Alnylam Escalates Liver Cancer Drug Candidate to Dose Levels Predicted to Yield Meaningful Knockdown

Alnylam provided an update on the ongoing phase I clinical studies for their RNAi Therapeutics candidate ALN-VSP02 for liver cancer. Clinical data such as these are widely anticipated by investors and potential partners alike, and Alnylam with recent comments have left themselves little choice but to be measured against the quality of the data.

The presentation provided at the Chemotherapy Foundation Symposium held this week in New York City follows a June 2010 presentation at the prestigious ASCO meeting at which point 19 patients had received the liposomal formulation containing siRNAs directed against VEGF and kinesin spindle protein (KSP) up to a dose level of 0.7mg/kg. The importance of the new data is that the trial has now reached a dose of 1.25mg/kg, a dose that based on pre-clinical studies for VSP02 is predicted to start to yield meaningful and widely distributed gene knockdown in the liver (compare ASCO pharmacokinetic data with AACR 2009 animal data). However, for VSP02 to reach a dose of at least 1.5mg/kg would be even better.

The ASCO data included preliminary data from 19 patients and strongly suggested that VSP02 has anti-angiogenic activity as measured by reduced blood-flow in the tumors and as might be expected from reduced VEGF activity. This response, however, was not dose proportional which could be due to small numbers. As to safety in this quite sick and heavily pre-treated patient population as of ASCO, the one patient death was remarkable, an event that was deemed possibly due to study drug. Since this patient had an extensive liver tumor burden it had been decided to exclude patients of comparable or worse cancer burden for the remainder of the studies.

Achieved dose bodes well for the entire LNP pipeline

The good news is that the 0.7mg/kg dose has been overcome and that there have been no dose-related elevations in liver enzymes, the expected dose-limiting toxicity for most LNP applications. If we assume that such general liver toxicity and potency of LNPs are independent parameters, in the case of the ionizable LNPs I believe a valid assumption, then the fact that 1.25mg/kg has been achieved in this challenging patient population, challenging especially in terms of liver health, this should bode well not only for VSP02, but also for the other D-Lin-DMA-based LNP drug candidates currently in clinical trials or close (ALN-TTR01, TKM-PLK1, and TKM-EBOLA), and even more so for the 2^nd gen LNP formulations with up to 100x increased potency for liver gene knockdown.

Among the 9 patients recruited since ASCO, one patient experienced transient grade 3 thrombocytopenia at 1.25mg/kg, a dose-limiting toxicity that necessitated a confirmation of the safety of 1.25mg/kg. Again, given the nature of the patient population and the importance of the liver also for platelet function, I would not read too much into this adverse event. With 6 patients in this cohort having been initiated on the drug, it looks like 1.5mg/kg is realistic.

Some patients have now received quite a number of LNP administrations, up to 13 (up to 5 at ASCO), and 3 with stable disease have entered the extension phase of the study. A trend towards increased disease stabilization with dose was noted. While this is promising, I would again caution that we are dealing here with very limited number of patients in an uncontrolled trial, and also the fact that some of this analysis could be confounded by the fact that a slightly different patient population is now being recruited at the higher dosages following the 0.7mg/kg adverse event. No tumor responses were reported.

In summary, the update is very encouraging for the success of VSP02, and even more so for the entire LNP pipeline. The next major milestone will be the presentation of biopsy data, possibly at ASCO 2011, which should critically tell us whether the drug is doing what it’s supposed to do (RNAi cleavage assay; mono-aster formation; RNA/protein knockdown etc). Beyond that, it will be interesting to speculate on the nature of the next clinical studies. A combination of VSP02 with a microtubule-targeting chemotherapeutic could be promising with VSP02 functioning as a sensitizer, thereby opening up MT cancer drugs also for liver cancer. Combinations trials with DNA-damaging agents often used for liver cancer would also appear to be reasonable to investigate.

Next stop for the clinical data-flow: Results from the ongoing ALN-TTR01 studies- this time hopefully with knockdown data.

Update on Silence Therapeutics

Yesterday seemed to be RNAi Cancer Therapeutics Awareness Day, with Silence Therapeutics highlighting a publication related to their lead candidate Atu-027, also in phase I studies for solid cancers. The publication concerns the activity and potential mechanism of action of the PKN3-targeting lipoplex formulation in mouse models for (hematogenous) lung metastases.

