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.

Journal Club: mRNA Levels are a Good Indicator of Off-targeting

Next to delivery and immune stimulation, sequence-specific off-targeting is considered one of the fundamental challenges in the development of RNAi Therapeutics. Such off-targeting largely occurs by microRNA-like gene silencing following recognition of off-target RNAs that have limited sequence complementarity to the guide strand of the siRNA. Since microRNA gene silencing had long been believed to inhibit translation without affecting mRNA levels, there has been concern that the true extent of RNAi off-targeting may be larger than originally reported in studies assessing changes in global RNA levels. By achieving the feat of simultaneously measuring microRNA-induced changes in thousands of proteins, two high-quality papers now find that, surprisingly, mRNA levels are indeed a very good indicator of off-targeting, validating the use of established high-throughput RNA assays during the siRNA selection stage of RNAi Therapeutics development.

Key to the success of the Bartel lab at MIT (Baek et al.: The impact of microRNAs on protein output) and the Rajewski lab in Berlin (Selbach et al.: Wide-spread changes in protein synthesis induced by microRNAs), was their use of the SILAC technique (Stable Isotope Labeling with Amino acids in Cell culture). In this procedure, newly synthesized proteins are labeled with amino acids containing different isotopes. To determine the impact of microRNAs on global protein synthesis (~5000 proteins were identified in the studies), the samples with and without a given small duplex microRNA were labeled with different isotopes. This results in peptides from the same protein exhibiting slightly shifted peaks during protein mass-spectrometry, and the ratio of the peak intensities belonging to a pair of peptides is taken as a measure of microRNA-mediated gene silencing (1= no change). Changes at the protein level thus determined were then compared to changes on the mRNA level by microarray analysis.

As expected, the authors found that among the down-regulated proteins were highly enriched those that contained microRNA seed target sites in their 3’ untranslated regions (UTRs). Consistent with our notion of microRNAs as regulators of a group of genes rather than individual genes, typically 200-500 proteins were changed by more than 30% following the introduction or deletion of a microRNA, with about half of them likely the consequence of a direct interaction with the microRNA. Remarkably, despite the introduction of rather large amounts of microRNAs into the tissue culture cells (25-100nM using lipofection), most of these changes were rather subtle, and very rarely were changes of more than 3-fold detected. While it is always possible that wide-spread subtle changes may lead to a directed cellular response or cause toxicity in the case of RNAi Therapeutics, this observation is in agreement with microRNAs often acting as rheostats to maintain cellular homeostasis and suggests that the introduction of random short duplex RNAs should not necessarily lead to gene regulatory chaos and distinct phenotypes.

The surprise came when it was found that changes in protein levels could for the most part be accounted by changes in mRNA levels. Only very few proteins were reduced without concomitant mRNA changes. Without going into the details, the studies nevertheless support a model in which translational repression is the primary mechanism of microRNA-mediated gene silencing and mRNA destabilization a consequence thereof. Consistent with this hypothesis,
in the rare instances where 2 microRNAs were located close to each other, silencing was synergistic, yet the mRNA level was not further reduced. This is quite interesting as it may explain how the early use of microRNA reporter systems in which multiple microRNA target sites were closely spaced and which consequently caused strong gene suppression could have led to the notion of translational silencing only. The 20-30% reduction in mRNA could have easily been dismissed as experimental noise.

For practical purposes, however, measuring mRNA levels are highly predictive of proteomic changes and this should allow for the selection of RNAi Therapeutics candidates that have an increased likelihood of not causing toxic changes in gene expression. Merck has long been a proponent of this approach and partly for this reason have closely aligned the systems biology expertise of subsidiary Rosetta Inpharmatics with their RNAi incubator Sirna Therapeutics, in addition to employing systems biology for identifying the right gene targets.

It is very comforting to know that the extent and severity of off-targeting on the protein level is not significantly greater than what we have observed at the RNA level. Moreover, the ability to use high-throughput RNA expression technologies for assessing off-targeting and chemical modification techniques which have proven to significantly further reduce off-targeting potential of siRNAs should minimize the risk of RNAi Therapeutics candidates to fail due to sequence-specific off-targeting. A good siRNA at high concentrations in tissue culture reduces about 5-10 off-target RNAs by more than 2-fold (and as we know now proteins to a similar degree, not more), while the target is the most suppressed gene. By acting at the RNA level, RNAi Therapeutics has the distinct safety advantage in that it allows for off-targeting to be assessed in such a comprehensive manner. Before investing $1 billion dollars in the development of a drug, it appears to be well worth taking advantage of this by trying to understand these changes.

Disingenuous Leerink Swann Report on Value of RNAi Therapeutics

What a difference 2 years can make…

• "RNA interference could be the next breakthrough technology and we believe RNAi is well positioned at forefront of development of novel nucleic acid based therapeutics." – Leerink, 2006
• "We believe RNAi's therapeutic technology platform could yield a robust source of pipeline candidates…" – Leerink, 2006
• "Alnylam is narrowly focused upon the development of a Nobel Prize winning technology called RNA interference (RNAi) as a drug development tool that we feel is more suited for research purposes than the development of therapeutics." – Leerink, 2008

Originally, I intended this blog to be positive and reflect my enthusiasm for RNAi Therapeutics. While my enthusiasm remains intact, I also learned that as the financial stakes in the industry have increased rapidly, it has not only attracted supporters hoping that one day RNAi will improve healthcare, but also an ever-growing list of detractors hoping to capitalize on the financial misfortunes and medical failures of what would appear to be a benign biotechnology. As the drug development and approval process is as political as it is about science and medicine, and requires significant investments, I see it within the scope of this blog to defend RNAi Therapeutics from some of these misconceptions that are being planted.

This week’s report by Leerink Swann analyst Jonas Alsenas, initiating coverage of Alnylam with a ‘Sell’ rating (the only one by Leerink Swann at that!) and a 2009 price target of $14-16, is probably the most amateurish, yet apparently effective attempt to sabotage the development of RNAi Therapeutics. This report which essentially states that nucleic acid therapeutics have never been a success, and therefore never will be, and that the recent interest in RNAi is merely a reflection of the poor state of Big Pharma in general (but what about all the other technologies that do not get similar interest in this climate?), is littered with so many factual mistakes that one has to come to the conclusion that a) either is the author utterly unqualified to comment on biotechnology and RNAi and should look for another job soon, or b) that it was driven by an agenda to hurt the share price of Alnylam for the benefit of the massive short interest in the company, or maybe c) just the a revenge of somebody having missed the boat. BTW, 2008 is a bit late for a “healthcare investment bank” to initiate coverage of the leading company in the RNAi space.

But now to the most important part of today’s blog entry. In what follows, a good friend of mine, scientist, and long-time follower of the RNAi Therapeutics space has volunteered to dissect the Leerink Swann report to expose the mistakes and intentions behind it. Not only is he very well qualified to do so, he also has a uniquely refreshing writing-style that cuts through the clutter which I’m sure you are about to enjoy as much as I do.

Leerink Swann Report on Alnylam- A Review

Preface:

Honestly, I’m not sure where to begin writing about the Leerink Swann (LS) report by Alsenas and Yoo. One does not need to look hard to find plenty of acceptable areas to take issue within this less-than-glowing assessment of RNAi. Is their report accurate? I will examine their assessment point by point. LS discusses some areas of RNAi technology that are, and will be, areas that will continue to progress, but overall the report is intellectually dishonest at best. Let’s face it, we’ve all seen examples in politics (on many sides in many issues) that are “true” but intellectually dishonest. For example, I could say that “history has taught us that airplanes don’t fly.” That is technically a true statement if one looks prior to December 17th, 1903 when the Wright brothers took the first flight; however, it’s clearly a misleading statement. There are a number of examples that read similarly to the fact that “history has taught us that airplanes don’t fly” in the LS article on Alnylam Pharmaceuticals, and I cannot understand how or why such instances would be presented as honest reporting by a reputable firm.

