Antisense or Guide Strand? That’s the Billion Dollar Question

As somebody who has spent the better part of my last decade in academic science, which in its ideal form is guided by the self-less pursuit of the ‘truth’, I must admit that I am somewhat irked when I read a paper on Argonaute 2 enzymology and find science being used also (though not exclusively) as a tool of corporate propaganda. You may think that this is nothing new at all. However, I’d like to use the latest publication by ISIS Pharmaceuticals on RNAi as an excuse to highlight a few of the issues that come up when ISIS Pharmaceuticals, by sheer repetition, claims that RNAi Therapeutics is an antisense technology.

In the paper by Lima and colleagues in the Journal of Biological Chemistry (‘The Binding and Cleavage Specificities of Human Argonaute 2’), the investigators study the effect of sequence length and modifications of what they call the ‘antisense’ strand on Argonaute 2 binding. What the authors call here ‘antisense’ is widely referred to by the rest of the field as the ‘guide strand’, that is the small RNA of the double-stranded RNA trigger that gets loaded onto Argonaute 2, the enzyme in RNAi that carries out the target mRNA cleavage. Admittedly, the ‘antisense’ is sometimes used in the RNAi literature to operationally describe the relative polarities of the RNAs involved (guide strand, passenger strand, target mRNA). However, when ‘antisense’ comes up 243 times within a manuscript, but ‘guide’ not at all, even as a verb, I believe it is a valid hypothesis that this cannot be a coincidence, but is calculated corporate speak.

I have explained before why RNAi by its very nature is a double-strand RNA technology, most tellingly illustrated by the fact that the discovery that what had been thought was mediated by antisense was actually mediated by the double-strand RNA contaminants of antisense preparations was awarded the Nobel Prize, so I will not repeat here myself. This is also not meant to criticize the scientific merits of the paper which furthers our understanding of the chemistries that could be used for the less efficient form of RNAi, namely single-strand RNAi and must be seen in the context of the recently announced ssRNAi initiative by ISIS and Alnylam, except maybe for the observation that the finding that single-strand RNA binds purified Argonaute 2 apparently better than double-strand RNA does not reflect Argonaute 2 loading in a living cell.

The point is that whether RNAi is labeled as an antisense technology or not can have significant consequences for the interpretation of the scope of patents, some of which date from before the discovery of RNAi in worms. It is all good for Alnylam for example to implicitly subscribe to ISIS notion as long as they have exclusive access to that IP as it helps constrain the freedom-to-operate of competitors such as Merck and Silence Therapeutics. However, when these early patents are ultimately not deemed as critical, can this leave Alnylam with patents that were pursued less rigorously so as not to conflict with the ISIS’ IP? One of the Tuschl patents comes to mind, I believe dubbed ‘Tuschl IV’, which for the first time explicitly described the use of single-strand RNAs for the induction of RNAi, but, to my initial surprise, had received very little attention among all the ssRNAi talk.

Aside from this political minefield, labeling RNAi Therapeutics as ‘antisense’ just does not do justice to the many ground-breaking discoveries this field has seen, and the people and institutions that made them and that you would think deserved their rightful place in RNAi history and a larger piece of the royalty pie. Specific modifications when used within an siRNA yes, but not the fundamental mechanism of action.

I leave it up to the reader whether this makes ISIS Pharmaceuticals an even better investment, and I must say they clearly deserve credit for their pioneering role in oligonucleotide chemistry and clinical application, and are now harvesting the financial fruits not only from their own drug development pipeline, but also from licenses for all types of therapeutic RNA approaches, including microRNAs (progress so far have exceeded my expectations, and according to a just published comments by Regulus’ CEO on HCV-miR122 this looks set to continue for the foreseeable future) and aptamers. Regardless, 243 times antisense does not make RNAi such.

ALN-RSV01 Update: The Drug is Safe- and Efficacious?