I would categorize these studies as being consistent with the previous scientific reports by Silence Therapeutics that suggest pleiotropic mechanisms to be responsible for the observed pre-clinical activity of Atu-027. Importantly, the results further seem to indicate that we may not see a response in the form of existing tumor shrinkage in the phase I studies as Atu-027's main activity appears to be in preventing the spread and seeding of new metastases. Similar to ALN-VSP02 the maximum tolerated dose will be the primary focus of the phase I trial with Atu-027.

Shareholders, albeit apparently pleased by the publication of these studies, will be even more interested in a Calando or Alnylam-like clinical update for Atu-027. And even more than that (!), they will want to know whether a takeover offer is finally forthcoming. In any case, it may be advisable to become more concrete about short-term financial plans in order to avoid a situation like last year when Silence Therapeutics was forced to merge with Intradigm as it was running out of funds.

This includes reporting milestones it is currently receiving, such as from Quark. Alnylam reported a Q3 increase in InterfeRx revenues of about $2M which I speculate largely comprises the Quark payment related to Quark granting Novartis an option on their kidney drug candidate. I speculate that Silence received a similar amount from Quark.

Alnylam Green Shoots After Corporate Restructuring

The latest quarter must have been the best quarterly performance by Alnylam in the last 3 years. After going through a rough patch that culminated in Novartis not adopting a widely expected $100M platform license with the attendant lay-off of a third of its staff, the results and also tone of the conference call struck me as if the company had re-discovered its old enthusiasm for RNAi Therapeutics drug development and is intensely focused on creating value by providing additional clinical proof-of-concept data over the coming months.

There is reason to be cautiously optimistic about the upcoming clinical data. For one, dose escalation for the liver cancer candidate ALN-VSP02 seems to have progressed beyond the magic 1mg/kg mark and is still ongoing! First generation or not, the fact that patients have now received and tolerated this kind of dosage, and on a repeat basis, is major de-risking for all of Alnylam’s, but also Tekmira’s pipeline candidates which involve LNP delivery (almost all of them). Similarly encouraging, ALN-TTR01 dosing is also ongoing and with 3 centers in Europe recruiting now, results may not be that far off (28 patients anticipated in this phase I study).

Although the first generation LNPs appear to be well tolerated, Alnylam seems to be even more excited about a potential quantum leap in LNP delivery: LNPs containing the next-generation MC3 lipid which not only silence in the unprecedented single micro-gram per kg range for gene targets in the liver, but also appear to be very well tolerated in animals. The MC3 lipids are derived from the DLin-KC2-DMA ionizable lipids developed by Tekmira, results from which were published earlier this year in Nature Biotech.

Besides the science, financials seem to be in better-than-expected shape, and I believe is what underlies today’s jump in share price (you can assume that the stock market wouldn't pay much attention to MC3). With over $370M cash/equivalents on the balance sheet, Alnylam is poised to close the year with well over the previously guided $325M cash/equivalents. Having slashed the work force, albeit probably at considerable personal cost, thus buys the company strategically valuable breathing room. In addition, there were a number of positive one-offs this quarter, including $2M for the successful award of 8 federal grants, a Quark milestone, and increased activities related to Novartis finalizing its 31 target picks. And with Novartis' relationship with Alnylam settled now, it should become easier to talk to other companies about partnerships and further add to the strong financial position.

Alnylam and Medtronic also stand to receive very significant funding from the Huntington’s Disease Foundation (CHDI), likely totaling over $10M, some of which has already been earned (agreement is retro-active). Somewhat dampening the excitement around that program was news that ALN-HTT is now a 2012 goal for entering the clinic, while LNP-delivered ALN-TTR02 and PCSK9 (both probably MC3) are poised to be the Company’s 2011 new clinical candidates.

An interesting tidbit on PCSK9 is that, faced with numerous competition from mainly monoclonal antibody candidates against the same target, some of which have already entered the clinic, it appears to be now Alnylam’s strategy to emphasize the fact that with RNAi you down-regulate both intra- and extracellular PCSK9 levels, which therefore is closest to recapitulating human genetics which has been driving the adoption of PCSK9 as a drug target for hypercholesterolemia in the first place. A good argument indeed.

Overall, my most important take-home was that this seems to be a re-energized company, and with some of the right clinical news, there may soon be a second honeymoon between Alnylam and its shareholders (of whom I’m btw not one of…yet).

