Voyager Therapeutics Sets Out to Fulfill Promise of AAV-directed RNAi Therapeutics (and More)

Finally!  Long overdue (because so obvious and compelling), with the founding of Voyager Therapeutics, the foundations have now been laid for a strong AAV (adeno-associated virus) platform gene therapy company, including DNA-directed RNAi Therapeutics.   Taking advantage of new AAV shuffling approaches to identify novel serotypes (Gao and Kay labs) to more efficiently and specifically target cell types of interest and the world-class expertise in RNAi trigger design of  two of its scientific co-founders (Kay and Zamore labs), the new company will make use of the best available science to realize the potential of AAV and ddRNAi technology for the many diseases of high unmet medical need in the CNS (e.g. Huntington's disease).

With $45M in start-up funding from Third Rock Ventures (the VC that has started the comparable lentiviral-based gene therapy company which recently went public, bluebird bio), they will deepen that expertise through in-house research and thereby sail by Benitec which sadly has refused to face the fact that in order to be a platform company and stay relevant you need to have such efforts.  If not at a time like this when interest is high, then when??  The latest publication by Benitec and Pfizer (Denise et al. 2014) was ironically the best illustration of that omission by showing that the first-generation ddRNAi expression technology of Benitec generates a plethora of small RNAs which obviously is less than ideal.  By contrast, work in the Kay lab in particular (e.g. Gu et al. 2012) has shown how to generate much cleaner expression cassettes.

Voyager strutting its RNAi expertise and related therapeutic development plans is further confirmation of the dramatic decrease in the value of Benitec's once-prominent IP position in ddRNAi (Graham patent).  With an expiration of ~2018-2020 and Baulcombe the real gate-keeper (although the clock on that one is also rapidly running down), I don’t see why there should be a need to take a license except for maybe the likes of Calimmune which may have something close to commercialization by then should things proceed without a hitch.

The start of a company like Voyager is always very exciting and I trust that the scientific founders have the expertise and wisdom to guide it along the way.  It is also a good time to acknowledge the likes of Dirk Grimm (Heidelberg), Shuo Gu (NCI), and most recently Leszek Lisowski (Salk) which I had the privilege of working next to in the Kay lab when they were critically contributing to the technology underlying Voyager.  The pharmaceutical industry and indeed the investor community is wasting a lot of potential by not tapping into the skills of such driven scientists just because they may not labor in one of the biotech hotbeds.

Mr. Anonymous Thwarted in Zamore US Patent Re-exam

Silence Therapeutics reported that a number of valuable RNAi trigger patents related to the Zamore Design Rules that were issued last year in the US were upheld following a re-examination request by an anonymous 3^rd party. Even stronger claims related to the same Zamore patent series have been issued in Europe, and unsurprisingly Alnylam, but also Novartis and Alcon are opposing them with the outcome to be decided (EP 1633890 B1). However, given the importance of both the US and Europe in the commercialization of innovative drugs, having a patent position in one jurisdiction alone can be considered a valuable strategic asset already.

The Zamore Design Rule patents are owned by the University of Massachusetts and exclusively licensed to Silence Therapeutics for medical uses. As described in more detail in other blog entries before (here and here), they cover methods of promoting the incorporation of the desired guide strand into the RISC gene silencing complex as well as of enhancing RISC turnover resulting in more effective and selective RNAi knockdown.

Especially the patent covering guide strand selectivity methods (‘thermodynamic end-stability rule’; US 7,750,144) is widely recognized in the art for greatly increasing the likelihood of finding efficacious RNAi triggers and is incorporated in essentially all bioinformatic sequence pre-selection algorithms. It has to be said though that the claims do not cover the entire spectrum of approaches of achieving differential end-stability. They do, however, cover chemical and structural approaches that have been reported by a few companies such as Marina Biotech and Sirna/Merck before. Moreover, because the coverage involves modified nucleotides, there will be the concern that even if such modifications were applied for other purposes (e.g. stability or immune abrogation), they may fall under the patents. Consequently, a company with a promising late-stage candidate may want to take a license instead of taking a chance in an infringement lawsuit.

This makes it the second time within a week (see PKN3 opposition by Alnylam) that important patents by Silence Therapeutics were upheld essentially unchanged following challenges. Alnylam’s management once laughed off the value of the Zamore patents in a conference call following their issuance last year. They obviously considered them serious enough to oppose them in Europe and Alnylam is certainly the most likely identity behind Mr. Anonymous (have your say by participating in the survey on the right). Less likely, but not entirely out of the question would be a Big Pharma company like Novartis which is considering taking a license to the Zamore patents, but first wanted to kick the tires before it did so (patents that have been unsuccessfully challenged are considered stronger).

With all these patent successes, and at least one more likely to come, Silence Therapeutics need to monetize their assets in the form of non-dilutive funding.

Hemophilia Gene Therapy Success Bodes well for ddRNAi Therapeutics

Following years of public scorn and derision, including by Alnylam which in 2006 waved off ddRNAi Therapeutics and gene therapy as ‘dangerous’, gene therapy is back with a vengeance. This week, a consortium of researchers reported in the New England Journal of Medicine that an self-complementary AAV8-delivered Factor IX transgene was able to significantly correct hemophilia B in a small clinical trial.