Alnylam provided this Monday an update on their phase II study results of ALN-RSV01 in lung transplant patients naturally infected with respiratory syncytial virus (RSV). RSV infection in lung transplant patients is linked to irreversible damage of the lung, decrease in quality of life, and sometimes even death. With no proven drug for the treatment of RSV, this remains an area of high unmet clinical need.

The 90 day data follow results announced in June that showed the drug to be well tolerated and adds to the growing evidence of safety obtained with this drug in various phase I and II studies. While safety was the primary objective, based on the initial 30 day data report no conclusions could be drawn on the efficacy of the drug as measured by either reduction of RSV titers or symptom scores due to differences in baseline characteristics between the drug-treated (N=16) and placebo control-treated populations (N=8).

The 90 day data confirmed the safety of the drug that was administered 3 times daily via inhalation. The longer time period also allowed for monitoring lung function. Remarkably, while lung function was significantly impaired in about one third of both the drug and control populations at the start of the study, most likely as a result of the RSV infection, at 90 days after drug treatment only 14% of the ALN-RSV01 patients experienced an FEV1 value 20% below baseline compared to 38% in the placebo group. While this did not reach statistical significance in this small study, when another related measure of lung health was considered, namely the incidence of new or progressive BOS (bronchiolitis obliterans syndrome), ALN-RSV01 did significantly better than control (1 in 15 patients for ALN-RSV01 versus 4 in 8 patients of placebo).

Taken together, the drug was shown to do no harm and there is intriguing evidence for clinically relevant efficacy of the drug in a randomized, double-blind trial, albeit somewhat tainted by the baseline differences. The difficulty of running such a trial is illustrated by the fact that 13 institutions around the world were involved and I cannot see a regulatory body requiring a 200 person lung transplant trial when there are only about 2000 lung transplants a year and only a fraction of those actually becomes infected with RSV. The results are certainly better than the standard of care today, ribavirin, which has unproven efficacy at least in this setting and is well known to be toxic. Transplant surgeons certainly would love to have another treatment option, even if only supported by such data. Therefore, is there a case for Alnylam and their co-development partners to talk to the FDA about approving ALN-RSV01 in this orphan patient population (compassionate use argument), maybe after further follow-up confirm the positive trend in improving lung function which has to be the ultimate measure of treatment success?

It would be ironic for the FDA e.g. to deny such a request due to the difficulty in interpreting the RSV biomarker results while the medically relevant outcome, lung function, showed positive results. Normally, the agency rejects drugs that although the biomarkers were positive (e.g. blood sugar and cholesterol), more direct clinical outcomes were not.

Needless to say, such an outcome would be unexpected, but it does not cost much to ask. In any case, for the wider application of RNAi Therapeutics in lung disease it is comforting to see that the inhalation of unmodified siRNAs even in this fragile population was so well tolerated and it will be interesting to see how the RSV program, sometimes criticized for its seemingly labyrinthine course, will unfold.

HBV Collaboration between Benitec and Biomics Indicates Shift in DNA-directed RNAi Therapeutics towards Asia

My four years as a post-doc here in Stanford, during which my advisor Mark Kay served a term as the President of the American Society for Gene Therapy, taught me that drug development is as much about politics and perceptions as it is about the science.

This is particularly true when it comes to gene therapy and it is no surprise that despite pre-clinical data that, taken together, often surpassed that obtained with synthetic siRNAs, DNA-directed RNAi (ddRNAi) Therapeutics is struggling for funding in the corporate world. Targeted Genetics is a prominent example of a gene therapy company that despite much scientific (AAV-delivered RNAi data for Huntington’s Disease) and clinical progress (saving patients from blindness) is now facing bankruptcy. The reason? The regrettable death of a patient in a Targeted Genetics-sponsored clinical trial that has now been linked by experts to a immuno-suppressive monoclonal antibody the trial participant had been taking. The case was taken to the level of an NIH RAC (recombinant advisory committee) hearing, and synthetic oligonucleotide therapeutics companies are quite right in being scared that they, too, may be subject to RAC review in the future. I wonder what the outcome was from a recent meeting to discuss just this matter.

