in days or weeks—the biological effect of inhibiting the target. This should facilitate better decision-making on targets destined for small molecule and/or antibody inhibition, and significantly increase the probability of success of these therapeutics in the clinic. Acute dosing of siRNA (e.g., four to six doses total) may also be useful for treating some illnesses, such as life-threatening viral infections.
In the longer-term, the goal is to commercialize RNA therapeutics for chronic use (e.g., 12 to 24 doses per year). In partnership with Allergan, Merck is currently developing an investigational RNA therapeutic called AGN211745 (Sirna-027) for age-related wet macular degeneration (AMD). This siRNA targets the mRNA of the vascular endothelial growth factor receptor, a well-established therapeutic target for AMD. In addition, Merck is integrating RNAi technology across all of its franchises and discovery platforms, anticipating that RNA therapeutics could be applied to most diseases, including diabetes, neurological conditions, and cardiovascular and infectious diseases. RNAi may be especially useful in cancer, because it offers the possibility of specifically targeting cancer-causing genes or decreasing, rather than eliminating, expression of normal genes that cannot be completely knocked out without causing detrimental effects.
The most significant impediment to the use of RNAi as a therapeutic is delivery. Delivery of siRNA is currently possible for some solid tumors and some accessible organs, such as the liver and the eyes. However, delivery to different cells, tissues, and organs will likely require multiple approaches optimized for each target.
Targeted delivery to specific organs and tumors is a critical milestone that must be achieved before siRNA therapies realize their full clinical potential. To achieve this goal, Merck is collaborating with external scientists on discovery and development of new delivery mechanisms. For example, Sirna is currently exploring the use of cationic liposomes as a means to enhance tissue uptake.
Much work also remains in optimizing the selectivity properties of siRNAs within cells. Once optimized, it is anticipated that their efficacy and safety profiles will be enhanced.
Once the hurdles of delivery are overcome, the focus for RNAi-based therapeutics will shift from first-in-class to best-in-class products. This will reduce the problem to one of lead optimization, much like small molecule therapeutics. Lead optimization will focus on answering the question, “How can we further modify siRNAs to obtain the best safety and efficacy profiles in patients?”
About the Author
Alan Sachs assumed leadership of Sirna Therapeutics and established the Merck RNA Therapeutics Department in January 2007. He is one of the leading figures in the field of mRNA translation and regulation and has made landmark contributions, particularly to the understanding of the role of the poly(A) tail in translation and mRNA stability.
This article was published in Drug Discovery & Development magazine: January, 2008, pp. 33-35.
Filed Under: Drug Discovery