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.

The RNAi Therapeutics Blog Will Take a Break

While on vacation (and far away from the financial markets mess…), I had plenty of time to re-prioritize my research and investment efforts.  To my pleasant surprise, the blog has turned out to be a very rich (not monetary) experience for me with many new contacts that allowed me to learn about the various aspects of biotech R&D and financing. Thank you for this! 

As I don’t know how long I will have the privilege to discover science with my own hands and knowing of some of the challenges of RNAi Therapeutics, I decided to expand my research efforts into looking at the molecular aspects of DNA-directed RNAi which I believe is very underappreciated by the investment community for its therapeutic potential.   What is lacking, is a champion similar to Alnylam in siRNA-mediated RNAi Therapeutics combining basic RNAi science leadership, business and therapeutic development know-how, and in the case of DNA-directed RNAi (viral) vector expertise for delivery. 

Should my plans change or should my boss boot me out of the lab for under-performing, I might be back, and then it would probably be full-time.  In the meantime, I hope everybody and RNAi Therapeutics in particular to get through the economic turmoil well.

RNAi Therapeutics and Innate Immunity- Keeping the Field Honest

As part of the RNAi Therapeutics review series in Human Gene Therapy earlier this year, former Protiva scientists Adam Judge and Ian MacLachlan (both now with Tekmira following the Protiva-Tekmira reunion) made some rather bold statements with regards to the interpretations of a number of pre-clinical RNAi Therapeutics validation papers (‘Overcoming the innate immune response to small interfering RNA’). As part of the same reviews series, in the risk section of “The Business of RNAi Therapeutics”, I also cautioned that some of the first RNAi Therapeutics candidates may show clinical efficacy, but not necessarily for all the right reasons.

The reason for this should not come as a surprise to anybody in the oligonucleotide therapeutics field: long known from the experience with antisense and other oligonucleotide therapeutics classes, oligonucleotides such as siRNAs have the potential to induce innate immune responses which can have antiviral and anti-angiogenic activity independent of their gene knockdown capacity. In fact, there are significant efforts to harness this biological property for therapy in its own right, particularly the TLR responses. Furthermore, the potential for inducing innate immune responses by synthetic and DNA-directed RNAi has been well documented since 2003 and many of the pathways involved elucidated. Nevertheless, one should not ignore the fact that while RNAi Therapeutics may actually be able to take advantage of such activity as part of synergistically acting immunostimulatory RNAi Therapeutics, the risk is that the oligo-dependent immune responses are quite complex and therefore often difficult to predict and in the worst case may cause serious adverse events.

Since many of the early RNAi Therapeutics validation papers involved antiviral and anticancer applications, it was therefore reasonable to suspect that some of the studies misinterpreted therapeutic effects as the result of RNAi gene knockdown when, in fact, innate immune responses accounted for the majority of the activity. In support, the Tekmira researchers now report that almost all of the unmodified siRNAs reported in a sample of such papers were immunostimulatory whereas a single siRNA that, somewhat disturbingly so, was used as the control siRNA in many of the studies proved to be the exception having no such detectable activity. I should add, however, that the assay conditions were rather stringent (types of cells used and high siRNA concentrations) and just because an siRNA may induce immune responses under these conditions does not prove that these were actually responsible for the treatment effect seen in each of the cited studies. Also, if TLR therapeutics history is any guide, oligonucleotides that elicit immune responses in small animal models, do not necessarily do so in primates.

Given its potential as a whole new class of therapeutics, the scientific and clinical bar for RNAi Therapeutics is set particularly high and reports like the effect of TLR3 stimulation by siRNAs on preclinical models for wet AMD and the present paper by Tekmira tend to get quite a bit of press. While they remind us of the complexities involved in establishing a functional new drug discovery platform, they should also be regarded as promoting that process. In fact, the handful of bona fide RNAi Therapeutics groups, pure-plays and Big Pharma subsidiaries alike, are already taking oligo-induced innate immune responses very seriously and have taken advantage of the rapid progress in the field by applying best practices for identifying and correcting these responses (modification, siRNA structure) in developing the latest crop of RNAi Therapeutics candidates.