4 out of the 6 patients treated were able to largely discontinue the standard frequent (often 2-3 times a week) and expensive use of prophylactic recombinant FactorIX protein therapy which accounts for estimated healthcare costs north of $20M a lifetime. This means that even in the limited duration of the trial (6-16 months of follow-up), the low cost of the gene therapy ($30k cost of goods for a treatment that is expected to last many years if not a lifetime) meant that this therapy is already saving money (and improving quality of life). If the results can be confirmed in a larger trial and the side-effects, including transient liver enzyme elevations can be controlled with similar efficiency as in this small one, approval may not be that far away.

The hemophilia results also bode well for ddRNAi Therapeutics. First of all, the study has validated the safety and efficacy of delivering the highly promising AAV vector family by peripheral vein infusion to the liver. This represents progress over a previous hemophilia gene therapy study which employed considerably more invasive direct hepatic infusion. In particular, it is likely that, should the ddRNAi collaboration by Tacere/Pfizer for HepC continue, it would involve not only the same vector family (AAV) and target organ (liver), but also the same serotype (AAV8) and self-complementary genome strategy as employed in this hemophilia trial.

(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).

Zamore Design Rules not Essential, but Highly Desirable and Strategically Important

As Silence Therapeutics is being issued one patent after another from the Zamore siRNA design rule patent families in the US, and possibly also soon in Europe, there has been some confusion about their value, both scientific and strategic. Silence claims that they are highly valuable additions to the RNAi Therapeutics toolbox; Alnylam dismisses them as something they chose to pass on as worthless. This controversy is part of the general debate of how easy it is to work around Alnylam’s historically strong IP position in RNAi triggers at a time that the Tuschl Tussle and a hearing on Tuschl II in Europe in early December are set to provide much-anticipated clarification of the RNAi trigger IP playing field.

As I have detailed before, I personally believe that while one of the Zamore patent families related to enhancing RISC turnover (mismatches between the 3’ end of guide and target mRNA) is quite useful and probably still widely underappreciated, it is the differential end-stability patent family that should be of Alnylam’s concern. Sure, going forward, you can easily get around it by simply not using the covered methods, but ignoring one of the most impactful siRNA design rules would be very much at the considerable expense of the siRNA discovery efficiency. Also, there is a good likelihood that some of the current RNAi clinical candidates have employed these rules and would require licenses at some point. Ask a handful of RNAi molecular biologists and I bet 4 or 5 out of 5 will attest you the importance of the end-stability rules which are quite comprehensively covered by the issued claims. Here is an exemplary main claim:

1. A method of producing a dsRNAi agent having decreased off-target silencing activity, the method comprising:

(a) identifying an off-target silencing activity mediated by a sense strand of a first dsRNAi agent, wherein the first dsRNAi agent directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA; and

(b) synthesizing a substituted dsRNAi agent comprising one or more substituted base pairs with respect to the first dsRNAi agent, wherein the substituted dsRNAi agent comprises a sense and an antisense strand, each strand having a 5′ end and a 3′ end, wherein the substituted dsRNAi agent directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′ end of the antisense strand (AS 5′) and the 3′ end of the sense strand (S 3′) and are selected from the group consisting of a mismatched base pair, a wobble base pair, a base pair comprising a rare nucleotide and a base pair comprising a base-modified nucleotide, such that the sense strand of the substituted dsRNAi agent is less effective than the sense strand of the first dsRNAi agent at entering the RISC complex;

You will see that it is a claim on a method detailing specific steps taken to increase the specificity of an siRNA, and in order to enforce it you would probably need to have insight into the development history of individual RNAi Therapeutic candidates. Since I was therefore curious as to how Silence thinks it may enforce such claims, I recently spoke about it to the CEO of Silence Therapeutics, Phil Haworth, on the phone.

Dr. Haworth stated that Silence’s current strategy was to raise awareness of the Zamore patents and have companies decide for themselves whether they ought to get a license to it or not, implying that getting a license now would be cheaper than waiting until products are close to commercialization. He agreed that it may be impossible to prove without doubt that the Zamore rules had been employed without direct insight into the RNAi trigger lead development of the various companies. He added, however, that after Silence Therapeutics and Intradigm merged earlier this year, they found that Silence Therapeutics would indeed have infringed the Zamore design rules. Although only an n=1, this is consistent with the notion that particularly the end-stability rules find wide application in the industry.

I also asked whether we may see composition of matter patents issued covering the end-stability rules, particularly since they may be more straightforward to enforce. For reasons that are not fully clear to me without studying the status of the entire patent family in detail, Dr. Haworth wished to leave this an open question. So it’s possible, I guess, and Silence may want to retain the thunder for any such issuance.

Whatever the case, I feel Silence has a strong case that it is likely that Big Pharma will license some of the Zamore rules on a non-exclusive basis, either broadly for companies interested in RNAi Therapeutics as a platform (Merck, Roche, Novartis probably highest on the list) or on a case-by-case basis.