The meat of the report:

Leerink Swann initiated a sell rating on Alnylam, expecting a valuation of $14-16 in 2009 based on cash (currently over $13 per share) and a valuation of $200 million for their IP and technology. LS claims that Alnylam is “narrowly” focused on RNAi and that RNAi is better suited for research than as a drug platform. Additionally LS investigates the respiratory syncytial virus drug ALN-RSV01 as “underwhelming” and evidence of a lack of systemic delivery capabilities for RNAi. LS states that RNAi delivery is impossible at the moment, and will probably be a hurdle never surmounted. Furthermore, LS believes that the number of collaborators working with Alnylam (and paying for the privilege) is a liability and not an asset. Moreover, LS states that Alnylam “will use its significant cash position of $538 million...to “diversify” or “forward integrate” by acquiring another technology or clinical product.

I will look at a small portion of the statements made by this report simply because documenting a group of clear errors is enough to toss out the report in its entirety.

Valuation:

LS values Alnylam’s IP and technology at $200 million. That’s all well and good, but how did they arrive at that number? They don’t say. However, we can ascribe a more realistic valuation than the professionals at LS by comparing actual dollars rendered for RNAi and similar technologies. First of all, when someone (Roche) is willing to pay $273 million dollars for non-exclusive access to 4 of 20 disease areas using Alnylam’s IP and technology, not counting the future milestones and royalties but an upfront $273 million, it’s difficult to realistically value Alnylam’s entire IP at $200 million. So far, Alnylam has over $500 million in the bank mostly derived from licensing (non-exclusively) their technology to a few Pharmas, namely Novartis, Roche, and Takeda. The authors disingenuously attribute these payments to “The pharmaceutical industry is often swept by new technology fads.” Well, even if that’s all Alnylam has to offer, wouldn’t those dollar amounts suggest that it’s a fad many of the other pharmas are going to try? Science has shown us that this is not the latest fad, but rather a monumental discovery that has the ability to revolutionize medicine.

Let’s look at a few other players that own IP space on platform technologies. First and foremost is Isis Pharmaceuticals that owns the IP on many RNA chemistries and more importantly the use of single-stranded oligonucleotides designed to repress messenger RNA, collectively grouped under the term “antisense.” The early days of antisense were plagued by disappointments and one can see the vestiges of the early adopters (Genta etc.), but Isis has overcome the initial troubles and developed a working and robust platform based on antisense. Reading the LS report, one comes to the conclusion that there are no oligo-based drugs that demonstrate efficacy. Isis’ pipeline is full of effective drugs. Recently one such drug was partnered for hundreds of millions upfront with potentially a more than a billion dollars in milestones and royalties of 30-50% of sales. Does that sound like nothing is working with oligo-based drugs? Perhaps the authors simply read only the investing reports on Isis and not the scientific literature. It is interesting to note that LS also downgraded Isis stock to hold at $14 from $17, it has since moved back up and closed yesterday at $18...

Another company that owns IP around a platform of potential drugs is Sangamo Biosciences. This company produces specific proteins (zinc-finger proteins ZFP) that bind to DNA and perform several possible functions mainly to either inhibit or increase the expression of messenger RNA and consequently increase or decrease the amount of that gene’s expressed protein. The design and production of these molecules requires human hands and many ZFPs are then tested to determine which has the ideal properties for the specific disease state being investigated. Subsequently, the molecules need to be delivered to the interior of the cell and then into the nucleus. (The production of one molecule is much more time consuming than the production of RNAi where a computer chemically assembles the individual bases into the drug.) Is this starting to sound like antisense and RNAi? Yep, there’s a reason for that: all three require delivery in the form of either chemical modifications, a chemical delivery vehicle (e.g., liposome/SNALP), or possibly through an engineered viral genome. Antisense is the smallest and therefore easiest to deliver naked, or without a carrier. RNAi is about twice the size of antisense (similar size, but 2 strands instead of one) while ZFPs are up to 20 times the size of antisense molecules. Does this sound like ZFPs are easier to deliver than antisense or RNAi? For the sake of the valuation of RNAi IP and technology, let’s assume that RNAi is equally valuable as ZFPs.

LS has a price target of $18 for Sangamo (SGMO) stock or about $650 million for the IP and technology. Why such a high valuation for SGMO IP compared to Alnylam? They must have a list of multi $100 million dollar collaborations right? Nope. But they do have about $76 million in the bank at the moment... Remember, LS thinks Alnylam’s IP is worth $200 million. Does that make sense? Not really, but as LS says, “Alnylam is narrowly focused on the development of a Nobel Prize winning technology called RNAi.” I am not saying Sangamo is not worth that much for fundamental IP, but clearly there is a rational disconnect between the valuations of these two companies by LS.

Which brings us to the next point: Alnylam is ‘narrowly’ focused on a technology that could be applicable to every gene in the genome (read this as potentially 10’s of thousands). Huh? Yep, you read that correctly. Alnylam owns the bulk of IP for RNAi, half of the company Regulus that controls the majority of IP for a closely related technology to regulate genetic pathways via microRNAs, and recently acquired the fundamental IP for RNAi’s cousin RNAa that can be used to increase production of gene products. Can anyone honestly say that this is narrow? Too broad would be a more believable tale to weave, but then they might have to back that up with data that would cast shadows on the remainder of their story.

Perhaps I should be generous and alter their statement to read that Alnylam is narrowly focused on respiratory syncytial virus. This is currently the only drug Alnylam has in Phase II clinical trials; however, I’ll be quick to point out that they have a number of other programs that will soon be entering clinical trials including liver cancer, hypercholesterolemia, Huntington’disease, Progressive Multifocal Leukoencephalopathy (PML), hepatitis C virus (HCV), and biodefense projects including Ebola virus. The authors state the results for the RSV trials as “difficult to interpret and undewhelming.” They are especially difficult to interpret when one leaves out a major portion of the positive data. Compared to the control group, approximately 1/3 displayed similar symptoms to non-treated individuals, 1/3 had reduced symptoms (by about 38%), and 1/3 had no symptoms (based on the controls, one would expect 12 subjects to not show symptoms but 24 were symptom free). The authors kindly neglected the group that displayed no symptoms but were shown to have a very low viral infection. Is this a fair approach to science? Additionally, Alnylam conclusively demonstrated that the viral reduction was due to specific RNAi mechanisms and not due to non-specific activation of the immune system. Read the actual data here, it is not difficult to interpret.

Was the study underwhelming? I guess if one has expectations of complete obliteration of all RSV virus, then yes it was underwhelming. But then again, every other drug trial would be underwhelming as well. To be fair, it was a difficult trial. Alnylam had to develop a model system for a virus that had never been done before. Because they were administering the virus to healthy volunteers, they used a mild strain of the virus that would be least likely to harm the test subjects. Would they have had stronger data if they used a strain that was much more likely to be lethal in elderly/infant populations? Maybe, but how are you going to sell that study to volunteers and the ethics board? LS also objected to an additional study in lung transplant patients where RSV is a serious disease that can lead to tissue rejection (to be sure, it is serious if one’s lungs are rejected...). This study provides an additional look at the ALN-RSV01 safety profile and a more optimized administration route of nebulized drug PRIOR to testing in infants. There can be no doubt that Alnylam has chosen to move into the pediatric tests in a cautious manner, but that is to be expected.

