Early clinical safety results for RNAi therapeutics are beginning to roll in, and the news is good. The latest generation of RNA-based therapies are well-tolerated and no safety issues are emerging. An estimated 1,500 patients are enrolled in clinical trial programs in RNAi. Alnylam and a Pfizer-Quark Pharmaceutical collaboration have Phase 2 trials for respiratory syncytial virus and diabetic macular edema and wet AMD, respectively. There are also Phase 1 trials from Quark, Alnylam, Tekmira, Calando Pharmaceuticals, Silence Therapeutics, Sylentis, Santaris, and a single-patient Phase 1b clinical trial by Transderm. Programs failing in preclinical or early clinical stages are being terminated due to lack of efficacy, not toxicity. The few toxicity issues that have emerged in the past with these molecules have been addressed through chemical modification and other technologies, leaving only the category of “unknown unknowns” to work through in clinical trials.
Unlike small molecules, RNAi therapeutics do not interact with P450, and are not involved in liver toxicity, prolongation of QT interval, toxic drug-drug interactions, or other organ toxicities. They also have not been found to affect ion channels as many small molecules do. There are a couple of reasons for these safety advantages. One is that small molecules are designed to distribute widely throughout the body, whereas oligonucleotides tend to be limited to smaller areas by their delivery mechanisms. Another is that small-molecule drugs have a broad diversity of structure, and it’s nearly impossible to predict what unexpected effects these structures may have. In contrast, RNA-based therapeutics have predictable structure and tend not to have totally unexpected effects.
The documented toxicities of RNAi therapies fall into two general categories: inflammation due to the innate immune response and off-target effects. Biotech scientists first began to stumble over the innate immune response during development of antisense technology. They found that antisense therapies triggered the innate immune system through toll-like receptor 9 (TRL9). The reason for this turned out to be that TRL9 recognizes CG motifs in DNA that are not methylated, which is characteristic of bacterial DNA, but not human. The receptor was an innate immune mechanism evolved to defend against bacterial invasion.
Similarly, RNAi therapies have triggered innate immune mechanisms that defend against viral infection. Toll-like receptors 3, 7, and 8 all react to certain types of RNA. The solution that has emerged has been to add certain chemical modifications that will prevent RNA from triggering these receptors. 2’ methylation and pseudouridine are some of the most common, naturally occurring RNA modifications and can be used as chemical modifications for siRNAs to prevent them from activating the innate immune response. “Yes, there is potential to activate the immune system and cause tolerability problems … we know how to avoid that,” says Stuart Pollard, PhD, vice president of Business and Scientific Strategy for Alnylam Pharmaceuticals. IDT Technologies (Coralville, Iowa) is one company that offers a portfolio of chemically modified oligonucleotides for RNAi research, specifically addressing the prevention of recognition by these receptors.
Off-target effects can also contribute to unacceptable toxicity in an RNAi therapeutic. One of the most serious safety problems to date has been interference with the microRNA pathway. In 2006, Stanford University researchers reported that several mice died during a study of gene silencing using shRNA in the liver.1 It was found that the shRNAs swamped part of the microRNA transport system, resulting in down regulation of native microRNAs originating from the liver.1
Through careful design and chemical modification, it has been possible to avoid off-target effects. Directed delivery of small RNAs has also been successful in avoiding off-target effects such as interference in the microRNA pathway. The T3 Targeted Transport Technology by Bioo Scientific (Austin, Texas), has been developed to deliver RNAi mediators to target locations, reducing the overall amount of RNA used, which in turn reduces off-target effects by increasing efficacy and specificity. The T3 carrier is conjugated to an antibody that is specific to a receptor or other extracellular domain of the targeted cell or tissue type. Says Lance P. Ford, PhD, vice president of Research and Business Development for Bioo Scientific, “Bioo Scientific’s T3 Technology can help reduce the potential risk of off-target effects and prevent the RNAi mediator from binding to the toll-like receptor by facilitating the fine targeting of siRNA delivery.”
Self-delivering RNAs, made by RXi Pharmaceuticals (Worcester, MA) and designated sd-rxRNA, are another strategy for precision delivery of RNA that reduces the complications of innate immune response and off-target effects of siRNA. As described by Anastasia Khvorova, PhD, RXi chief scientific officer, sd-rxRNAs are a novel class of RNA reagent, which behaves more like a small molecule and has real, drug-like properties. “Standard oligonucleotides are highly negatively charged entites, and do not get inside the cells and tissues without a delivery vehicle. Our novel proprietary chemistry enables us to see efficient cellular uptake of those compounds within minutes of exposure to the cells,” says Khvorova. So far, no safety issues have arisen in RXi’s preclinical development program.
The use of microRNAs as targets is yet another strategy for avoiding the early problems in RNAi therapeutics. There are approximately 500 known microRNAs, and this group represents a completely new class of druggable targets. Regulus Therapeutics (Carlsbad, Calif.) is a company spun out of Carlsbad, Calif.-based Isis Pharmaceuticals and Alnylam Pharmaceuticals of Cambridge, Mass. for the purpose of developing therapies based on microRNA targets. According to Regulus, one of the many advantages of using microRNAs as therapeutic targets is an expectation of safety as compared to small molecule drugs or standard siRNA therapeutics. As well, the once uncertain regulatory pathway for oligonucleotide-based therapies has been ironed out by the large number of cases the FDA has already handled. Says Kleanthis Xanthopolous, PhD, president and CEO of Regulus, “Because the FDA has seen more than 50 different protocols and IND’s, we don’t anticipate any issues … there are now more than 10,000 patients in clinical trials that have experienced oligonucleotides.”
Although RNAi is looking good in Phase 1 and Phase 2, toxicity continues to be a realistic concern. Bioinformatics can be used to eliminate known safety hazards, but full clinical trials are necessary to rule out unknown safety issues. “No amount of thought and foresight is going to replace empiric testing,” says Mark Behlke, chief scientific officer of IDT Technologies.
About the Author
Catherine Shaffer is a freelance science writer specializing in biotechnology and related disciplines with a background in laboratory research in the pharmaceutical industry.
1. Grimm D, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature. 2006; 441(7092):537-41.
This article was published in Drug Discovery & Development magazine: Vol. 13, No. 3, April 2010, pp. 28-31.
Filed Under: Drug Discovery