Big Pharma Interest in RNAi Therapeutics Often Poor Indicator of the Science

The current perception is that large pharmaceutical companies have become quite a bit more conservative in their approach towards RNAi Therapeutics. This stands in stark contrast with only 3 years ago when some of the same companies topped each other in their efforts to securing a piece of the RNAi Therapeutics action. Clearly, given that the development of any new class of drugs is a gradual process and facts do not change as fast, Big Pharma must have been very wrong not too long ago, or it is now. So today I try to put Big Pharma’s mood swings into the perspective of the big picture progress in the underlying science.

In brief, while some players in the RNAi Therapeutics sector are partly responsible for the current Big Pharma RNAi conservatism, a lot can be explained by the herd mentality prevalent in Big Pharma where the actions of a fellow company rather than the primary scientific data guide the decision making, since standing up for your beliefs and out from the crowd has rarely proven to be good for climbing the corporate ladder. At the moment, the overpowering mantra in Big Pharma from which RNAi suffers from as being considered too early to know is that, with the exception of diagnostics, investments in R&D and especially innovative technologies generate deficits. This is not helped by the fact that the healthcare sector does face a few economic uncertainties leading to a state of paralysis where RNAi clinical development is put on hold and investments in RNAi technology development are reduced to rather mundane pharmacological assay development projects instead of real enabling technology development.

The 2006-2008 gold rush

About 4 years ago, Merck set on a collision course with Alnylam and bought rival Sirna Therapeutics for more than a billion US dollars. Now, RNAi Therapeutics was firmly on the radar of Big Pharma with Roche firing the next volley through a $300M+ platform deal with Alnylam the following July, the same month that Silence entered into a relatively broad RNAi development deal with AstraZeneca. Heightening the excitement was the Nobel Prize to Fire and Mello later that year for having discovered, only a decade earlier, that it is double-stranded, not single-stranded antisense RNA that triggers highly potent homology-dependent, post-transcriptional gene silencing.

Consequently, and despite the cracks in the economy that were starting to surface then, Takeda spent $150M for limited access to Alnylam’s IP estate, to at least secure an RNAi leadership position among its fellow Japanese pharma companies. All this left Pfizer scrambling not to be left behind in RNAi. Pfizer made the unorthodox decision to acquire Coley Pharma, which was working on TLR therapeutics and with which Pfizer had a collaboration, and use their oligonucleotide therapeutics expertise to form the basis for Pfizer’s RNAi platform effort. In addition, Pfizer also licensed a ddRNAi Therapeutics candidate for HepC from Tacere. The price tag: $164M for Coley alone. Despite all these investments, Pfizer has not formulated an outwardly cogent RNAi Therapeutics strategy, with no significant access to leading delivery technologies (this after having lost Mirus to Roche in 2008) and RNAi trigger IP.

Now, do I believe that Big Pharma overpaid for RNAi Therapeutics in that period? If you consider how RNAi has already revolutionized biomedical research and feel, as I do, that it also has the potential to do the same as a therapeutic platform, then the multi-million dollar deals should not come unexpected. I do believe, however, that some of these investments certainly did not find the right targets, the Sirna Therapeutics acquisition probably being the most egregious example. In addition, when it came to delivery, Big Pharma largely behaved penny-wise, pound-foolish, treating it almost the necessary evil of RNAi Therapeutics, or worse, ignoring it altogether. Why for example would anybody want to spend over a billion dollars for essentially RNAi triggers only, when delivery had only just reached the non-human primate stage (Alnylam-Tekmira 2006 Nature SNALP/LNP paper)? And even for LNP delivery then, scale-up, immune stimulation and the ability to repeat administer were still very much in doubt. Like building an aircraft and forgetting that you need fuel to fly it.

So RNAi Therapeutics investments at that time had still to be regarded visionary investments that could pay off hugely, driven by the belief that humanity would not fail to exploit such an elegant natural gene-regulatory pathway for therapeutic purposes, and I am convinced that Roche and Merck conducted some careful analysis of whether the attributes of RNAi Therapeutics would fit into the pharmaceutical business model of the future (‘personalized medicines’). Nevertheless, the actual trigger for the nature and timing of these investments in many cases must have been some mild panic of maybe missing the RNAi Therapeutics train about to leave the station, a technology that may have come around just in time to help the industry through the worst of the patent cliff that it was just starting to face. And if Merck invests $1.1B in the technology, maybe they know something we don’t know?