One additional point to make about the authors contention with ALN-RSV01: they claim that it will not be worth much money and that they will have competition with Synagis, a monoclonal antibody for the PREVENTION of RSV infections. First of all, the market is large enough that Synagis is a billion dollar drug and secondly ALN-RSV01 is a TREATMENT for RSV infections. Synagis does not enter the cells where the virus replicates - thus it is used as a prophylactic in premature infants where RSV infections are quite dangerous. (I like analogies, so how about “antibiotics are worthless because condoms exist?”) ALN-RSV01 does enter the cells and shut off the viral replication capacity of those cells. It is tempting to speculate on the potential synergism between the two drugs.

ALN-RSV01 is as simple as an RNA-based drug can be. It is a non-modified RNA oligo pair dissolved in saline water. It is literally misted into the lungs and absorbed by the cells it contacts. There is no delivery vehicle and it is not a systemic drug. Why not? It works without either chemical modifications or a delivery vehicle. Simple enough.

Systemic delivery:
This seems to be the Authors’ main objection to the RNAi platform. They claim that systemic delivery is not currently possible with RNAi (but remember, delivery of a much larger molecule with Sangamo should be no problem and thus Sangamo should be valued much more highly than Alnylam...). There is no doubt that delivery is the rate-limiting step for achieving the potential that lies within RNAi. In fact, John Maraganore (the CEO of Alnylam) has stated as much when he said that Alnylam would be working on delivery for many different tissue types for years. Does that mean that they can not effectively deliver RNAi? They can, they have demonstrated so with Tekmira using technology developed by Alnylam, Protiva (now part of Tekmira), Tekmira, and Robert Langer’s lab at MIT. Yes, Alnylam delayed their IND filing for VSP and PCSK9 last year, but in hindsight it appears to have been a wise move. They had liposome (stable nucleic acid lipid particles - SNALPs) formulations that were effective, but near the date of filing the aforementioned technology came together to achieve a 10 fold increase in RNAi activity. I’d wait a few months and file for an IND using those formulations as well. The beauty of the SNALP system is that it is easily tunable to different sized particles and each can have different properties determined by different lipid or lipidoid formulations. This is where the MIT lab has aided in the production of tissue-specific and/or property optimization of Tekmira’s SNALPs. SNALPs can be engineered to be stable in serum for long periods and be slowly delivered or be rapidly absorbed by the liver depending on the desired pharmacokinetics. Each drug may have differing desirable characteristics and with SNALPs it is easier than ever to design such properties into delivery.

The process of producing a delivery vehicle is not easy, but the assessment of such technology is simple. The only two questions that matter are: does enough of the drug get in the cell to be effective? and is the formulation toxic? Tekmira/Protiva developed delivery vehicles that were effective and not toxic, the newer formulations are better. End of story, I won’t even bother with their quotes here. If you want to see data and read about the advances made at Tekmira, please read the Prospectus (type in 'Tekmira' under company name, then select 'management information circular dated 6 May, 2008).

This system is currently being used to actively and specifically target RNAi to the liver. Read that again, EFFECTIVE DELIVERY TO THE LIVER. Oh, fine, you say. That’s only one organ...with dozens of known targets. Many of them would be major blockbusters - cholesterol reduction, hepatitis C, cancer etc. Personally, I don’t think Alnylam needs to be able to deliver to any tissue besides the liver to become a major player in pharmaville; however, Alnylam and its collaborators are actively targeting many other tissue types (CNS, eye, lung, tumors, heart, smooth muscle) with both SNALPs and many other technologies. Delivery will not be a one-size-fits-all endeavor, and it doesn’t matter. Each cell type has specific differences and these will call for different delivery vehicles. Each cell type will probably have multiple targets so delivery optimization will result in many additional drugs for these tissues. To convince yourself of the value of RNAi with delivery to only one area, read the list of major drugs that each big pharma has currently. They are not long lists of major drugs.

Leerink Swann states that “Alnylam has approximately 25 drug delivery collaborations...which we see as signs of desperation rather than strength.” Technically this statement is true, but it’s true in the sense that historically airplanes don’t fly. It is a sign of desperation. Now before you get your shotgun, hear me out. Big pharma and small biotech companies alike are so desperate for innovation they are clamoring for RNAi and have partnered with Alnylam. The authors did not mean it this way, but instead meant it as an act of desperation for Alnylam. Perhaps they didn’t bother to read which direction the money was flowing. Investing is simple. Follow the money. If Alnylam had paid Roche to help, it would be Alnylam that was desperate; it was, in fact, Roche, Takeda, Novartis, etc. who paid Alnylam, implying that they were desperate enough to pay for the privilege of using Alnylam’s IP on a non-exclusive basis. Again, I feel compelled to point out that we are talking over a half a billion dollars in cash, not including milestones and royalties. Additionally, the list of collaborators has been altered recently to include a number of 50/50 splits with Alnylam. Alnylam renegotiated the contract with Medtronic from a small royalty with milestones to a 50/50 split. Alnylam is allowed to pick at least one of the drugs Tekmira is developing for 50/50 splits. Alnylam has the right to a 50/50 split on 4 drugs of their choice with Takeda. Does this sound like Alnylam is desperate? Domineering maybe, but they are not desperate.

From the tone of the article, it has not escaped our attention that there may be desperation from investors on the short side of the trade rather than those who have researched and paid for the right to use RNAi.

LS ends their “investment thesis” section with “When Alnylam uses its significant cash position and high share price to “diversify” or “forward integrate” by acquiring another technology or a clinical product, we will take it as a sign that the management team has acknowledged the difficulties with its current approach as well.” I know you’re wondering if a reputable financial analyst would say this in their research report, or if I made it up. The answer is no, a reputable analyst would not say this, and no, I didn’t make it up. See other articles on this blog for additional information on the pipeline.

RNAi is not Antisense

From the abstract of an important paper in RNAi:

"Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.
Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA. fire@mail1.ciwemb.edu

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts...Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference..."


RNAi is not antisense- This very finding formed the basis for awarding the 2006 Nobel Prize committee to Andy Fire and Craig Mello. Despite this authoritative decision, as the promise of the RNAi Therapeutics platform is getting closer to reality by the day, the promise also of financial fortunes has led to efforts to re-brand RNAi as yet another antisense technology. Although this in a way could be considered as some kind of validation of the competitive nature of RNAi, it has important implications for RNAi intellectual property.

In the past, ISIS Pharmaceuticals, undoubtably a leader in RNA therapeutics and with nucleic acid modification IP very relevant for RNAi Therapeutics, has made a number of statements I cannot agree with. Essentially, the company claims having discovered RNAi and has long regarded Alnylam as one of their many “satellite companies”. This, however, does not reflect reality in as much as repetitively claiming that RNAi is an antisense technology does not make it a true statement.

The latest example can be found in ISIS Pharmaceutical’s press release on the issuance of an ApoB-related antisense patent summarizing the claims as covering “the use of both single-stranded and double-stranded (siRNA) antisense drugs complementary to any site of the mRNA of human apoB regardless of their chemistry or antisense mechanism of action.” “Double-stranded antisense drug”- quite an oxymoron. But note that nowhere in the claims of the patent are duplex RNAs mentioned, instead “12 to 30
nucleobases in length targeted to a nucleic acid molecule encoding apolipoprotein B wherein said compound is 100% complementary to the nucleic acid sequence set forth in SEQ ID NO: 3” are claimed.

RNAi being antisense, having discovered RNAi first, and Alnylam being a satellite company, just does not agree with the fact that the very reason why Fire and Mello’s discovery is considered a major breakthrough, is because they found that RNAi is induced by double-stranded RNAs and not by antisense molecules as had been widely believed, and this indeed was the very working hypothesis that Fire and Mello famously disproved and for which they were awarded with the Nobel Prize. Come to think of it, RNAi as an “anti-antisense” technology may be a more apt description.