The acquisition of former TLR therapeutics company Coley Pharmaceuticals by Pfizer for example may be interpreted as Pfizer investing in solving siRNA-induced innate immune responses as one of the main challenges for RNAi Therapeutics they had identified. Similarly, Sirna Therapeutics and Protiva in their prominent 2005 Nature Biotech paper on RNAi delivery in a mouse model of hepatitis B recognized the potential of unmodified siRNAs to elicit non-specific viral suppression and solved the issue by appropriately modifying the siRNAs. Around the same time, Alnylam somewhat quietly generated IP related to double-strand RNA immune stimulation that it then exclusively licensed to Tekmira. Clearly, the main players in the field have not chosen to ignore the issue, but have invested considerable efforts with tangible results.

But what about the current RNAi Therapeutics clinical candidates that have already entered the clinic? There are one phase III (Opko Health) and two phase II (Sirna/Merck-Allergan and Quark-Pfizer) siRNA candidates for the treatment of wet AMD that obviously have naturally come under increased scrutiny. As far as I am aware, all three of these are ‘unformulated’, intravitreally injected siRNAs with one of them, Opko’s, being an unmodified siRNA. While it is not clear how well the mouse TLR3 studies translate into humans, they certainly raise the concern that non-specific responses might be responsible for any thus far clinically observed therapeutic effects, particularly since in the recent Nature study gene knockdown by this route was very limited at best (cholesterol-conjugated siRNAs, however, administered by the same route were shown to mediate functional gene silencing in the same study).

As is the case with Alnylam’s lead candidate ALN-RSV01 for the treatment of RSV infection which has raised similar concerns, it will be important to be forthcoming in the interactions with the regulatory agencies such that safe trials can be designed based on our best understanding of the mechanisms of action of the different siRNAs. While I haven’t read the documents, it certainly wouldn’t be the first time if such non-specific effects were noted as potentially contributing to treatment. In the future, it would not surprise me at all to see openly declared immunostimulatory siRNA drug candidates enter the clinic. If, however, these issues are not addressed upfront, and should adverse events occur as a result, this could easily backfire and future trials rendered much more onerous- something that should be in nobody’s interest. As for the prospects of the individual drug candidates in question, even if non-specific effects contributed to the therapeutic efficacy of these candidates, as long as they are safe and well tolerated they may very well be viable drugs.

Finally, it is curious as to what exactly motivated Tekmira to re-test an entire battery of published siRNAs for their potential of inducing non-specific effects. It is possible that Tekmira has evaluated siRNA therapeutics for a number of the same applications like flu and wet AMD and were frustrated to see publications come out that according to their experience should have been artefacts (scientists tend to measure themselves by the number of publications and their impact factors and don’t like to see their own published work de-valued this way). Another part of the answer may also have been to keep the field honest at this early stage of RNAi Therapeutics drug development before long-term damage is caused: “However, surprisingly few of the reported studies have adequately tested, or controlled, for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.” By setting a rigorous new standard, Tekmira also signals their expertise not only in RNAi delivery, but also in siRNA chemistry and safety (like Coley, Tekmira has a long-standing interest in the use of immunostimulatory oligonucleotides for therapy). Supporting their claim, Tekmira/Protiva’s publications on abrogating TLR7/8 responses and SNALP RNAi delivery have proven to be extremely reproducible in many different laboratories.

The road to RNAi Therapeutics reality won’t be smooth. As much as it is important to tackle the scientific hurdles head-on, investors and the press should also make an effort to discriminate between ‘good’ and ‘bad’ science.