Alnylam may be in a more tricky situation. Although they would probably also be invited to take a license, such rights are unlikely to come together with rights to sub-license the Zamore rules to Alnylam platform licensees. As such, these potential partners may now feel that even if they pay for a RNAi trigger license from Alnylam, they still would not have all the desirable siRNA design tools at their disposal. Silence, of course, is in a somewhat similar situation, namely that while they can probably work around Alnylam’s RNAi triggers, particularly for blunt-ended dsRNAs 22 base-pairs and longer with some related composition of matter patents to boot ('AtuRNAi'), potential partners may not want to sacrifice on the use of 3’ overhangs that, although dependent on the Tuschl litigation, are likely to be controlled by Alnylam in the future.

Alnylam is arguably in the better position. It could just continue and bury the $20M market cap Silence Therapeutics under patent litigation costs and diverted management attention. One example that struck me here particularly was Alnylam’s opposition to Silence’s PKN3 patent which I feel is built on solid data and given the 20000 or so genes available, Alnylam should have no reason to oppose except to hold up Silence’s efforts with IP costs. On the other hand, as its investors grow impatient about long-promised deals, Alnylam does not have the luxury of there being increased uncertainties not only on its gate-keeping position, but also whether the scope of its freedom-to-operate is scientifically satisfactory. There is, of course, also the distinct possibility that Merck may see Zamore a relatively cheap way to gain leverage over Alnylam and may therefore choose to be Silence’s white knight. With Silence at a $20M market cap and Alnylam having $400M in cash waiting to be invested in RNAi Therapeutics instead of litigation, you’d think Zamore would be a good investment for Alnylam to make.

Studies on siRNA Stability to Increase Adoption of RNAi Therapeutics

A study by the Zamore group published in Science (Ameres et al.: Target RNA-directed Trimming and Tailing of Small Silencing RNAs) provides intriguing insights into an important mechanism impacting siRNA stability by showing that guide strands that are perfectly complementary to their target mRNAs are subject to tailing and subsequent degradation. By better understanding siRNA stability, such work has the potential to decrease the frequency and dose with which synthetic siRNAs have to be administered, thus improving the overall clinical and commercial profile of this class of RNAi Therapeutics.

Actually, siRNA Therapeutics turned out to have a better pharmacokinetic profile than I had thought when I first started to work with siRNAs in late 2002. Most experiments in these early days for mammalian RNAi were conducted in rapidly dividing tissue culture systems where, somewhat depending on the natural turnover of the target mRNA/protein, silencing would be maximal between day 1-3 after siRNA administration and then quickly subside by ~day 5. Luckily, it was then found that it is cell division that is responsible for the relatively rapidly diminishing silencing effect observed in many tissue culture systems by diluting the siRNA-loaded RISC silencing complex between daughter cells. By contrast, silencing in non-dividing tissues, particularly in whole organisms typically persists for about 2-4 weeks after single administration- and on top of it is btw also typically more potent there on a per molecule basis as well!

Especially since many of the first batch of RNAi Therapeutics are likely to be intravenously and intravitreously (needle injection into eye) administered, the longer the interval between drug administrations, the broader the adoption of the technology should be, particularly for non-lethal, chronic diseases. 2-4 weeks is at the lower-to-mid-range for what is practiced for many monoclonal antibodies (subQ and IV) and in my opinion quite acceptable for most diseases where it is worth going to seek treatment for. On the other hand, extending this to something like 6-10 weeks, could in some cases quite significantly impact the adoption of RNAi Therapeutics, especially in situations where the administration procedure itself is associated with cumulative risk e.g. as is the case for the intravitreous route. This realization should also be the motivation for the recently formed collaboration between ophthalmic drug company Surmodics and Egen for ocular controlled-release siRNA formulations.

What Ameres and colleagues show in their paper is that small RNAs such as microRNAs and siRNAs are destabilized in the presence of target mRNAs with perfect complementarity, especially with regard to the 3’ end of the guide strand. For reasons that remain to be determined, the cell somehow recognizes this configuration triggering a polymerase to add a few nucleotides to the guide strand 3’ end which in turn is recruits the ubiquitous RNA degradation complex, the Exosome.

This finding has direct practical implications for the design of siRNAs. Because RNAi gene silencing molecules tolerate mismatches towards their target mRNAs without losing efficacy, especially at the 3’ end, it should be possible to increase siRNA stability by introducing such mismatches. Incidentally, such mismatches should have the added benefit of increasing RISC turnover rates, a finding made by the same group earlier and covered by the first Zamore siRNA design rule patent issued in the US a few months ago and exclusively licensed to Silence Therapeutics.

It is, however, also likely that instead of introducing 3’ mismatches, (blocking) nucleotide modifications at the guide RNA 3’ end would have the same effect by not being suitable substrates for the polymerase.

It will now be important to test how these findings translate to silencing in large animals and develop in vitro assay systems that faithfully recapitulate such small RNA destabilization. If the stability of the guide strand within the RISC complex is indeed a major determinant of the gene silencing longevity in mammals (rather than stability of Argonautes e.g., in which case slow-release strategies would gain in relative attractiveness), then this line of work should have significant potential for improving the eventual clinical and commercial profile of RNAi Therapeutics.