The claim why RNAi is supposed to be an antisense technology is largely based on the observation that during RNAi, the double-stranded siRNA is unwound, and the single-stranded guide RNA strand incorporated into RiSC which it then guides to target mRNA partly, but not exclusively, based on base-pair complementarity. Just because base-pairing interaction is involved, however, does not make it antisense. What is important for a biotechnology that harnesses an endogenous biological pathway are the features that make it being recognized as a bona fide substrate. In the case of RNAi, this clearly is double-stranded RNA with a couple of additional characteristics, and not single-stranded antisense RNA. This is the result of RNAi having evolved as a way to recognize and fight RNA viruses which are characterized by double-stranded RNA intermediates. Re-branding RNAi after one of the components of its inducers would be like saying the wheels of a car are the car.

While single-stranded antisense RNAs may also induce RNAi, research by ISIS itself and others has shown that they do so much efficiently, about 20- to 50-fold less efficient than double-stranded RNAs, that is about as efficient as a conventional RNaseH antisense molecule (probably another reason for ISIS’ growing interest in RNAi).

With so much “new fundamental” trigger IP being claimed, RNAi now becoming an antisense technology etc., are we about to see a different approach by the RNAi Therapeutics companies in possession of the real fundamental IP? I would not necessarily expect legal proceedings as long as it does not start to affect their business dealings. However, such rhetoric has the potential to lead to market inefficiencies, hurt smaller companies with a less active PR department (see Tekmira and their ApoB program), and lead to a situation in which as the first RNAi Therapeutics are approved and patent disputes to be resolved, the debate will have been re-framed to the disadvantage of RNAi Therapeutics companies.

PS: The RNAi-is-Antisense claims also get to the important issue of target IP. Target-specific IP should be encouraged as it incentivizes the identification of suitable RNAi Therapeutic gene targets, as long as certain minimum validation standards are satisfied. I would therefore agree that if the ISIS antisense patent had been a milestone in demonstrating the suitability of apo-B inhibition for the treatment of hypercholesterolemia (note: ribozymes targeting Apo-B were specifically exempted from the claims which already suggests that the present patent is not the seminal ApoB-hypercholesterolemia patent), this should also have consequences for drugs based on other mechanisms of actions such as RNAi, monoclonal antibodies etc. In cases where there is a proven benefit in targeting at the RNA level, the royalty rate should climb commensurately, although I do not believe that in practice this means that an antisense therapeutic will be able to stop an RNAi Therapeutics from reaching the market and vice versa.

Note: The abstract of the Fire and Mello paper has been added after the first publication of the blog entry.

Bristol-Myers Squibb Deal Further Positions Tekmira as RNAi Therapeutics Delivery Hub

Yesterday, Tekmira added Bristol-Myers Squibb to an already impressive line-up of RNAi Therapeutics delivery partners that further include Alnylam Pharmaceuticals, Roche, Merck, and Takeda. The new research collaboration specifically aims at developing SNALP RNAi for delivery beyond the liver. It follows a previously undisclosed target validation collaboration between BMS and former Protiva (now merged into New Tekmira), and is yet another vote of confidence for the clinical viability of SNALP RNAi delivery.

SNALP RNAi delivery is well validated for gene knockdown in the liver, including in non-human primates. Just this week, Alnylam published in the scientific journal PNAS the lowering of PCSK9, a cardiovascular disease target, in rodent and monkey liver using SNALP liposomes. For clarification purposes since there has been much confusion on the issue of SNALPs and lipidoids, while the particular SNALP formulation used in the publication contained lipidoids, lipidoids are just one of the various other lipids that could be used with SNALP technology. Moreover, Tekmira has non-exclusive access to lipidoids from the MIT via Alnylam in case they wanted to incorporate them into their own SNALPs.

Investing in SNALP delivery outside the liver (Tekmira expects to receive $450k from BMS for the second half of 2008) should therefore expand the therapeutic potential of this technology and directly aid the development of a broad and balanced Tekmira RNAi Therapeutics pipeline. Strategically, it should also provide Tekmira with a potential future development partner and access to disease know-how and gene targets.

Cancer is the most obvious non-liver target given what we know from the passive targeting of SNALP/liposomal particles to solid cancer tissues. Further validation for the concept of using liposomal RNAi delivery for cancer comes from plasmid delivery data from Tekmira itself and various liposomal RNAi publications by others. Some of that research made use of active tumor targeting, and it is not far-fetched that the research collaboration will also consider such active targeting strategies. These may also hold promise for indications outside solid cancer such as hematological malignancies, blood vessel-related disorders (e.g. cardiovascular/metabolic disorders), and immunological diseases.

Cancer and metabolic/cardiovascular disease would also make sense as the two focus areas of BMS research and development. While the scope of the Tekmira-BMS collaboration may indicate that the returns of investing in SNALP delivery to the liver are diminishing with first INDs expected to be filed within the next 12 months, it may also be interpreted as accommodating Alnylam or even point towards a follow-on deal with Alnylam which may not want to further dilute the perceived value of what it has granted its previous platform licensing partners.

Cancer on the other hand is a genetically highly diverse set of diseases, and with SNALP and RNAi, Big Pharma must feel like kids in a candy store with more than enough targets and unmet needs for everybody to pick from. One note of caution though is that while SNALP RNAi has demonstrated potent activity in pre-clinical models of liver cancer, much less is known about its actual knockdown activity in other cancer types. It will therefore be important to confirm the clinical feasibility of SNALP RNAi for cancer through conference presentations and scientific publications.

BMS’ decision, of course, has also to be seen as part of the rush by Big Pharma to transform themselves into developers of innovative biotech drugs. Offers by Roche and BMS itself for Genentech and Imclone, respectively, are just two recent high-profile examples. The deal also demonstrates BMS’ increasing commitment towards the RNAi platform. Only last year, BMS entered into an antisense collaboration with ISIS Pharmaceuticals for PCSK9 knockdown in cardiovascular disease. Interestingly, this was largely based on BMS’ successful validation of PCSK9 as a cardiovascular target using RNAi (and as we now know most likely SNALP-enabled as part of the BMS-Protiva target validation collaboration), data of which was presented at last year’s OTS meeting in Berlin. When asked why BMS decided on antisense, not RNAi in light of the impressive RNAi data presented, the response was that ISIS’ mipomersen represented clinical proof-of-concept that antisense could knock down genes in human liver. It now seems that like other pharmaceutical companies, positive experience with RNAi as a target validation tool has translated into embracing it as a therapeutic platform in its own right.

The one-million-dollar question weighing on the minds of Alnylam investors will be whether yesterday’s development heralds the next Alnylam platform alliance deal. In the past, partners tended to come to Tekmira as Alnylam sub-licensees. Although details of the deal are lacking, due to otherwise too many potential conflicts-of-interests and the importance of Alnylam to Tekmira, it is unlikely that BMS will turn out to be a Merck-like star in the Alnylam-Tekmira constellation. Given that Tekmira has inherited a number of additional technology feasibility partnerships with Big Pharma and biotech from Protiva, further such deals are likely to materialize with Tekmira functioning as one of Alnylam's baits.

An interesting aspect in this context is that the corresponding author of this week’s paper, Kevin Fitzgerald, came to Alnylam (leader of the oncology and cardiovascular/metabolic disease group) from BMS. At BMS, he had been part of the target validation group, very likely the same group that had been involved in the BMS-Protiva collaboration. Obviously, RNAi has not disappointed him, but beyond that, is this part of a larger puzzle?

Now that Tekmira has RNAi delivery partnerships with Alnylam, Roche, Merck, Takeda, and BMS, and more to come, the 100-employee strong Tekmira is slowly emerging as the RNAi Therapeutics delivery hub. This also starts to pay off financially with essentially no appreciable decrease in cash reserves over the last quarter. It will also be interesting whether Tekmira will pursue interests beyond liposomal RNAi delivery. Combining various delivery technologies into one operation, whether as an independent company or not, would have a number of synergies as many of the same questions need to be addressed with the different technologies.