Johnson & Johnson Likely Tekmira’s Next Official SNALP RNAi Delivery Partner

Since I know that many readers of this blog share a keen interest in Tekmira and the fate of SNALP delivery, one if not the leading systemic delivery technology for RNAi gene knockdown in the liver, and likely with use for solid cancers and immune-related diseases as well, you may like what I dug out today from the long-neglected junk mail pile on my desk: An abstract for the 6th Annual CHI conference on ‘RNAi for Therapeutics’ (Boston 22-23 October) by Pieter Peeters from the Johnson&Johnson company entitled ‘LIVER-SPECIFIC KNOCKDOWN USING SNALP-FORMULATED siRNAs TO STUDY PATHWAYS IN LIPOPROTEIN SECRETION AND STEATOSIS’.

The abstract body, which can also be found online here, as follows:

“As the application of RNA interference in vivo further develops, we are pursuing several promising technologies for systemic delivery. The goal of the current study was to evaluate the SNALP (“Stable Nucleic Acid Particles”) systemic RNAi delivery platform to knockdown genes encoding for key enzymes in triglyceride synthesis. SNALP-Formulated siRNAs were found to effectively knockdown mRNA levels by >90% in liver compared to a SNALP-Delivered scrambled siRNA control. The effect knock-down in liver was further investigated following a 3-week high-fat feeding challenge. The results demonstrated the efficient in vivo SNALP-mediated delivery of siRNA by systemic route and the utility of targeting novel targets to reduce fat storage in liver and improve hepatic steatosis.”

It is thus apparent that J&J is one of the as yet undisclosed SNALP evaluation partners of Tekmira, and has extensively used the delivery technology for target discovery/validation purposes as well as with a view of using it as a therapeutic modality itself. Given the obvious success of their efforts (note that J&J chose to present SNALP from among the other 'promising' delivery technologies it has evaluated), I would not be surprised for J&J to soon join the ranks of Alnylam, Merck, Takeda, Bristol-Myers Squibbs, and Roche, as the next major Tekmira SNALP RNAi delivery partner.

While I do not say that SNALP is the end-all, be-all for gene knockdown of the liver with new technologies likely to emerge over time, the extensive successful use of the technology by third-parties is great validation of its reproducibility and clinical relevance for advancement into the clinic at this time, and a testament to not just the liposomal patent position of Tekmira, but also the RNAi-related know-how of its scientists (see also Tekmira’s publication on the immunostimulatory potential of siRNAs and how to avoid it here).

Beyond the use of SNALP-RNAi in target validation, where will J&J likely be interested in applying the technology clinically? My first bet certainly would be on the treatment of hepatitis C infection. Not long ago, we learnt from a report in RNAiNews that J&J was about to contribute to a $25M fund-raising round for the DNA-directed RNAi company Nucleonics before the deal was called off on questions about scientific misconduct. I honestly could not believe that J&J would even consider collaborating with a company that has entered an RNAi phase I clinical trial for hepatitis B that was so patently destined to fail because of the known inefficiency of liposomal plasmid (not siRNA) delivery.

Developing an RNAi Therapeutic for HepC has also to be seen within the context of building upon J&J’s imminent franchise in this hard-fought market. Together with marketing and development partner Vertex Pharmaceuticals it is currently conducting phase III studies for telaprevir, a protease inhibitor that has all the looks of becoming the next big breakthrough drug in HepC (my other favorite biotech drug story is Tysabri, in case you wanted to know). With predicted cure rates of 60-70% for type I after the introduction of protease inhibitors, RNAi antivirals should be able to enhance cure rates due to their complementary mechanism of action.

Two years ago, J&J awarded RNAi discoverer and Nobel Laureate Craig Mello the Dr. Paul Janssen Award for Biomedical Research. This alone leads me to believe that J&J should have broad ambitions in RNAi as a platform technology. As the number of conference abstracts related to RNAi in the liver (especially fibrosis, metabolic disease) suggests, the liver alone should yield sufficient targets to keep SNALP scientists busy for now.

PS: Since I know that this blog is also read by some larger investment companies, despite the apparently low volume of Tekmira on the Toronto Stock Exchange, it should be possible to acquire shares in size without moving the price too much as the overhang of Protiva stock following the merger is noticeable. End of advertisement.