The Ripple Effects of the Dark Days of 2008-9 Still Being Felt Today

The worst financial crisis in decades was made worse still for the industry as it became clear that some of the early results that may have led Merck to believe that RNAi was quite close to reality, particularly in the antiviral, wet AMD, and cancer areas, were indeed too good to be true. Innate immune stimulation reared its ugly head, and soon every in vivo efficacy result was assumed to be an immunostimulatory artefact. Could innate immune stimulation be the fatal fundamental flaw of RNAi Therapeutics?

Here, the industry proved resilient and some high-quality studies came out that showed that in vivo efficacy can be achieved in the absence of immune stimulation and rules how to avoid them emerged. I would like to highlight here the efforts by Tekmira which in many ways have proven to be the forward-looking savior of the industry a) by developing the most advanced systemic delivery technology, and b) for having addressed immune stimulation almost as soon as they entered the field. In early 2009, Silence Therapeutics also provided high-quality pre-clinical proof-of-concept for non-immunostimulatory RNAi for cancer in various animal models. This was nice also because this validation occurred outside the Alnylam-Tekmira space.

Still, Big Pharma interest in RNAi Therapeutics as a platform hit a low. Merck-Sirna Therapeutics strangely made it their PR policy to question the platform, Roche after their merger with Genentech became noticeably more cautious about RNAi Therapeutics (also probably due to a change in personnel), and Pfizer just last week said that maybe, although we still have to test it, antisense is great after all? Contrary possibly to Pfizer, I had always believed in investing in drugs for diseases where treatment decisions are not influenced by whether having to go for a half-hour infusion every 2-4 weeks is sufficiently convenient to patients. With all due respect, I don’t understand a number of comments that were published in an interview on Pfizer’s RNAi efforts last week.

Considering the publications and conference abstracts from Big Pharma, one may speculate that Big Pharma’s PR strategy for RNAi Therapeutics may be considerably informed by lack of access not only to IP, but especially enabling delivery technologies. Lack of access not because such IP and technologies don’t exist, but because they cost something. Moreover, RNAi champions within these organizations are likely frustrated by being held on a short corporate leash due to the general economic uncertainties of the pharmaceutical industry and the fundamental loss of Big Pharma’s confidence in the power of innovation. This means that Big Pharma’s internal efforts in RNAi Therapeutics are largely limited to more mundane pharmacologic assay development, which albeit certainly useful, cannot substitute for investments in delivery technologies with essentially all the innovative, ground-breaking work happening outside their walls.

This situation is not helped by the fact that the high-ranking decision-makers are typically too busy to read the scientific literature to properly inform their own opinion and instead rely on the conventional wisdom which at the moment says that RNAi has disappointed as a therapeutic modality and now it needs to prove that it is more than just a useful laboratory tool. And it does not matter whether the current scientific literature has well moved past this existential angst phase.

I know that this is a rather scathing critique of Big Pharma’s RNAi Therapeutics philosophy, one driven by herd instinct and PR rather than an open-minded assessment of the latest primary data. There must be many scientists in Big Pharma, too, that are frustrated by the constraints and lack of scientific leadership in those companies. In a way, I sometimes feel sorry for the criticism that Merck gets for its $1.1B purchase of Sirna Therapeutics. Others are now well aware of the consequences of sticking out from the crowd as RNAi visionaries.

It is now up to the industry to carefully manage its way through this funding desert and, over the next 12 months provide a series of human proof-of-concept data with Alnylam’s ALN-TTR01 and ALN-VSP02 coming up first, then followed by Silence Therapeutics’ Atu-027 phase I results in H2 2011. In addition, there should much to be gained for the negotiating position of pure-play RNAi Therapeutics companies by aligning some of the fundamental IP and pushing back efforts by Big Pharma to talk down the price of RNAi.

(Very) Broad Zamore End-Stability Patent Issued in Europe

Last week, Silence Therapeutics announced that the European Patent Office has granted a patent from the Zamore RNAi trigger design IP estate (EP 1633890 B1). This follows the issuance of related patents over the summer in the US. This IP is assigned to the University of Massachusetts and exclusively licensed to Silence Therapeutics.

What is newsworthy in this latest patent issuance is that very broad claims were allowed which would almost require a company with RNAi Therapeutics platform ambitions to take a license. As I have discussed here before, Zamore made the highly influential finding that it is both the absolute and relative base-pairing strength (relative to the base-pairing strength on the other end of an siRNA duplex) at the 5’ end of the guide strand that determines its RNAi effector complex (RISC) incorporation as well as discourages passenger strand incorporation. Accordingly, the rules have implications for both efficacy and specificity of RNAi gene silencing. It has to be assumed that the end-stability rule figures in one form or another into the siRNA design algorithms used by companies as part of the siRNA screening process, and it should also be an important guiding principle in optimizing an initial candidate siRNA.