I am aware that gene therapy carries risks. Western society, however, needlessly deprives itself of potentially life-saving treatments when it chooses to suppress the entire field following isolated, albeit very unfortunate cases of adverse events linked to gene therapy. Then there are the so called ethical concerns of changing the human genome by introducing DNA into our cells as if sick patients had the luxury of worrying about this. By contrast, news of drug-related deaths in clinical trials of small molecules, many of which unlike the commonly used viral vectors have never been introduced into the human body, hardly ever reaches the wider public.

This week’s memorandum of understanding between Australia’s Benitec and China’s Biomics to collaborate on a DNA-directed RNAi therapeutic for chronic hepatitis B virus (HBV) infection may be a sign that the near to mid-term future for ddRNAi may instead lie in the economically vibrant parts of Asia. Here, practicality and an eagerness to adopt innovation means that gene therapies fall on much more fertile ground, including funding. Just last year, Benitec spin-off Tacere signed a similar deal with Japan’s Oncolys for the development of an ddRNAi Therapeutics for another viral disease of the liver, hepatitis C virus (HCV) infection.

Funding and access to R&D may have been financially struggling Benitec’s main motivation to reach out to Biomics. Also, Biomics provides Benitec with a foothold in a country that faces end-stage liver failures and hepatocellular carcinoma caused by chronic HBV that are of epidemic proportions. On the other hand, while Biomics, a biotech company with locations also in the US and that, with the help from some former Nastech employees, strives to transform itself from a mainly RNAi research-reagent company into an RNAi Therapeutics developer, certainly appears to enjoy better economic health and brings with it RNAi know-how, ideally it would have complemented Benitec ddRNAi patent estate and insights into shRNA design by providing an advanced delivery technology, maybe AAV. Although it is possible that they have such a technology, this is not apparent from the company's website which describes a range of delivery modalities that they are apparently working on.

For RNAi Therapeutics in general, Asia not only provides a growing market, but also an enormous R&D opportunity with many highly trained, detail-oriented chemists and increasingly also biologists to draw from. I am often surprised for example how many chemistry- and gene therapy-based publications on RNAi delivery come out of a country like Korea which is relatively minor in terms of biomedical research budgets, yet is little capitalized on due to lack of risk capital there. As for the IP situation in a country like China, I believe that once it becomes relevant, that is in maybe 7-15 years, China should be more aligned in this regard with the rest of the world and it would be a mistake not to make an effort of protecting your RNAi Therapeutics IP there, too.

HBV played a prominent role in the history of RNAi Therapeutics. Both synthetic (SNALP RNAi) and DNA-directed approaches proved successful in repressing HBV replication in mouse models. Since suppression of viral replication is a well-accepted measure for predicting HBV treatment success, RNAi Therapeutics should very well be able to complement current interferon-alpha and nucleoside analogue-based standard of care that result in unsatisfactory treatment success rates of only 20-30%. Due to the nature of the disease, however, it is unclear to me whether synthetic siRNAi or ddRNAi would be preferable. However, since treatment success by nucleoside replication inhibitors requires long-term treatment, probably due to the persistence of viral DNA in hepatocytes, a gene therapy approach has certainly theoretical justifications.

And finally, following Nucleonic’s fall and the situation around Targeted Genetics and Benitec, it is time for the entire RNAi Therapeutics field to think about creating a strong ddRNAi Therapeutic company before much of the IP is squandered. Consolidation of these efforts into a re-capitalized Benitec (disclosure: no current investments) may be one, although not the only option.

Journal Club: Unraveling the Substrate Specificity of RIG-I and what it Means for RNAi Therapeutics

A paper by the Hartmann group in Bonn, Germany, takes the mystery out of the structural features of RNAs that activate the cytosolic viral innate immune sensor RIG-I and offers simple design rules for either avoiding, or in some cases intentionally inducing such activities with RNAi triggers. It is good news for essentially all types of synthetic siRNAi as practiced today, but requires re-evaluation of some (but not all!) DNA-directed RNAi approaches, and those approaches that depend on the preparation of RNAi triggers by in vitro transcription rather than by synthetic means.