Silence Therapeutics Issued Important Zamore siRNA Design Rule Patent

Silence Therapeutics announced yesterday the issuance of a United States patent related to the design of gene silencing siRNAs. This is the second such patent issuance in little more than a month for which Intradigm, Silence’s merger partner earlier this year, had exclusively licensed from the University of Massachusetts and that is based on fundamental work by the Zamore lab on the enzymology of RNAi. These and other recent patent issuances related to sequence/gene-specific siRNAs address some of my concerns that I had with regard to Silence’s RNAi trigger strategy that I felt was at the risk of being too narrow and, as a consequence, becoming outdated. They should complement Old Silence's lipoplex delivery technology for vascular endothelia with valuable siRNAs in the fields of oncology and retinal disorders. Together, these developments provide evidence that the merger is starting to prove synergistic, with Silence providing the technical know-how and Intradigm complementary IP.

The two Zamore patents that were recently issued in the US relate to guidelines on how to design siRNAs with increased efficacy and selectivity. The first one, announced last month (US patent 7732593) teaches the use of mismatches between the 3’ end of the siRNA guide strand with the target mRNA so as to enhance the ability of the RNAi gene silencing complex RISC to detach from a target mRNA once cleaved and seek out new target mRNAs (note: one of the reasons RNAi is so potent is because one siRNA can destroy many mRNAs). The biochemical experiments on which these claims are based have well stood the test of time. The value of this particular patent for Silence Therapeutics is that it gives them and potential partners optionality for eventually replacing the somewhat restrictive Atu-siRNA design with next-generation siRNA structures based on solid scientific evidence. It has to be said, however, that while many current siRNAs have in fact two such mismatches, or ‘non Watson-Crick base pairs’, namely the classical Tuschl dTdT overhang that was originally conceived as stabilizing the siRNA from exonucleolytic degradation, the claims specify at least three such mismatches in the last five nucleotides of the guide strand. Moreover, the benefit of such mismatches in living cells remains to be determined, as some of the helicases and nucleases that may facilitate RISC detachment from a cleaved target mRNA in living cells may have been missing in the test tube experiments by the Zamore lab.

So while US7732592 is certainly a quite useful patent to have control over, it is the Zamore patent US7750144 of which the issuance was announced yesterday (‘Methods and Compositions for Enhancing Efficacy and Specificity of RNA Silencing’) that should provide for some interesting discussions. This patent is based on one of the classic findings in RNAi molecular biology, namely that the efficacy and selectivity of small duplex RNAs, both siRNAs and miRNAs, is critically determined by the relative thermodynamic stabilities of the two ends of an siRNA (Schwarz et al., 2003: Asymmetry in the assembly of the RNAi enzyme complex). Accordingly, it is the strand of which the 5’ end is less stably base-paired that is preferentially loaded into the RISC gene silencinng complex to become the guide strand, while the non-incorporated strand is discarded as the 'passenger strand'.

This has a few implications for siRNA design. First of all, it increases the absolute loading and consequently silencing efficacy of an siRNA. Secondly, by preferentially loading just the desired strand to become the guide, the potential undesired/off-target activity of the passenger strand can be virtually eliminated. These asymmetry rules have also been extensively validated in living cells and are part and parcel of most siRNA design strategies and algorithms. They are also consistent with structural X-ray crystallographic findings that show that the 5’ end of a guide strand within RISC is unpaired. As a result, it is very likely that a number of siRNA therapeutic candidates currently in the clinic could be interpreted to fall within the scope of this patent. This should also encourage some of the research & reagent companies that sell synthetic siRNAs to take a license from Silence Therapeutics.

The actual issued claims relate to a method of decreasing passenger strand activity by introducing one or more changes in the interaction between the 5’ end of the guide strand with the passenger strand such that the loading of the guide strand is enhanced. The broadest interpretation, and the one that I would expect Silence will take, is that any siRNA containing such a modified nucleotide or mismatch in the specified region (the first 5 nucleotides of a guide strand) would infringe. Most siRNAs should fall within this definition. In some cases, such modifications may have in fact been motivated by other things like stabilizing an siRNA against degradation or reducing innate immune responses, but this remains to be tested, unless the companies involved choose to settle without resorting to yet more exhausting and extremely tedious legal battles. I should note here that when one follows the patent prosecutions especially in Europe, there does not seem to be a single patent that gets issued that will not be opposed by the other party, no matter the scientific merits (my personal opinion). It is enough that some in Big Pharma routinely abuse the patent system to bully smaller competitors into bankruptcy, essentially a way of doing business, and pure-play RNAi Therapeutics companies should be smart enough to know that by adopting these practices they only destroy the size of the shared pie they all depend on: patent-protected innovation. But I digress...

In summary, the recent events have lessened my concerns that it was largely Intradigm that was the beneficiary of the merger as their historical focus on IP is now bearing fruit in the form of some quite fundamental patent issuances and should nicely complement Silence’s strength in the science and clinical translation of RNAi which I had considered to be the stronger of the two. So while I still await data that similarly support the company’s claim that the merger also brought synergies in siRNA delivery, the combined company appears to be quite undervalued here with a market cap of around $20M. The patent issuances should also bode well for the extension of the RNAi trigger-focused part of Silence’s relationship with AstraZeneca. Because of this and because the markets appear to be on summer vacation with the stock virtually unchanged following such strategically important patent news for the company, I’ve decided to buy a few shares of Silence here.