A strong patent, of course, does not necessarily follow such fundamental biological insights. In this instance, it could well turn out to be the case. The US claims cover methods focussed on the reduction of off-targeting effect, including first assessing the off-targeting of a first siRNA, and then changing it according to the end-stability rules. As the recent Merck paper illustrates, companies in the field undertake such modification-RISC incorporation studies. Since a given siRNA structure can theoretically be arrived at via a number of different routes, such methods papers are more difficult to enforce. In addition, the direct value of the US claims may somewhat affected as they emphasized the reduction of off-targeting aspect of the design rules rather than the enhanced efficacy aspect which might be considered the more attractive feature of the invention.

What is therefore different in the European patent issuance is that not only does it emphasize the efficacy aspect, but it also importantly includes very broad composition of matter claims relating to the structure of an siRNA. It should be very straight forward to enforce these.

The breadth of the claims is striking: siRNAs with small features already that lessen the base pairing at the 5’ end of a guide RNA are covered in these claims. This can be a mismatched base pair, relatively widely employed for example at the very 5’ end of the guide RNA, or a single nucleotide modification. One of the methods claims even covers siRNAs solely characterized by having fewer G:C base-pairs at the guide strand 5’ end compared to the 3’ end. I would expect many if not most siRNAs to fall into that bucket.

In a phone conversation last night with Phil Haworth, the CEO of Silence Therapeutics said that Silence Therapeutics are naturally excited of having been granted these broad claims. When asked, he added that similar efficacy and composition of matter claims derived from the original Zamore patent application are also being considered in the US (note: due to a restriction requirement, the off-target reduction elements of the invention were initially pursued in the US and the efficacy aspects put on the back-burner). I also agree with him that given the strength of the claims and because this is a European patent prosecution, competing RNAi Therapeutics companies can be expected to challenge the validity of the patent. This should also be a good indicator whether Alnylam really meant what it said when it stated that it saw nothing of value in the Zamore siRNA design IP estate.

Given the importance of the siRNA end-stability rules and broad-ening claims, will we therefore see Silence Therapeutics soon swim in cash? Here, Phil Haworth was a bit more cautious and said that Silence’s RNAi trigger IP estate would be just one element in the discussions they are having right now with pharmaceutical companies.

As you will be aware, Silence Therapeutics has been ‘approached’ by a company a few weeks ago, an approach that could lead to an offer, and Dr. Haworth confirmed that these discussions were still ongoing. Without going into any more details, he also said that they are conducting a number of platform partnership talks in parallel and that the ‘approach’ and platform conversations would be separate discussions.

Phil Haworth did not disagree when I speculated on the potential strategic value of the Zamore end-stability IP to particularly Merck, given the one billion dollar+ Merck spent on Sirna Therapeutics for access to the 3’ overhang IP which it now stands to lose (see coverage on the 'RNAi Litigation Blog'). He emphasized, however, that the company does not spent much time speculating internally what other companies might be scheming and instead focus their limited resources on building strong science and IP. In the end, the value of the Zamore siRNA design rules will be closely tied to advancements in the delivery of RNAi triggers and in that regard they are pleased with the continued dose escalation of Silence’s first clinical candidate Atu-027 (6^th of planned 11 dose cohorts ongoing).

2 Short Stories: An siRNA Delivery Paper by Merck, Pharmaceutical Interest in Silence’s Gene Target

The most enjoyable part in following RNAi Therapeutics is to look at the rich stream of scientific data and determine the absolute maturity and competitive position of the technologies and companies involved, as well as getting a glimpse at relationship dynamics. I therefore thought to share today two examples of this that I picked up recently. One is a paper by Sirna Therapeutics/Merck shedding some light on their approach towards RNAi pharmacology and RNAi trigger design. The other is some intriguing evidence that Silence Therapeutics’ most important gene target, PKN3, is gaining traction in the pharmaceutical space.