RNAa- Alnylam Continues to Gobble Up Fundamental RNAi-related IP

Earlier this week, Alnylam announced the consolidation of intellectual property related to RNA activation (RNAa). RNAa is a method of sequence-specifically activating genes by the introduction of cognate double-stranded small RNAs. It is similar to siRNAs, but in this case the dsRNA corresponds to certain gene promoter elements instead of the mRNA in the case of siRNAs. In addition to once again positioning Alnylam up to be the gate-keeper of a potentially new class of small RNA therapeutics that harness natural gene regulatory mechanisms, as RNAa touches upon RNAi biology, this development has also to be seen as further cementing Alnylam’s grip on fundamental RNAi and siRNA IP thereby creating immediate value.

RNAa quietly emerged over the last 3-4 years in the shadows of the RNAi revolution. When it was discovered that RNAi-related mechanisms function in transcriptional gene silencing (TGS) in fission yeast (explanation: in TGS, siRNAs mediate chromatin modifications that prevent a gene to be transcribed in the first place, rather than being targeted for degradation after having been transcribed as in RNAi), researchers around the world, including myself, started to look at whether a similar mechanism operated in mammalian cells. While initially met with some skepticism and controversy, the overwhelming evidence now strongly supports that TGS indeed exists in mammalian cells.

One branch of TGS research introduced siRNAs targeted to gene promoters with the aim of silencing that gene by recruiting chromatin silencing machinery to the targeted DNA elements. As was subsequently reported, in some cases they do. But to the surprise of many, some of these siRNAs had just the opposite effect, namely they activated expression from the corresponding gene. As detailed in Alnylam's press release, this does not appear to be an isolated effect, but was shown by a handful of labs to be applicable to a number of genes looked at.

While the mechanism of action of these small RNAs, referred to as ‘antigene RNAs’ (agRNAs) to distinguish them from their better known cousin, remains spotty, it appears that low-level non-coding transcription around the targeted promoters are recognized by the agRNA in a process that involves Argonaute proteins and leads to activating chromatin modifications. In addition to sharing with RNAi the Argonaute proteins, good agRNAs appear to follow similar design rules as siRNAs: 19-21 base-pair dsRNAs with seed-match dependency. 3' overhangs have also been used, although the utility of this structural feature has not been explicitly demonstrated. Consistent with an epigenetic mechanism, the effect can be quite long-lasting (~10-14 days of gene activation following agRNA introduction in tissue culture cells, and likely to be even longer lasting in primary cells).

Moreover, agRNAs appear to tap into a natural gene regulatory pathway as endogenous small dsRNAs had previously been shown to switch on the activity of cognate promoters. MicroRNAs, some of which are known to be localized to the nucleus, have also been implicated here.

From Alnylam’s press release and the Q2 '08 conference call, the consolidation of RNAa IP that is based on the first reports of RNAa in the literature, was explained as representing yet another small RNA-related business opportunity for the company, similar to how it views microRNAs. Due to the early nature of RNAa, it is likely that while Alnylam will pay for IP-related expenses, it will be the academic labs that licensed the technology that will conduct much of the initial research. It also remains to be seen when we will see a Regulus-like RNAa spin-off.

I would argue that if RNAa and RNAi had little overlap, Alnylam would have been better advised to let others invest instead of diverting focus and resources from RNAi Therapeutics that should yield a much higher return on investment. RNAa is still at an comparably early development stage, and the number of therapeutic opportunities should be much restricted compared to RNAi Therapeutics. Initially, I would expect the re-activation of tumor-suppressor genes for cancer therapy to be the major attraction.

One factor, however, that could have tipped the economic scales in favor of an investment in RNAa is the fact that RNAa and RNAi seem to intersect both with respect to biology and technology. One paper suggests that Argonaute 2, the Slicer of RNAi, is indispensable for RNAa. As the functions of the 4 human Argonautes slowly emerge from the research labs around the world, this adds to the notion that siRNAs, once introduced into cells, could function in more than one way in regulating gene expression. As such, RNAa broadens the methods of using siRNA structures protected by Alnylam IP.

This synergy in IP may also at least partly explain why Alnylam and not ISIS Pharmaceuticals, its Regulus partner, embraced RNAa in such a comprehensive manner. This is despite of ISIS’ contribution of research reagents to one of the underlying papers and a RNA therapeutics business strategy that has been broader than that of Alnylam’s. Still, with Alnylam securing fundamental IP related to harnessing natural small RNA gene regulatory pathways and ISIS in possession of an impressive and complementary nucleic acid IP portfolio and a near-term drug opportunity in mipomersen, I would not count out a future involvement of the Carlsbad company. I also wonder at which point it may even make sense to just combine the two companies.

PS: Should RNAa turn out to be widely applicable and if RNAi Therapeutics history is any guide, odds are that competing companies will come up with Atu- and RXagRNA designs that vary slightly in structure, claiming to have superior activity and enjoying freedom-to-operate. Nucleonics and MDRNA, however, should serve as stark reminders that this may not be the best strategy of creating shareholder value. The so very demonstrative nature of the RNAa IP consolidation this week should serve as a useful reference against which competing claims will have to be measured.

Tysabri “News” Highlights Value of RNAi Therapeutic Against JC Virus

Tysabri made the headlines again late Thursday when Biogen Idec and Elan Corporation revealed the first two cases of progressive multifocal leukoencephalopathy (PML) following the re-launch of the multiple sclerosis (MS) drug almost two years ago. Although this had been widely expected, a sensational media and stock market reaction that wiped out $10B off the market cap of the two biotech companies, gives me an opportunity to discuss ongoing efforts to develop an RNAi Therapeutic against JC virus, the etiologic agent of the disease. Such an RNAi Therapeutic has the potential to play a crucial role in the risk management strategy of Tysabri and beyond.

Most of us are carriers of JC virus, a double-stranded DNA polyoma virus. PML is the result of re-activation of the virus following immunosuppression. PML really only became a medical problem with the AIDS epidemic, but cases of organ transplant-related and other immunosuppressive drug-related PML infections are increasing. In fact, it perplexes me why a drug like Tysabri that I believe, based on impressive clinical trial results and countless patient and physician testimonials, is the one in a dozen drugs that really makes a big difference for patients, has been singled out for this type of special treatment.

This is, however, not to downplay the potentially deadly nature of the disease. Tysabri, a monoclonal antibody aimed at neutralizing VLA-4 integrins, prevents the migration of leukocytes from the blood vessels into tissues, thereby preventing effective immunosurveillance of infections. In what the drug label estimates to be 1 in a 1000 cases, this allows the latent JC virus to become reactivated which may lead to de-myelination linked to viral replication.

An unparalleled surveillance program instituted by the companies with guidance by the regulatory agencies ensures that everybody involved in the treatment of Tysabri is fully aware of the risks. The heightened awareness should lead to early detection by PCR and MRI and the discontinuation of the immunosuppressive drug. In many cases this is enough to allow the immune system to catch up with the virus. An RNAi Therapeutic should increase the likelihood for that to occur both by decreasing viral load as well as potentially inflammatory viral protein expression which may cause the demyelination.

Clearly, in the absence of a proven antiviral, the potential of an RNAi Therapeutic targeting JC virus in the brain has not been lost on Biogen Idec. Three months after the re-launch of Tysabri in June 2006, Biogen Idec said that it would collaborate with Cambridge, Mass, neighbor Alnylam on the development of such a treatment. The last update was provided at the Keystone RNAi meeting earlier this year in Vancouver, and the data were quite encouraging and clinical trials may not be too far off.