PS: For the various reasons that you can also find in Silence’s regulatory filings, investments in RNAi Therapeutics in general and Silence Therapeutics in particular have to be considered very high risk. Moreover, although I consider myself fairly familiar with the molecular biology of RNAi, I am not trained in intellectual property and my interpretations of the Zamore patents have to be read in this light. Please also read the disclaimer at the bottom of the page.

Follow the Court Proceedings of the Tuschl Tussle

The ‘RNAi Litigation Blog’ is a service by John Leavitt and his colleagues Doug Naab and Scott Lloyd from the technology Research and Advisory firm Nerac that provides a great deal of background information on the Tuschl case and real-time summaries and insights of the court proceedings. As you will remember, this case touches on the ownership of the fundamental Tuschl I and II RNAi trigger patents and of which the outcome could decide what kind of economics Alnylam will be able to extract from its IP and what type of workaround strategies Alnylam’s competition will have to adopt (primers on the Tuschl Tussle and the potential fallout can be found here and here).

The most recent entry on the RNAi Litigation blog was on a hearing held on April 12 about Whitehead’s and UMass’ (the defendants) motion to dismiss the plaintiffs’ (Max Planck and Alnylam) First Amended Complaint. A lot of the hearing seem to have concerned Zamore’s assignment of his rights to the Tuschl I invention to UMass which the plaintiffs strongly feel Whitehead was contractually obligated not to have allowed. After all, it is UMass’ involvement in all of this which makes this case so important because UMass then decided to go it alone and essentially licensed all of the Tuschls most importantly to Sirna Therapeutics (now Merck). This could very well substantially deprive Alnylam of the economic benefits of its, what it believed to be exclusive rights to Tuschl II. To me, it actually seems quite fantastic how UMass believes that the one month that Zamore worked at UMass until the first filing of Tuschl I would now entitle them to the entire Tuschl inventions. Should the plaintiffs prevail in the assignment question alone, then much of the risk to Alnylam’s future business dealings would be taken off the table, unless of course events would escalate in such a way that both the Tuschl patents explode because the USPTO declared the patents invalid because of mishandling of inventorship. The defendants first line of defense is to claim statute of limitations on the assignment question to which the plaintiffs responded that they only became aware of the fact that Whitehead allegedly deceived them in their recent discovery and that they were first damaged in 2007 as Tuschl II ran into problems at the USPTO because of the way Whitehead prosecuted it.

There was an awkward moment in the hearing when the judge asked why Sirna/Merck was not part of the case as it appears that overlooking for a moment the few million in royalties that UMass may enjoy, it is Merck that stands to lose their $1B investment should they end up with a therapeutically useless Tuschl I. It appears, however, that UMass will have to bear the brunt instead because it apparently told Sirna/Merck that they were able to provide access to the inventions described in both the Tuschl patents. Maybe not surprisingly, Merck (‘outside pressures’) also appears to be the reason why attempts to settle this case have failed miserably.

The next important milestone in the case seems to be which counts will eventually be admitted. Unfortunately, it seems as if the judge is not keen at all to delve into the technical details of the case and would rather let the USPTO agonize over it. I am afraid, however, that in order to understand and solve any of the counts at hand, she would eventually have to refresh her high-school biology...

Well, instead of my second-hand account, why not bookmark and visit the 'RNAi Litigation Blog' here directly.

The RNAi Trigger Marketplace in the Post-Tuschl World

This entry is the second of a 2-part series on the upcoming decision of who will control key intellectual property for therapeutic applications of RNAi. In the first part, I tried to provide an outline of the developments causing ownership of certain data in the Tuschl patent applications to become such an important issue. Here, I will try and delve more into the technical details of the scientific milestones that made RNAi a conceivable new class of human therapeutics, and based on that understanding make an educated guess about the outcome of the Tuschl Tussle and how this could shape the RNAi trigger IP marketplace in the future.

Back to Science.

Could RNAi be used as a therapeutic? That was the sort of topic of wild speculation in the lab where I worked as an undergrad in 2001 on a plant gene silencing project. Hey, Fire and Mello reported this cool stuff in worms 3 years ago and as we can see double-stranded RNAs can trigger the same process so beautifully also in plants thanks to some nice work by the Baulcombe group and another one in Australia. But humans? Well, unfortunately vertebrates seem to represent the exception when it comes to the existence of RNAi. That darn interferon response system...All this would change in a watershed moment when Tuschl and colleagues at the Max Planck reported in Nature the very existence of RNAi in human cells and taught a captivatingly simple technology to induce it there: siRNAs. The story therefore seemed quite simple until then. First it was Fire-Mello, then Tuschl's siRNAs.

Fire-Mello

Fire-Mello coins RNAi. The critical contribution by Fire and Mello in 1998 was their realization that it was in fact double-stranded RNA that was the effective inducer behind a variety of strange gene silencing phenomena in worm genetics and quite likely beyond (e.g. variegated Petunia flower color). While it was not necessarily obvious at the time that this would be applicable to humans as it was still very much doubted that RNAi existed in humans, the deeply influential nature of this eureka moment of the field of gene silencing and the non-exclusive licensing approach taken by the Carnegie Institution, the owner of Fire-Mello, established it as a widely respected patent. Add to this the endorsement by the scientific community as evidenced by the Nobel Prize in Physiology and Medicine this work entailed, there should be little doubt in the mind of patent examiners about the therapeutic relevance of that work. Prohhhhbably a fundamental patent.