Studying the pharmacology of siRNA delivery

Pei and colleagues from Merck published in RNA a nice paper on better understanding the pharmacology of siRNA delivery [Pei et al. (2010). Quantitative evaluation of siRNA delivery in vivo]. Unlike small molecules or even antibodies, the pharmacology of RNAi Therapeutics is more complex as simply measuring the raw tissue abundance of an RNAi trigger is a poor indicator of successful RNAi delivery. This is because functionally inactive siRNAs may vastly outnumber the active siRNAs loaded into the mammalian Argonaute 2 protein (Ago2), the nuclease responsible for seeking out and destroying complementary target messenger RNAs.

Not surprisingly, the Merck researchers employed the LNP/SNALP delivery technology in rodents and monkeys as their system of choice. After intravenous delivery of these LNPs, siRNA abundance was determined by quantitative PCR both at the tissue (mainly liver) and Ago2 level.

For the LNP aficionados among you, the 1mg/kg ED50 lipid nanoparticle used in this study still involved the CLinDMA lipid that was shown previously by Merck to be associated with immunostimulation (Abrams et al. 2010), something that Tekmira has interpreted as being the result of the strong positive charge of such LNPs.

Although playing too many number games carries the risk of missing biology sometimes, a number of quite interesting findings were made. One is that the vast amount of siRNA in the liver (>99%) is lost in the first 24 hours upon which a slower tissue elimination phase sets in that is apparently dominated by the turnover of guide strand incorporated in Argonaute.

It is generally thought that the longevity of gene silencing often seen in vivo, often on the order of 1-2 months following a single administration, is due to the stability of this complex. Despite that, there was still an approximately 3-5 fold decrease in the abundance of such complexes over a week. Not too fast, but fast enough to make it worthwhile studying in more detail whether the stability of these complexes is limited by Argonaute protein half-life or by a selective removal of the guide strand. If the latter, siRNA structure-chemistry may be able to increase silencing duration still. Such studies should also shed light on what pharmacological advantages siRNA depots might have which could be of particular interest to ocular and oncology applications.

Merck employed Zamore rule in siRNA design

The paper also allowed for some interesting insights into the siRNA trigger design process employed by Sirna/Merck. Supporting the importance of the Zamore end-stability patent recently issued in the US and exclusively licensed to Silence Therapeutics (Intradigm) from UMass [note: this corrects an earlier version that improperly stated the IP had been assigned to Silence], the authors first determined the relative Ago2 incorporation efficiencies of guide and passenger strands and then studied how this was changed following chemical modification of the same sequence. As a reminder, achieving a high ratio of guide to passenger strand in RiSC is widely considered to be beneficial both for reducing passenger strand-mediated off-targeting as well as enhancing siRNA efficacy.

Indeed, the authors find that chemical modification changed (in this case enhanced) guide strand incorporation over passenger strand incorporation. However, the authors argued that this was not due to the application of the Zamore rules, but due to having added inverted caps to the ends of the passenger strand. I agree that since the 5’-modification of the guide strand plays a major role in Argonaute loading, these caps, as also employed e.g. by mdRNA, should have a considerable effect on loading the passenger strand. However, since the modified siRNA with which the comparison to the unmodified siRNA was undertaken contained additional modifications besides the caps, it is not possible to argue that it was only the caps that had the effect on differential loading. In fact, the differential strand loading efficacies of 2 modified siRNAs, distinguished only by the nature and position of backbone modifications, differed by a factor of 2, clearly indicating that siRNA modifications besides the cap have a major influence on differential strand loading.

Wyeth/Pfizer shows interest in PKN3 in cancer

I had always considered it the wrong strategy for Silence, even more so before their merger with Intradigm, to focus so much of the company’s resources on a single drug target: PKN3. One reason is that Silence claims to be an RNAi platform company, and resources would have been better spent on building on their early pioneering position in siRNA design which then seemed to be at the risk of getting stuck in an early 2000’s 'dead-end'.

Even more worrisome is that Silence was essentially the only group really working on the PKN3 gene, and at that early stage it is always a very real possibility that it might turn out to be a useless artifact of no commercial value. However, the scientists stuck to their guns and it now seems that additional data confirms PKN3 to be an interesting oncology target in the angiogenesis field with the rest of the pharmaceutical world slowly paying attention.