Some of the first wave of RNAi Therapeutics candidates that were rushed into the clinic were most likely based on pre-clinical efficacy results due to the induction of non-specific innate immunity. As an aside, it always ‘surprised’ me for example how companies with no track record in nucleic acid therapeutic development would suddenly claim to be the first ones to enter an RNAi Therapeutics candidate into the clinic. Innate immunity is based on the recognition of pathogen-associated molecular patterns, PAMPs, by cellular receptors that then initiate a powerful signaling cascade leading to the successful defense against viral and bacterial infections, often resulting in the death of the infected cell itself. Therefore, to avoid mis-interpretation of RNAi data and to ensure safety, the RNAi Therapeutics field needs to take into account the structural and sequence-specific signatures of nucleic acid triggers of innate immunity.

There are two classes of nucleic acid receptors relevant to this discussion. The first one are the toll-like receptors (TLRs) 3, 7, and 8 which recognize certain types of single- and double-stranded RNAs mainly in the endosomes. This is important when RNAi triggers are delivered from the outside, but not for DNA-directed RNAi. The second one is comprised of cytosolic PAMP receptors that both synthetic and DNA-directed RNAi triggers may encounter. While PKR was initially thought to be the main cytosolic receptor relevant to RNAi, it more and more emerges that the RNA helicase RIG-I is what the field needs to be mindful of, and is the subject of the present paper by Schlee and colleagues.

Before these findings, it had been thought that any RNA with a triphosphate chemical group at the 5’ end would induce RIG-I. Blunt-end double-strand RNAs of the size of siRNAs were also thought by some to have this capacity independent of a 5-triphosphate modification. The present findings, however, show that the confusion about the exact RIG-I substrate structural features arose from the origin of the RNAs used in those studies. Since 5’-triphosphate modifications are not routinely offered by synthetic RNA vendors, it has been convenient to use RNAs generated through in vitro transcription by recombinant, purified phage polymerases which leave a triphosphate group at the 5’ end. Unfortunately, these phage polymerases have the property of generating additional species of RNAs aside from the desired one. It turns out that double-stranded RNAs, still with a 5-triphospate group, are one of those and that these are the ones actually recognized by RIG-I. The authors were thus able to show that well-defined synthetic single-strand RNAs with a 5’-triphosphate alone were not sufficient to induce RIG-I.

For RNAi Therapeutics the findings mean that RIG-I should not be a concern for essentially all synthetic siRNA therapeutics, as almost all of them are administered in a 5-hydroxylated form which are then 5’-monophosphorylated. Both modifications would abolish RIG-I activation. This is also good news for blunt-end ‘Atu RNAi’-type siRNAs as practiced by Silence Therapeutics which may have been previously suspected to trigger RIG-I. It is true, however, that in the context of 5’-triphosphates, the more traditional double-stranded Tuschl-type siRNAs which contain 3’ overhangs further diminish RIG-I activity even in a 5’-triphosphate context.

The picture is somewhat more complex for DNA-directed RNAi Therapeutics approaches. Historically, perfect complementary hairpin RNAs driven by RNA polymerase III promoters (Pol III) have been used. As these hairpins are destined to be exported into the cytoplasm, the cellular location of RIG-I, the minimally or not at all modified 5’ ends of such shRNAs, i.e. 5’-triphosphates, run the risk of triggering RIG-I responses. However, a simple mismatch of the 5’-triphosphate nucleotide with the opposite strand should abrogate most RIG-I activity and some of the widely used Pol III expression cassettes fortuitously carry such mismatches which do not appear to affect gene silencing. For RNA Polymerase II-driven DNA-directed RNAi Therapeutics, RIG-I should not be a concern for the reason alone that Pol II transcripts exhibit a 5’ modification that does not induce RIG-I. And finally, for trans-kingdom RNAi Therapeutics where the bacteria expresses the RNAi trigger, one may want to design shRNAs that are similar to the Pol III strategies. It is also good news that not any 5’-triphosphate RNA induces RIG-I, since bacterial transcripts are quite rich in 5’-triphosphates.