Biochemical work in fly cell extracts by the inventors behind Tuschl I (Tuschl, Zamore, Bartel, Sharp) and involving the MIT, the Whitehead, UMass, and Max Planck on the same gene silencing phenomenon in flies aimed at the elucidation of the molecular fate of these long dsRNA RNAi triggers. Their main finding was that during RNAi, long dsRNA gave rise to 21-23 nucleotide small RNAs and that target RNA was cleaved at 21-23 nucleotide intervals also. This strongly indicated that it was the 21-23 nucleotide RNAs that were guiding the destruction of the target RNA. This, however, is different from demonstrating that the 21-23 nucleotide small RNAs are able to trigger RNAi themselves, something one would think would be important for claims to this effect to be considered enabled. To test this hypothesis, they therefore isolated and then reintroduced the 21-23 nucleotide mix of RNAs into fresh fly cell extract and asked whether those were able to induce RNAi gene silencing, too.

What may come as a surprise to a few: the silencing with these purified 21-23nt RNAs was actually quite mediocre, about 50% silencing compared to >>95% silencing with the long dsRNA (Figure 12 of US Tuschl I application). Similarly, when the dsRNA length dependency of RNAi was tested, the shorter the dsRNA, the worse the silencing. Together, these types of findings described in Tuschl I seriously calls into question claims that Tuschl I technically enabled human RNAi. Some may even cite such data as proof to the opposite, namely that this work made it even less likely that short RNAs would be useful RNAi triggers.

What is the explanation for this somewhat surprising finding? In retrospect, it is most likely the fact that when the 21-23nt small RNAs were introduced they were single-stranded and not double-stranded and demonstrates that at that time, the authors did not know about the requirement for double-strandedness also of the small RNA intermediates for triggering RNAi . Consequently, the patent contemplates both single-stranded and double-stranded RNAs as candidate RNAi triggers. Thus, while an important piece of the puzzle of RNAi molecular biology history with ~1600 citations to the underlying Year 2000 paper, it by no means was the catalyst leading to the adoption of RNAi in humans. I would not even be surprised if the authors did test the hypothesis of whether such isolated 21-23nt small RNAs were able to silence genes in humans cells (not very difficult to do) and came up short. It is also worth noting that their discovery of small RNAs during RNAi was not entirely new to gene silencing scientists then, a year after Hamilton and Baulcombe reported such an observation in Science, something that also has not escaped the patent examiner.

Tuschl II

Tuschl II coins ‘siRNA’. The critical insight that, first of all, proved the existence of RNAi in Man and even more importantly in terms of enablement, taught a straightforward method for triggering this process in humans, came from very elegant work led by Tom Tuschl at the Max Planck in Goettingen and forms the basis for Tuschl II.

The spark of ingenuity by the people at Max Planck, not involving those at the MIT, Whitehead, or UMass, was that the small RNAs had to be in double-stranded form to serve as useful triggers of RNAi. To prove this, they generated short dsRNAs, which they coined siRNAs, through chemical synthesis, also a first, and found them to be potent triggers of gene silencing not only in fly lysates, but subsequently also in human cells. The fact that the fly lysate work was reported separately by the Max Planck group and temporally between the Tuschl I 21-23 nucleotide RNA paper and the human RNAi findings, further illustrates the temporal, geographic, and intellectual separation of Tuschl's work in Massachussetts and then as a group leader in Germany.

Importantly, these siRNAs allowed for gene silencing that was specific and independent of the interferon response, again something speculated about, but not clearly proven in Tuschl I. The Tuschl siRNA-template is now used by thousands of laboratories around the world, with an amazing 6000 citations to the underlying paper further illustrating its importance.

In the Max Planck vs Whitehead case, the Whitehead argues that 3’ overhang siRNAs that are at the core of the Tuschl II patent application were already part of Tuschl I. On the surface this is true. This is because for some strange reason and that is the biggest mystery to me in all of this and that I hope the next months will shed some light on, the human siRNA data miraculously appear at the end of the Tuschl I application, as does the term ‘siRNA’ emerge without prior definition. If this data were to remain part of Tuschl I, there is the real possibility that Tuschl II could be declared invalid on a technical basis because of Tuschl I’s priority status (in a temporal sense) and double-patenting laws. Not good for Alnylam!

The two related questions of which the answer will rock the RNAi Therapeutics universe are therefore: a) Has the human siRNA data that in light of the weak activity of the ’21-23 nucleotide RNA’ in fly lysates and very uncertain translation of those results into humans now form the inventive basis for the broad human RNAi claims in Tuschl I, been rightfully included? b) In doing so, has the Whitehead, responsible for prosecuting Tuschl I also on behalf of Max Planck, fulfilled its fiduciary duty towards all its partners?