The data supported those reported by others (here and here) that siRNA treatment is able to inhibit viral replication in vitro, both when administered before the initiation of viral replication, and more therapeutically relevant, after the initiation of viral replication. Since a reliable in vivo model for JC viral infection is lacking, further proof for true therapeutic potential came from demonstrating 55-75% target-specific knockdown of an oligodendrocyte marker gene (CNP) in rodents and non-human primates. Oligodendrocytes were chosen as the primary site of JC virus infection.

Given that localized delivery of RNAi to the brain appears to be closer to the clinic than systemic alternatives, with the exception of perhaps the intriguing publication last year on the use of rabies peptides for the delivery of siRNAs across the blood-brain barrier following systemic administration, the main question should be whether such delivery can not only get into the right cell types, but also achieve sufficient coverage to stall the virus.

Early detection will help by decreasing the size and number of viral replication sites that need to be treated. Unlike the name would suggest, PML infection is as often unifocal as it is multifocal, and localized siRNA/DNA-directed viral vector administration to limited numbers of infection sites based on the PML-diagnostic MRI seems reasonable.

Finally, a word on the stock market reaction to the Tysabri “news”. It is quite remarkable that the report of the expected PML cases was able to trigger 30% and 50% declines in the stock prices of Biogen Idec and Elan, respectively. Sensational media and scare-mongering analyst reports, some of them fabricated, accompanied short-selling tactics aimed at suggesting rock-bottom valuations. At one point for example, Elan traded down over 75% in relatively light German trading. Also noteworthy was that 5 minutes before the market close and announcement of the PML cases, 30,000 August '08 Elan put options were traded for what must have been an amazing $30M-dollar-in-5-minute gain.

As the financial world is starting to realize, abusive short-selling aimed at destroying the ability of companies to grow, which in the case of biotechs is designed to undermine their ability to fund drug development, is very real. This should be of great concern to the biotech industry, and it is hoped that trade organizations such as BIO seize the opportunity to put pressure on the SEC to address this problem not just for the benefit of a select number of financial companies.

Pfizer and Quark Progress RNAi Therapeutic for Diabetic Macular Edema into Phase II

Quark Pharmaceuticals today announced that development partner Pfizer has initiated dosing of the first patient in a phase II trial of the RNAi Therapeutic candidate PF-4523655/RTP801i-14) for diabetic macular edema (DME). The start of the phase II trials is a vote of confidence in RNAi Therapeutics for eye diseases by a major pharmaceutical company with a considerable eye care franchise and growing ambitions in RNAi Therapeutics.

The 160-person safety and efficacy trial follows phase I/II studies conducted by Quark that showed the drug to be safe and well tolerated in patients with wet age-related macular degeneration (wet AMD). Similar to DME which affects about 10% of type I and II diabetics, wet AMD is caused by the growth of leaky blood vessels in the back of the eye leading to blurred vision, and in some cases blindness. For this reason, it is not uncommon that a given compound is being tested for both indications.

The reason that Pfizer has chosen DME instead of wet AMD as the indication for this trial may be related to the fact that the wet AMD RNAi Therapeutics space has become crowded with Opko Health and Sirna Therapeutics/Merck having wet AMD RNAi Therapeutics in phase III and II studies, respectively. Opko Health also has a phase II study for DME using the same compound. It may also be related to the recent controversy caused by a Nature paper that showed that dsRNA-triggered immune responses alone may be anti-angiogenic in a mouse model for wet AMD and that this may have led to mis-interpretations of data from related pre-clinical RNAi Therapeutics studies.

However, PF-4523655 is unlike Sirna’s and Opko’s compounds in that it is intended to prevent abnormal blood vessel growth and leakage in a VEGF-independent manner. It is also a 19bp blunt-ended compound and therefore unlikely to trigger non-specific TLR3 responses (21bp seems to be the cut-off where you have to take TLR3 signaling into account). I trust that the former Coley Pharmaceutical’s folks with their deep understanding of TLR biology and that are now running the RNAi Therapeutics show at Pfizer will have closely looked at the issue.

Quark received an undisclosed amount for the initiation of the trials, Silence Therapeutics which had originally licensed the AtuRNAi technology to Quark collected $1.9M, and Alnylam from which a technology license was subsequently obtained on Pfizer’s insistence another undisclosed amount. Clearly, with today's allowance of Silence Therapeutics’ 10/633630 patent in the US which specifically claims AtuRNAi molecules of 18 or 19 base-pairs the IP battles are about to begin, and will be addressed in a follow-up blog entry. In any case, it is an interesting coincidence that the initiation of the phase II studies and the patent allowance occurred on the same day.

Independent of all these patent issues, I look forward to the results of this compound which has the makings of a safe and differentiated RNAi Therapeutic for DME.

Comments Section of the RNAi Therapeutics Blog

I have been very impressed by the quality of the comments left below some of the blog entries. If somebody hasn't noticed them, I would highly encourage you to read them for interesting insights, fresh perspectives, and discussion.

I think this small forum here exemplifies that not only is drug development undergoing profound changes, from a sales-force and reformulation-driven model towards one of evidence-based medicine and innovation, but so is the practice of science and investment. Freely accessible scientific publications, realtime quotes and news, and online forums bringing together people with complementary backgrounds mean that everybody has now a chance to participate on a level playing field. Appropriately, RNAi Therapeutics has in the same way empowered the individual scientist to conceive novel drug candidates without the need for large-scale automation, and I would also hope that emerging economies that have thus far not participated in the development of innovative therapeutics will grasp the emerging field of RNAi Therapeutics as an opportunity to be at the cutting-edge of medicine.

Before I digress further, I just wanted to say that I will try and do my best to answer questions if directed to myself, but may not always be able to do so. A sincere “thank you” to all the contributors.

RNAi Therapeutics delivery: not a zero-sum game

Shares of Tekmira briefly fell last week below 80c, a ~20% decline, following Roche’s purchase of fellow RNAi Therapeutics delivery company Mirus Bio. Initially, I did not draw a connection. If a reaction at all, then I would have expected the $125M price-tag to point out the value attached by the industry to promising RNAi delivery technologies in general, which “should have” resulted in a share price increase for Tekmira.

To my surprise, however, it then transpired that the Roche acquisition had been interpreted by some as a vote for DPC and against SNALP RNAi delivery, leading some to sell in a panic. This blog will briefly outline why, unlike is the case in the battle for core RNAi trigger IP, RNAi Therapeutics delivery is not a zero-sum game with room for more than just a handful of technology platforms.

As is becoming more evident by the day, RNAi Therapeutics offers the opportunity to address a very wide range of diseases. As all disease has a genetic element, regulating gene expression should always be able to modify or even correct a disease. To do this, however, one has to be able to knock down the desired genes in various different organs, tissues, and cell types. If there was a universal RNAi delivery technology, it would have to be able to physically contact every cell in the body, yet only knock down genes in the disease-related subset of cells.

Based on the limited publicly avaibable data, Mirus’ DPC technology actually promises to come close to that dream. It is very small in size (~20nm) and therefore satisfies at least one of the pre-requisites of achieving a broad biodistribution. Moreover, it has been shown to be capable of differentiating between the different cell types within an organ, in this case the liver, depending on which sugar had been added- all in the absence of apparent toxicity! It will clearly be exciting to learn more about the delivery profile of DPCs. Nevertheless, I doubt that DPC will necessarily be the delivery system of choice for every indication.

This is because the choice of a delivery system is not only determined by the ability to knock down a gene in a given cell, but also by factors such as overall maturity of the technology, safety as determined by the dose required to achieve such gene knockdown, the biodistribution and cell type-specific gene knockdown on a systemic level, as well as by cost, route of administration, and stability of the formulation.