Curiously, it is research by Wyeth, now part of Pfizer (!), that confirms that PKN3 plays a role in endothelial biology and that it is upregulated in a number of cancers. Even more intriguing is the fact that one of the co-discoverers of PKN3 as a cancer drug target (Anke K.-G.) is also named as an inventor in a PKN3-related patent application by Pfizer published this year (WO 2010/105128 A2). This patent application is about methods of using PKN3-containing protein complexes for cancer diagnostic purposes, e.g. determining patients with high PKN3 levels which would be candidates for a PKN3-targeting drug just like Silence’s Atu-027 (this candidate has been reviewed here with Tobias Wolfram). A nice validation of Silence’s own results and demonstrating just how close Pfizer’s PKN3 science seems to be to that of Silence is that a number of experiments described in the patent application were based on the same rodent cancer models previously employed by Silence showing that PKN3 silencing leads to an inhibition of tumor growth in mice.

With the PKN3 gene patented by Silence as a cancer drug target, it would make sense for Pfizer to gain access to Silence’s IP and maybe even take on the clinical development of Atu-027 itself for which Pfizer could use their methods as a response biomarker. Maybe Silence’s belief in PKN3 will be financially rewarded after all, and it might also explain why Alnylam seems to be so keen in weakening Silence’s PKN3 patent estate. Until now, I had come to believe that the only purpose of fighting that patent estate was to frustrate Silence by engaging them in yet another patent skirmish. It is interesting to speculate that the strength of PKN3 science and IP could critically inform whether Pfizer will partner with Silence or Alnylam.

Tuschl Litigation Decidedly Shifting into Max Planck-Alnylam’s Favor

As often in life, involve money, and you will soon see who your true friends are. This rule also seems to apply to the fate of the Tuschl patent applications which more and more seem to go in Max Planck/Alnylam’s favor, and against the interests of UMass and Sirna Therapeutics/Merck, according to the latest coverage on the Tuschl Litigation Blog.

It has always been a mystery to me why Whitehead and the MIT would want to side with UMass in the first place, since as parties to the Therapeutic Use agreements between Max Planck, MIT, and Whitehead, Whitehead and MIT had nothing to gain, actually much more to lose, from UMass’ decision to go it alone and license the therapeutic rights to their part in the Tuschl invention to Sirna Therapeutics (now Merck) and to some degree also RXi Pharmaceuticals. Even more so given that Zamore's assignment of his interest in Tuschl-I to UMass is questionable in the first place.

It has equally been a mystery to me why Wolf Greenfield & Sacks, the patent law firm engaged by Whitehead to prosecute the Tuschl-I patent application on the behalf of Whitehead, Max Planck, MIT and UMass would seek to gain the benefit of inventive subject matter that obviously belonged to Max Planck only.

It is therefore no surprise then that MIT, Wolf Greenfield & Sacks, and finally The Whitehead have all decided after all that the legal exposure from the Tuschl Litigation does not make it worth to them any more to continue to support UMass' insistence on ownership over the use of 3’ overhangs in RNAi triggers and the discovery of efficient RNAi gene silencing in mammalian cells using short double-stranded RNAs by claiming the benefit of the '325 priority application filed by Max Planck in Europe.

Unless Zamore’s testimony will shock the field of RNAi, the testimonies of the 2 more impartial inventors named in Tuschl-I, namely Sharp and Bartel, make it clear that the 3’ overhang work was the accomplishment of Tuschl after he set up his lab at the Max Planck. This is also consistent with my view of RNAi history that is not only based on the publication history and authors on key papers, but has also been critically influenced by how the RNAi field in general has always felt about who was to be acknowledged for that body of work: Tuschl. Science is such a gossipy endeavor after all that I would have expected to have heard rumors if Tuschl wasn't the inventor of the 3’ overhung siRNA.

So now it seems like UMass is the last man standing, and unable to move forward with the Tuschl I patent application. The fact that UMass has not surrendered yet let’s me speculate that not only do they stand to lose future benefits under the Tuschl patents, but that they feel considerable pressure from Sirna Therapeutics-Merck. Understandably, given that the $1.1B value Merck placed on Sirna Therapeutics much depended on perceived access to the mammalian and 3’ overhang data. Should UMass never have been allowed to license IP which erroneously cross-referenced the patent application of another party? Or might the pressure be one day on former Sirna Therapeutics, many of who have left Sirna after the acquisition, should Merck believe that they have been misled?

A less conspirational explanation for this mess, of course, would be honest human mistakes, such as misunderstanding the rules for citing priority documents or scientists signing off on legal declarations that they do not have the time to read, much less fully understand. But with billions of dollars at stake, what started as honest mistakes may quickly become interpreted as, and actually also have led to ‘malpractice’ and ‘deceptive behavior’. Money.