First of all, as I explained in my previous post, the data critical for the siRNA claims of Tuschl I had been generated by the inventors behind Tuschl II (most importantly, in addition to Tuschl, Elbashir and Lendeckel; both of them also at the Max Planck then), but who are not named as inventors on Tuschl I. On this technical ground already, Tuschl I in its present form is invalid. Beyond that, there appears to be early communication in which Max Planck confirmed with the Whitehead that the human siRNA data were the domain of Tuschl II. So even if Max Planck and the inventors of Tuschl I had been wrongly convinced by the Whitehead and their hired patent attorneys that this should not pose a problem for the approval of both patents, any patent attorney worth his salt should have known this to be a fundamental omission. It would therefore seem to be wise to remedy this deficiency either by including the inventors on the Tuschl I patent or by leaving out the data as stipulated by Max Planck, before the specter of 'malpractice' was raised. And obviously, Whitehead now is clearly not acting on Max Planck’s behalf and this should be sufficient cause to give back Max Planck de facto veto power in Tuschl I by confirming that the Whitehead does not have Max Planck's power of attorney any more.

Because some of these issues are civil ones that are not the domain of the USPTO, it is important to sort them out before it goes back to the patent office and can cause lasting damage to the patents. For the stated reasons, I am quite confident that Max Planck and Alnylam will prevail and regain control of the human RNAi data and some sort of declaratory judgement that the way that data had been used in Tuschl I cannot be construed to contest the validity of Tuschl II in the future.

Before I consider the ramnifications of the two main outcomes of the Tuschl Tussle for the RNAi Therapeutics RNAi trigger IP space, it should be noted that Tuschl II already disclosed the observation that blunt-ended siRNAs can silence, too, just not as efficiently as 3' overhung siRNAs on average. 3' overhangs were therefore taught to be a preferred characteristic of siRNAs when used for mammalian RNAi applications. Hence, with many more reports confirming that RNAi in fact is so robust that all sorts of exogenously introduced small dsRNAs can efficiently induce RNAi in humans, it will become more and more difficult to convince the patent offices of a proprietary nature of not only overhung siRNAs, but also those without overhangs. One exception may be Silence Therapeutics’ blunt ‘Atu-siRNAs’ which because it was a relatively early disclosure Silence/Atugen was able to convince the US and European patent offices of their arguably surprising stability, an important feature for most RNAi Therapeutics approaches.

Outcome 1: Tuschl II becomes dominant, Tuschl I essentially irrelevant

The most likely outcome. This will confirm Alnylam to be the most desirable partner based on RNAi trigger IP alone and leave Merck and RXi Pharmaceuticals empty-handed. Clear freedom-to-operate and exclusivity for the most efficient RNAi trigger that is also highly competitive with regards to other challenges such as innate immune activation. However, since Tuschl II does not claim blunt siRNAs, something I believe should have been done at least initially and maybe wasn’t because of an integrated Tuschl I-II strategy that now clearly has fallen apart, there remains scope for plenty of blunt-ended workarounds in the important 19-24 base-pair range. These workarounds, however, are not very attractive for licensing purposes if they cannot be protected by patents. Again, the exception here is Silence Therapeutics which, assuming that Kreutzer-Limmer's staying power is questionable, would be a beneficiary of such an outcome since it would now free Atu-siRNAs from the Tuschl I threat. There are, however, some significant limitations with Atu-siRNAs, since the scope of the patents is quite narrow in terms of allowed siRNA patterns and chemical modifications. Thus, while Tuschl II will offer a platform that should be applicable to RNAi Therapeutics for many years to come, Atu-siRNAs may not be able to adapt to the evolution in cutting-edge siRNA modification technology and hence its value should decline over time relatively quickly.

The IP position of Dicerna is probably least affected by the Tuschl outcome among the synthetic siRNA Therapeutics companies. There may be some uncertainties with whether and how the Tuschl patents may be applicable to Dicer substrates, but unless there will be a messy outcome in which both Tuschl’s go up in fire (highly unlikely), neither outcome 1 or 2 should change this much.

Under outcome 1, the market would have to balance the luxuries that Tuschl II offers, namely patent protection and overhangs, with the lower price, but added liabilities of the Silence Therapeutics and Dicerna platforms or even non-patented siRNA workaround designs. Last but not least, due to its use of overhangs, mdRNA’s overhung ‘usiRNAs’ would be a loser under this scenario.

Outcome 2: Tuschl I becomes (almost) gate-keeping, supersedes Tuschl II

If Tuschl I were allowed in the US in the form now proposed by Whitehead, then Tuschl II may go up in flames with Tuschl I covering blunt and overhang siRNAs comprising RNA strands of 21-23 nucleotides. Under this, albeit very unlikely scenario, Alnylam would have to share gate-keeper privileges for the most direct route to RNAi Therapeutics with Merck and RXi. Further risking to put pressure on price would be RXi selling such rights for a pittance as well as uncertainty about UMass’s ability and willingness to further grant rights to Tuschl I. Not all would be lost for the rest of the field even under this scenario. Silence Therapeutics, for example, would still be able to operate in the 15-20 base-pair range, with maybe 19 and 20 base-pair offering quite good opportunities of discovering efficacious and non-immunostimulatory siRNAs with acceptable efficiency. Similarly, 19 to 20 base-pair siRNAs may also become the preferred space for other non-patented siRNA designs, though all of this is dependent on what happens to Kreutzer-Limmer. Again, Dicerna would be little affected by all of this, and mdRNA may be well advised to try its luck with ‘usiRNAs’ outside the 21-23 nucleotides range, although I still feel chances are slim that one or two supposedly ‘non-nucleotide’ nucleotides will allow them to call what look and behave like siRNAs by another name.

