Back to Basics: Does RNAi Exist in Humans?

Whenever I try to explain the promise of RNAi for human biology and medicine to friends and family, I feel torn by a conflict of staying scientifically accurate or running the risk of losing my audience by burdening them with too many details. You may ask “why” now- isn’t RNAi this elegant straightforward mechanism in human cells whereby double-stranded siRNAs silence complementary mRNAs? How would you react when I said that a number, if not the majority of notable molecular biologists hold that RNAi does not exist in humans?

RNAi is most easily explained by starting with long double-stranded RNA (dsRNA) that is then chopped up by Dicer into short interfering RNAs which then get incorporated into the RiSC complex. The siRNAs loaded in RiSC consequently seek out and destroy their targets by a slicing mechanism. However, long double-stranded RNAs are not suitable for specific gene suppression in humans due to an innate immune system called the interferon response. The interferon response recognises dsRNA greater than 30bp in length and consequently shuts down almost all protein translation in a cell. This is a mechanism by which the cell protects itself from viruses which often produce dsRNA intermediates as part of their life-cycle.

I therefore vividly remember myself in an elective class (“RNA World”) as an undergrad student at Edinburgh University, when my lecturer, Dr. David Tollervey, predicted that it would only be a matter of time that RNAi would be discovered in humans- 1 year before Tuschl and colleagues published their seminal paper on siRNAs. In retrospect, it is clear that his confidence must have come from the fact that the human genome was littered with RNAi-related genes, such as some coding for Dicer and Argonaute proteins. He also may have been familiar with recent publications on the discovery of RNAi-related small RNAs in plants (Hamilton and Baulcombe) and the finding that double-stranded RNAs were processed into similarly sized small RNAs in an Drosophila in vitro system (Zamore, Tuschl, Sharp, Bartel).

Of course, we all know now that it was Tuschl who had the genius to conclude that siRNAs would be the natural effectors of RNAi in humans without inducing the interferon response. Except that they were not. Of course, synthetic siRNAs would work, but years of searching for bona fide naturally occurring siRNAs in humans derived from long dsRNA failed to convincingly prove their existence. It turns out that the RNAi enzymes are all present in vertebrates for another, albeit related reason, namely microRNA-mediated gene regulation. MicroRNAs are a major class of small RNAs that are not derived from long dsRNA, but hairpin precursors and that have turned out to be ubiquitous mediators of post-transcriptional gene regulation and that require RNAi-related proteins for their biogenesis and function. It is in this pathway that experimentally introduced siRNAs perform their gene silencing function.

It therefore appears that in humans the interferon system may have rendered antiviral roles for RNAi unnecessary. This is unlike in many invertebrates which lack an interferon system and where the ancient antiviral function is still alive and kicking. A fascinating story of evolving overlapping biological pathways with a happy ending for keeping the therapeutic promise of RNAi alive, but when you ask a scientist about RNAi in humans- beware!

Market Watch: Nastech reported “positive non-clinical study results for [their] siRNA program for treatment of influenza [in tissue culture in vitro studies].” Following an announcement this Monday of a webinar on flu RNAi to be hosted by Nastech this Friday, frantic trading led to big gains in Nastech stock in anticipation of major RNAi news. Nastech and Alnylam vie for a potentially lucrative government contract for flu preparedness and progress in this area will be closely watched as flu RNAi may turn out to be the first commercial RNAi Therapeutics product. It is likely that NSTK will give up some of its gain in the wake of today’s news, while ISIS may gather momentum as their RNA modification IP estate is starting to yield a rich harvest. Alnylam is to follow with their report tomorrow.

The Promise

The development of RNA interference (RNAi) into a technology that is poised to revolutionise medicine has been breathtaking. Only 9 years after its discovery in worms by Fire and Mello, the 2006 Nobel Laureates in Physiology and Medicine, in 1998 and the subsequent realisation by Tuschl et al. that so called small interfering RNAs (siRNAs) can induce the same process in human cells in 2001, there are already 5 phase I clinical trials ongoing with many more to enter in the coming years. Scientists and clinicians alike seek to harness the power of RNAi for conditions ranging from age-related macular degeneration, viral infections (RSV, HIV, Hepatitis B and C, pandemic flu etc.), Huntington's Disease to cancer. This is because RNAi allows the specific down-regulation of gene products called mRNAs, and since it is safe to assume that virtually any disease interfaces with a gene regulatory network we should be able to modify, if not cure it by tweaking this network. These networks and the role of individual genes therein are being worked out by thousands of laboratories around the world and has been greatly aided by the sequencing of the human and other genomes and technologies to measure and modify gene activities (among them RNAi itself). As there is consequently no lack of validated gene targets, the main hurdle towards its wide application is thought to be the safe and efficacious delivery of the RNAi inducing agent to the site of disease. Luckily, RNAi can draw from the experience of antisense oligo and gene therapy and the exciting prospect of RNAi has energised some of the best minds to come up with innovative strategies. I will address these and other challenges in the next post before introducing the main players in the RNAi Therapeutics space.

For those who want to understand more about the basic mechanism and discovery of RNAi, a popular documentary can be found at PBS Nova: http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html