Consider for example an RNAi Therapeutics against the kinesin spindle protein (KSP) that is being developed by Alnylam as a treatment for liver cancer. Although directly interfering with cell division by down-regulating KSP is seen as a very promising anti-cancer strategy, anti-KSP small molecule programs have indicated considerable dose-limiting toxicities that appear to be target specific. This, however, should not come as that much of a surprise since interfering with spindle function would be predicted to affect normally proliferating cells as well, particularly those of the hematopoietic system. Well, this dilemma is not new at all to the cancer field, and often the benefits outweigh the side-effects, but this example illustrates the value of a delivery technology such as SNALP-RNAi that can be tuned to deliver around 95% of the injected dose to the liver, including hepatic tumor tissue.

Certainly, Roche may elect DPC delivery over SNALP delivery (note: Roche has access to both) for some liver indications, but that type of competition has actually only decreased with Mirus Bio out of play. This is because all the other two dozen or so pharmaceutical companies equally interested in RNAi Therapeutics, will now have access to one less viable delivery technology. The incentive to gain access to the still fairly accessible RNAi delivery platforms with clinical potential has therefore never been greater. Roche, of course, enjoys the luxury of being able to choose between two technologies that, maybe in an effort to avoid the appearance of favoritism, it has valued essentially identically based on their $5M equity investment in Tekmira at $2.4 per share(Tekmira now has 51.6M shares outstanding).

Bottom line, due to the ever growing attraction of RNAi Therapeutics, any company, and that goes beyond Tekmira-SNALP, with clinically relevant RNAi delivery technology and a good IP package to protect it can rest assured that they own a very hot commodity.

RNAi Therapeutics portfolio update: With Roche’s validation of the value attributed to clinically relevant RNAi delivery technologies and with increased clinical visibility, I have slightly increased my position in Tekmira while paring back on the overweight ALNY position. In another attempt to capitalize on the idiosyncracies of the stock market, I have converted the RXi Pharmaceutical holdings back into CytRx shares (CytRx owns about 50% of RXi, but none of that is reflected in CytRx’s market cap) and added some more at the cost of largely cashing out of RNAi trigger competitor Silence Therapeutics (a token $1 investment is maintained to monitor stock performance).

Disclosure: Long Tekmira, Alnylam, Targeted Genetics, Oxford Biomedica.

Roche Nabs Mirus Bio’s RNAi Therapeutics Delivery Technology

Roche again...before this week will have come to an end, Roche has drastically changed the RNAi Therapeutics playing field leaving many heads in the RNAi space spinning.

You may think it is because I live in an RNAi Therapeutics bubble, but the Roche-Genentech press release and conference call to me clearly shows that a major motivation for the proposed taking private of Genentech was to broaden their RNAi Therapeutics efforts by bringing in a company with deep immunology and personalized cancer know-how, as well as being able to leverage Genentech’s monoclonal antibody capabilities for targeted RNAi Therapeutics delivery.

To this they now add for $125M, a sum that makes related companies look very cheap in comparison, the privately held Madison, Wisconsin, nucleic acid delivery company Mirus Bio. The jewel of Mirus Bio is their Dynamic PolyConjugates (DPCs), small, flexible designer particles for the targeted systemic delivery of siRNAs. Although the technology is relatively young and data scarce, from the PNAS publication last year (reviewed here in the RNAi Therapeutics blog) and conference presentations, DPCs are very competitive with liposomal technologies for delivery to the liver. Also very attractive from a safety and efficacy point-of-view is their apparent ability to selectively target silencing either to hepatocytes or Kupffer cells in the liver, depending on whether glucose or galactose-derivatives were attached. Although I haven’t seen data beyond the liver, the small size and modularity suggests that with the appropriate pharmacology it could well have applications for a number of other tissue types and organs and nicely complement larger nanoparticle delivery technologies.

It’s unlikely to be a coincidence that Roche is making all these moves in such short order. What has started with an IP license from Alnylam for basic access to RNAi mechanism of action, within 2 days they have now added to that one of the most coveted delivery technologies and scientific depth. The rapid moves by Roche means that fellow Big Pharmas like Pfizer, which had a non-exclusive license to DPCs, and Merck which had probably also been very interested in DPCs, now risk falling behind on delivery while their core RNAi IP has either not been secured yet (Pfizer) or is at best uncertain (Sirna Therapeutics/Merck). From a strategic perspective, it will be interesting whether due to their close relationships there will be any sharing/coordination of DPC technology with Alnylam and Tekmira, and for which indications Roche will employ the two leading delivery technologies (DPCs and SNALPs) both of which it has now immediate access to.

Today's acquisition is yet another piece of evidence that Roche is building their future on RNAi Therapeutics in a big and bold way. It's also encouraging that this comes a year after the Alnylam platform licensing agreement and suggests that they must have been pleased with what they have seen since.

Speaking of the Devil: Genentech Receives Full Takeover Offer from Roche; Stands to Become Alnylam’s Partner

Just as I was speculating that Genentech may become Alnylam’s next major RNAi Therapeutics partner, Roche makes an offer to buy the rest of Genentech’s shares that it does not own already. I’m sure the pundits will be all over what this event will mean for drug development, so just a few notes on its relevance for RNAi Therapeutics.

From the press release and based on historical comments against the notion of mega-mergers with fellow Big Pharma and against entering generics, it is clear that Roche is very keen on leading the development of innovative medicines, particularly personalized medicine. What better candidate than Genentech with all its know-how in this arena as well as size to sufficiently impact Roche's top and bottom lines? Just to demonstrate that personalized medicine is more and more becoming a reality, Roche appropriately also announced today European approval for an interferon-ribavirin treatment regimen for hepatitis C based on individual viral titers and rapid viral response. Interestingly, Alnylam disclosed really for the first time in the Pharmaceutical Executive report its interest in hepatitis C which might be best addressed by SNALP delivery. And how much more personalized can hepatitis C treatment get than to sequence the virus in a given patient and then deliver the appropriate siRNA combination?

Reassuringly, the press release prominently mentions RNAi Therapeutics as a central component of the innovation engine of the combined company:

“The structure of the combined company will allow for a diversity of approaches in research and early development, while also strengthening cross fertilization between the companies, leading to enhanced overall innovation within the Group. Roche's recently adopted Disease Biology Area approach, which allows five diverse groups to manage their innovative portfolios, will be maintained and strengthened. This, together with recent moves into RNAi (Ribonucleic Acid interference) and delivery technologies, as well as licensing activities, continues to provide a stimulating
environment for the creation of medically differentiated medicines.”

And on the “enhanced ability to innovate”, the press release goes:

“The transaction will over time significantly enhance cooperation and cross fertilization among all research hubs inside and outside of the combined company. Sharing of technologies (e.g. RNAi, novel protein architectures), assets (e.g. chemical libraries), intellectual property(e.g. antibody production), unique capabilities (e.g. exploratory development, modeling and simulation) and know-how of the combined research organization will strengthen the Group's ability to innovate.”

I wonder how much Genentech will be surprised by the offer. In this regard, it is also interesting to speculate whether Alnylam had been in negotiations with Genentech or not. If so, would an unsolicited takeover of Genentech by Roche mean that Alnylam has to forego platform license fees that it would have otherwise received if Genentech remained independent? On the other hand, could it be that Genentech’s moves into RNAi Therapeutics had long been co-ordinated with Roche’s efforts, relying on being able to access RNAi know-how and IP through Roche? I guess the answer will come whether the $89 a share offer will be recommended by Genentech management to shareholders or not. Given the close ties between the companies a Ventana-like takeover battle is difficult to imagine. The weak dollar may also aid Roche in increasing the price a little bit to satisfy all Genentech shareholders.

Today’s development promises to be a defining moment in the history of drug development. With the recent acquisitions of diagnostics company Ventana and now Genentech, Roche has now fully committed to embracing the future of personalized medicine. Based on comments by the new CEO Severin Schwan who has now more than amply demonstrated that he really means what he says, RNAi Therapeutics is to play a key part in this.