Where does Big Pharma stand in all of this? Those interested in taking broad platform licenses to RNAi trigger IP can probably be classified into into two categories: 1) those like Pfizer and GSK that have diligently done their homework and will already have made up their minds about what type of RNAi triggers are required, including whether they consider overhangs to be an essential feature or not. Such companies can simply await the outcome of the trial and then choose the most economical option everything else (e.g. access to delivery and other know-how) being equal; 2) those companies that have shied away from heavy investments thus far and would prefer to get started with a pure-play RNAi Therapeutics partner providing patent-protected siRNAs and other basic RNAi capabilities. These companies may be most swayed by the outcome of the Tuschl Tussle, since they may be more relaxed in terms of what they consider acceptable siRNA designs.

Tekmira is a pure-play RNAi Therapeutics company that should be uniquely affected by the outcome, because it does not tout having invented unique siRNA triggers, although it certainly could make up such claims to the same degree that others do in the space, and because of its complex relationship with Alnylam. For one, it may determine whether potential partners consider it to be necessary to access SNALP delivery via Alnylam or whether they can go directly to Tekmira and get the same for probably considerably less. Moreover, in the unlikely case of a Max Planck/Alnylam loss, it may become even more difficult to insist on controlling SNALP delivery for RNAi Therapeutics all the while it is obvious that Alnylam is intent on minimizing the importance of Tekmira for their delivery efforts (the whole issue of what is called a SNALP which clearly differs between Alnylam and Tekmira). If SNALP is old and first-generation where is the harm in letting Tekmira fully exploit this technology by partnering it out ex-ALNY instead of letting it wither on the vine? I guess something ought to be worked out here to the satisfaction of both companies.

After 8 years of spending enormous efforts on confusing the investor world with what is valuable RNAi trigger IP, it looks like everything will come down to basic science. The collective scientific community based on the number of citations a paper gets and how it has recognized critical inventions for example in the form of scientific awards, would have been a much more straight-forward, fairer, and infinitely cheaper way of determining ‘good’ RNAi IP. Who else was better suited to spot critical contributions in technically demanding areas such as RNAi than scientists themselves? One would hope that the judge will concur, also in the interest of what RNAi Therapeutics could do for society.

Disclaimer: The above are my own interpretations of the case, based on publicly available documents from the USPTO and court sites, interviews, and press releases. Accuracy cannot be guaranteed as I may have overlooked critical elements of the case, and am neither trained in intellectual property nor contract law. Information provided herein cannot be relied upon for making investment decisions. Investments in RNAi Therapeutics are very risky and not suited for most. Consult with your own professional advisor before doing so.

Next-Generation RNAi

Listening to a webcast by Rosetta Genomics last weekend, I noticed their concerted effort to brand themselves as the Next-Generation RNAi company. Although this is a misnomer as their efforts are really centred around microRNAs, and is likely driven by a desire to get the attention of Wall Street, it made me reflect on what I expect from the next generation of RNAi drugs, specifically the design of RNAi triggers.

Ideally, the next development cycle will yield siRNAs with higher specificity and potency. This should allow for the use of lower amounts of drugs in the clinic for obvious reasons of safety, but also cost. At the moment, algorithms can pretty well predict siRNA sequences that will give a decent knockdown in tissue culture experiments in the low nanomolar range. However, once in a while, we stumble across those “super-silencers” that have IC50s in the mid-to-low picomolar range, yet we do not understand what makes them so good.

I expect that the intense study of the RNAi-related pathways in both model organisms and human cells will ultimately explain their behaviour and reveal rules for designing better and better siRNAs. Exemplary are recent studies by the Zamore group in the fruit fly system that showed that small RNAs are partitioned into separate RNAi effector complexes based on their structure as double-stranded precursors prior to loading into the activated RNAi effector complex. Similar to flies and most other multicellular eukaryotes, there are also a number of related RNAi effector complexes in human cells. It is, however, still unclear how much they differ from each other or what their functional overlap is. It is therefore intriguing to speculate that it were possible, similar to what has just been demonstrated for flies, to introduce small RNAs that would specifically harness the RNAi-cleavage pathway, while remaining invisible to the complexes responsible for the non-cleavage silencing pathways. This is because the microRNA-like non-cleavage pathways are responsible for most RNAi off-target effects, and it would further minimise competition with the endogenous microRNA pathway.

The use of different RNAi triggers (PolII::sh-miR; PolIII::shRNA; Dicer-substrate; Tuschl siRNA; 3-stranded siRNA) or, possibly even more exciting from a drug development perspective, chemical modifications and structural variations to the siRNAs may allow us to introduce the desired bias into which effector complex the small RNA will be incorporated. Along these lines, Dharmacon reported not long ago the use of chemical modification at the 2nd nucleotide position of the guide RNA that would still allow for on-target cleavage activity, but almost eliminated microRNA-like off-target silencing by the siRNA in tissue culture. Although a recent abstract by Alnylam scientists for the Annual Meeting of the Society for Neuroscience suggests that this particular modification may not always be neutral to on-target activity, a combination of chemical modification guided by a deepening understanding of RNAi pathways in humans should yield next-generation RNAi molecules with higher clinical success rates.