RNAi Delivery to Vascular Endothelium Increasingly Validated

Less than a month after Napoleone Ferrara from Genentech was recognized with a 2010 Lasker Prize for identifying VEGF as the central actor in blood vessel formation, a prize widely regarded as the stepping stone towards the Nobel Prize in Physiology or Medicine, a press release by Alnylam seems to suggest that RNAi delivery to the vascular endothelium is appropriately reaching critical mass. Before that, the most important body of work in this area probably came from Silence Therapeutics, which, despite its apparent quality, got me a bit worried given what I perceived as a certain lack of enthusiasm in the commercial RNAi Therapeutics space and, more importantly, third party scientific validation.

It is particularly exciting that the silencing of endothelial gene markers following a single dose persisted for two months. Since the duration of silencing is critically dependent on the cell type, for example its proliferation rate, this bodes very well for all delivery technologies targeting the vascular endothelium.

Alnylam mentions that the new LNPs (liposomal nanoparticles) that work for endothelial siRNA delivery stem from their collaboration with the MIT which as we know has involved positively charged ‘lipidoids’. Seen in light of the data by Silence (cationic lipid-siRNA)/Intradigm (RGD-targeted PEI polymers), the picture that is emerging is that LNPs that comprise positively charged lipids have a natural propensity of being taken up by vascular endothelial cells. Although I haven’t seen the details yet, it is to be expected that a number of parameters beyond positive charge, such as the method of formulating the particles, e.g. SNALP-like siRNA encapsulation versus Atuplex-like lipoplex formation, determine the efficiency of the functional uptake of these particles.

Beyond Alnylam and Silence Therapeutics, which with Atu-027 already has an endothelial cell-targeting RNAi Therapeutics in the clinic, Tekmira should also have considerable expertise in this area. Similar to optimizing LNP delivery to the liver, leadership in endothelial RNAi will depend on first empirically determining the structure-function relationships of these particles and then the biological pathway by which the uptake occurs. To my knowledge, it is still unclear whether receptor-mediated uptake, non-specific macropinocytosis ('cell drinking'), or the limited capacity of endothelial cells for phagocytosis is involved. This will also be important to understand as more targeted technologies will be developed.

One trade-off that current technologies might suffer from is that their positive charge at physiological pH may cause them to be slightly more toxic compared to negatively or neutrally charged formulations. This may also tie in with Phil Haworth’s comments in my last interview with him that Silence Therapeutics will initially focus on acute indications with high unmet needs. But again, everything is toxic at sufficiently high concentrations and it is encouraging that Atu-027 is still in its dose-escalating phase according to the last clinical update by the company.

Progress also reported for systemic RNAi delivery to immune cells

Following up on their declared pursuit of vaccine opportunities, Alnylam also highlighted progress in the systemic delivery of siRNAs to immune cells. Previously, LNPs comprising lipids derived from the KC2 series of next-generation ionizable lipids had been described to silence the immune cell marker CD45 with an ED50 of approximately 1mg/kg following intravenous administration (see RNAi delivery roundtable). The new report suggests some improvement over those formulations, stating that a 95% knockdown of CD45 was achieved, with an ED50 of as little as 0.2mg/kg. It will be very interesting to see the scientific details and discussions on these experiments (note: this last paragraph was corrected from an earlier version that mistakenly stated that the 95% knockdown was achieved with 0.2mg/kg).

Uptake of RNAi triggers by immune cells per se is not entirely new. It is a case of turning lemons into lemonades, as the non-specific uptake of nanoparticles by phagocytic cells has been long lamented. However, these cells play a central role in at least as many important diseases as endothelial cells do- so why not harness the efficient uptake of particulates in those cells for therapeutic purposes? Sure, being taken up and being functionally released into the cytoplasm are 2 separate issues, but Tekmira’s Ebola work has shown already that LNPs can trigger gene silencing in macrophages. Again, it will be important to delineate the functional uptake pathways and to employ chemistry to increase the efficiency with which LNPs can harness them.

Once thought of as liver-only formulations, LNPs are showing more and more their considerable versatility. I remember well a talk given by Tekmira’s CSO Ian MacLachlan at Stanford two years ago when he demonstrated that by even only slightly changing the formulation parameters, strikingly different patterns of biodistribution can be achieved. Now it ‘only’ takes one of the LNP/SNALP programs in the clinic to show some efficacy, and the pharmaceutical industry to change its approach towards drug target selection, for the liposome to finally shed its image as the unloved, but necessary stepchild of the industry.