Genentech and RNAi Therapeutics- Part 1

A very enjoyable, yet detailed feature article by Walter Armstrong on the state and prospects of RNAi Therapeutics in the Pharmaceutical Executive this month, concludes with a somewhat bold prediction of mine: Genentech will partner with Alnylam to develop RNAi Therapeutics. I would like to use the next two blogs to expand on this speculation, explaining why such an alliance would not just make Alnylam shareholders happy (Part 1), but also the ways that Genentech’s participation in RNAi Therapeutics should benefit the entire sector (Part 2).

First off, all of this is conjecture and not based on inside information. I think it is fair to say that Alnylam is the bellwether of the RNAi sector. At the same time, however, Alnylam has made it no secret that it in turn is following the example of another biotech company- the iconic Genentech. For example, when Alnylam won the prestigious biotechnology James D. Watson Helix award for the mid-cap category in 2006, it was particularly proud to join Genentech as that year’s winner of the large cap category. References to Genentech’s business development strategy can also be heard at regular intervals in its conference calls and interviews like the one by Nature Biotech with Alnylam CEO John Maraganore in 2007 from which this excerpt is taken:

“JM: By that, I mean good old-fashioned, peer-reviewed, published research, appearing in top journals like Nature, Science and Cell. In this, we have a prominent role model: Genentech (S. San Francisco, CA, USA).When I was starting my career in biotech in the 1980s, Genentech wowed both academic and industrial scientists by blazing a trail in genetic engineering. Every week, it seemed, there
was a paper in one of those top journals describing an exciting new advance—Axel Ullrich making fusion receptors with cytoplasmic tails that helped us understand cell signaling, the cloning of tissue plasminogen activator—all of which were amazing feats at the time. Just as importantly,Genentech captured the imagination first of academic scientists, then of public-market investors and then of Hoffmann-
La Roche (Basel, Switzerland), all of whom profited handsomely on their decisions to place their trust and their capital in the hands of that company’s leaders.”

Also note the reference to Roche and Alnylam's deal with Roche announced 3 months thereafter. It was then also a statement by Roche made in the wake of Roche-Alnylam expressing their hopes that Alnylam may be their second Genentech (Roche is the majority owner of Genentech), that then confirmed that there may be more to Alnylam-Genentech than just the respect and admiration of an emerging company for the industry powerhouse. I’d like to think that at the time the statement was made, it was not merely hopes of making lots of money that where on Severin Schwan’s mind, now the CEO of Roche.

When as part of the Tekmira-Protiva reunion it was mentioned that out of the four technology evaluations by unnamed companies there was a Big Biotech, it was possible to start seeing the scientific dimensions take shape. Appropriately, in addition to Alnylam upping their stake, Roche joined by buying 4% of New Tekmira. As a reminder, one of the most promising therapeutic areas for SNALP RNAi delivery technology is oncology, and when it comes to Roche and cancer, we really mean Genentech and cancer.

Genentech not only pioneered recombinant protein therapeutics, but is also leading the pack in developing personalized cancer medicines. As I mentioned in an earlier blog, Genentech is now looking at small molecules as monoclonal antibodies cannot address the numerous targets it has discovered and accumulated considerable know-how in as part of their oncology efforts. These targets are downstream of the much more limited upstream components of cancer signaling pathways (downstream in general also means more specific/safer). This is particularly problematic when resistance mutations occur that render targeting the upstream ones futile. While RNAi Therapeutics had not been officially mentioned, I have to admit that I would be very surprised and quite frankly disappointed if Genentech did not resort to RNAi to harness its oncology target know-how by non-monoclonal means.

RNAi-related microRNAs should also be of great interest to Genentech as they could be developed as companion diagnostics for personalized cancer drug treatment, and possibly as therapeutic targets themselves to which Alnylam could provide access via Regulus.

Building on publicly disclosed RNAi screening know-how, the following job advertisement by Genentech earlier this year suggests that its RNAi Therapeutics plans are rapidly materializing:

“Responsibilities: We are seeking a highly motivated Research Associate or Senior Research Associate to conduct a range of research activities to enable delivery of an exciting new class of therapeutics based on RNA interference (RNAi). As a key member of our research team, this scientist will use a variety of methods (molecular biology, cell biology and biochemistry) to: (a) develop technologies to effectively deliver RNA-based drugs for a variety of therapeutic indications and (b) discover and elucidate the mechanisms of delivery and gene silencing.”

Whether an alliance is announced this year or not, the above circumstantial evidence and the secretive Genentech conference participants suggest to me that srtl RNAi Therapeutics will not only be validated as the innovation outlet for Big Pharma, but even biotech.

To be continued…

Journal Club: Structural Basis for Double-Stranded RNA Recognition by TLR3 and Activation

The recent Nature paper by Ambati and colleagues raised some concerns about whether non-specific inflammatory responses due to activation of TLR3 by double-stranded RNAs, the inducers of RNAi, would represent a significant, if not insurmountable obstacle towards the development of RNAi Therapeutics. While the short answer was no, since most in the field had already recognized the need to screen against the capacity of RNAi triggers to induce immune responses, determining the rules activating TLR3 and similar molecular patterns should facilitate the more efficient design of safe and potent RNAi triggers. Recent papers on TLR3 structural biology should do just that.

In an April edition in the journal Science, Liu and colleagues from the NIH report on the structure of mouse TLR3 with its double-stranded RNA substrate (note that mouse and human TLR3 are very similar). The structure suggests that a 40-50 base-pair double-strand RNA is optimal for binding by TLR3 thereby inducing TLR3 dimerization and downstream signaling. The fact that one of the dsRNA binding patches contains a number of pH-sensitive histidines further suggest why TLR3 signaling is most robust following uptake into endosome which provides for an acidic compartment. Overall, the structure supports previous observations that dsRNAs longer than what is typically used for siRNAs are better inducers of TLR3; however it does not explain well how smaller siRNAs may also induce such signaling.

An explanation for this is provided by structure-based mutagenesis studies by Pirher and colleagues from the University of Ljublijana (Slovenia). In this paper, they identify two dsRNA-binding patches within a TLR3 monomer and also find and explain why B-type helices, as typically found in DNA, only bind to one of the two binding patches and therefore fail to induce, and even competitively inhibit TLR3. This is in contrast to dsRNAs that typically assume A-type helical formation and bind both patches. With this the authors come up with a model to explain how TLR3 dimers bind to shorter double-stranded RNAs by assuming alternative conformations with ddimerization on the shorter dsRNAs being less efficient.

These findings immediately suggest various ways to avoid TLR3 signaling. The simplest would be to stay below 21 nucleotides, and it is well known that 20 nucleotide siRNAs are equally potent inducers of RNAi. Unlike the somewhat disgruntled 1st commentator following my previous blog would like to suggest, 15-21 nucleotide siRNAs as covered by one version of Kreutzer-Limmer in Europe are therefore therapeutically highly relevant. Generally, keeping it short is the probably easiest way to avoid non-specific immune responses. Next to sequence length, limited modifications of the siRNA at sites where they interact with TLR3 as shown by the structure should abolish any TLR3 activation and possibly serve to antagonize TLR3, similar to what has been found for 2’o-methylation and TLR7. Likewise, changing the helical shape at one end which may also have the added benefit in encouraging asymmetric RiSC loading may be a third strategy of circumventing TLR3 activation.

It is clear from reading the papers that much of the motivation for performing these is related to RNAi Therapeutics. These are findings that are not just theoretical in nature, but very much of practical relevance. The speed with which this progress has been achieved illustrates the vigor and the many tools brought to bear by the scientific community on making RNAi Therapeutics become a reality.