In summary, the paper by Schlee and colleagues indicates that innate immune activation by siRNAs in the cytosol is no show-stopper by any means. By contrast, since 5-triphosphates do not necessarily abolish the RNAi activity of double-stranded RNAs, bi-functional immunostimulatory siRNAs can be designed such as for antiviral and cancer applications- as demonstrated late last year by the same group in a collaboration with Alnylam. Of course, 5’-triphosphate dsRNAs may also be used independently of RNAi for the same reasons.

The RNAi Therapeutics Blog is Back

After taking a break for almost a year to finish up my post-doctoral studies and to figure out what to do with my future, I have decided to pick up writing this blog again. In addition to what I hope will be a small contribution to explaining RNAi Therapeutics to the scientifically and financially interested public, it will also help me to better feel the pulse of RNAi Therapeutics through my interactions with you. So please feel free to comment. Initially, I will only comment when there is highly significant breaking news and will become more active again starting mid-September. There should be much to write about with first clinical data from the Alnylam and Tekmira SNALP-siRNA trials coming up which in my opinion could be a real game changer. Fingers crossed!

Alnylam 1500, GSK 800

In what was possibly greeted in investor circles with a slight yawn, GSK and Alnylam announced today that Alnylam would add 1500 issued or pending RNAi Therapeutics-related patents to GSK’s 800 patent filings into an IP pool designed to facilitate the development of drugs for neglected tropical diseases. So what was this all about?

I do not want to dismiss the value of being a good corporate citizen per se or how it may win Alnylam and RNAi Therapeutics some political goodwill when it comes to charting their way through treacherous regulatory waters. I suspect, however, that there was another message hidden in today's press release. What struck me was that the joint press release emphasized the breadth and quality of Alnylam’s patent portfolio, including by juxtaposing Alnylam’s 1500 patents next to GSK’s 800 and the following quite friendly statement by GSK’s relatively new CEO, Andrew Witty (photo):

“We are delighted that Alnylam will join GSK in this important programme by adding their unique RNAi technology [emphasis mine] to the patent pool.”

This indicates to me that Alnylam and GSK are getting along pretty well. Both managements should be familiar with each other following the GSK-Regulus deal for the development of microRNA therapeutics for inflammatory disease (Regulus is the microRNA joint venture of Alnylam and ISIS). A positive experience there with small RNAs may have given GSK extra incentive to join the ranks of Roche, Takeda, Novartis, and Pfizer in considering RNAi Therapeutics as a bona fide remedy, if not cure for Big Pharma’s stuttering innovation machine and oncoming wave of expiring blockbuster. And yes, Merck is also one of them, and it is likely that when they bought Sirna Therapeutics in 2006, it left GSK looking for a new RNAi Therapeutics partner. Just before that in 2006, GSK and Sirna signed a major RNAi alliance for respiratory disease, and since then Merck has not made the impression that it likes to share its mysterious RNAi know-how.

You know where this is going, and regardless of whether an Alnylam-GSK RNAi platform alliance will actually be announced this year or whether this is just another example of my RNAi delusion, it is worth speculating about the scope of such a potential deal of which we expect at least one this year from Alnylam. Similar to the 2007 Roche platform deal, my guess with regards to therapeutic areas would be oncology, respiratory diseases, metabolic diseases and certain liver diseases. Since these are all areas in which significant improvements have been made in siRNA delivery in the last 2 years, the terms may be even more favorable.

And yes, it would be nice for GSK and Alnylam to combine their expertise in malaria drug development and liposomal siRNA delivery to the liver, respectively, to translate promising pre-clinical results by Alnylam and collaborators into a much needed weapon for a disease that disrupts the lives of up to 500 million people a year mostly in impoverished countries. That RNAi is even considered for such purposes is also a sign that the eventual cost of goods for RNAi Therapeutics should be well below that of recombinant proteins, including monoclonal antibodies, which in turn may be more suitable for vaccination approaches.