Alnylam Once Again Clutches at IP Straws to Support Valuation Gap (with correction)

(21Aug14) Yesterday, I mistakenly stated that Alnylam wrongfully concluded that Dicerna was infringing on a newly issued Tuschl patent.  Following comments in the comment section below, it came to my attention that indeed there was a claim that I missed, claim 81 (and some contingent claims), that covers RNAi triggers of 25 base-pairs as follows:

81. An isolated double-stranded RNA molecule, comprising:

(i) a sense strand and an antisense strand that form a double-stranded region of up to 25 
base pairs, said sense strand having an identity in the double-stranded region of at least 85 
percent to a target RNA molecule; and

(ii) at least one strand having a single-stranded 3’-overhang, wherein said 3’-overhang 
has been stabilized against degradation; and

(iii) at least one nucleotide analogue, 


wherein said RNA molecule is capable of target-specific RNA interference. 

Note that Dicerna's RNAi triggers make use of the 2'-O-methyl modification which sometimes is found in the 3' overhang and can also have stabilizing activity.  Taken together, this claim indeed questions Dicerna's RNAi triggers, and although I would expect vigorous debate around whether 25 base-pairs are covered by the patent's description requirements should it come to a patent litigation, the assumption is that Alnylam's new patent rightfully questions many, if not most of the RNAi triggers used by Dicerna currently.  

Since I'm at it, the new patent also comes awfully close to the asymmetric RNAi trigger designs by RXi Pharmaceuticals and others (asiRNAs).  RXi e.g. uses dsRNA lengths of below 15bp with the guide strand having a long 3' overhang.  I am a bit surprised that Alnylam got just enough extension both below and above their traditional 19-23bp stronghold to start overlapping with some asiRNA and Dicer-substrate designs.

Regardless, I stand by my point that Alnylam has re-invigorated their patent-related press releases in order to explain the valuation gap to its peers in the public markets.  The original blog entry follows here:



This morning, Alnylam greeted the competition with another IP-related press release.  It wrongly claims that a patent it just obtained covers competing technologies.  This suggests that it either lacks an understanding of RNA technology basics or that it is afraid that the market will come to understand that the valuation difference to its peers has no basis in either a commercially more attractive clinical pipeline, a superior patent estate, or simply better technology.

Dicerna’s Dicer-substrate technology not in 14-24bp range

Today’s press release concerns US patent application 13/725262 which is part of the Tuschl patent estate covering certain RNAi triggers with 3’ overhangs.  Although the patent has not finally been published, based on the latest submitted claim set, the RNAi trigger covered by the main claim should comprise the following features:

a)      a dsRNA length of 14-24 base-pair; 

b)      at least 1 3’ overhang;

c)       at least one ‘nucleotide analogue’;

d)      and the dsRNA is non-enzymatically processed.

Clearly, in citing the Rose et al. and another paper by Dicerna (actually their scientific founders from the Rossi lab at the City of Hope) as proof of Dicerna’s infringement, Alnylam hopes that its investor and business development audience does not actually read scientific papers. 

‘Specifically, the newly allowed patent application broadly covers small interfering RNA ("siRNA") molecules of various designs, including so-called "dicer substrate" RNAi triggers (Amarzguioui et al., Nat Protoc.2006;1(2):508-17; Rose et al., Nucleic Acids Res. 2005 Jul 26;33(13):4140-56)…’

Otherwise, it would quickly become apparent that Dicerna’s version of RNAi triggers have a dsRNA length of 25 base-pairs and, well, are enzymatically processed: Dicer substrates!

[Note: in the original entry I mistakenly said Dicerna's triggers were 27 base-pairs; to be precise, they are 25/27 designs with 25 base-pairs and a 2 nucleotide 3' overhang on the guide.]

So as the actual clinical pipelines of Arrowhead Research and Tekmira are about to look more attractive in terms of commercial value (HBV alone), look forward to more Alnylam patent-related press releases to help the market understand why Alnylam has a market cap of $5 Billion and its competition only about 1/10^th of that.

PS: the claim that usiRNAs infringe on this and other patents by Alnylam largely depends on the definition of ‘nucleoside analogue’ and ‘modified nucleotides’.

PPS: this patent does not change Alnylam's position as very similar ones related to 3' overhangs have already issued.  However, by slicing and dicing a patent application, it is possible to get issued a set of highly similar patents which, of course, is great fodder for the PR department.

Tuschl Patents Stage Remarkable Comeback in US

[please note the changes below after I discovered that I missed the last claim amendments, as correctly pointed out by a commenter]

Alnylam announced today that the US patent office had issued 3 Notices of Allowance in recent weeks pertaining to the Tuschl patent estate.  Before your eyes glace over, read on, it's actually one of the few important patent news.  

The granted composition-of-matter claims cover very broadly 19-25bp RNAi triggers, independent of structure.  This is a stark departure from the trend that had materialized in the Tuschl patent applications where Tuschl I seemed to get relegated to treating diseases of fly lysates and Tuschl II suffering from double-patenting issues over Tuschl I.

Claim 1 of allowed patent application 12/537602:

1. Isolated double-stranded RNA molecule, wherein each RNA strand has a length from 19-25 nucleotides, wherein said RNA molecule is capable of target-specific nucleic acid modifications.

1.  An isolated double-stranded RNA molecule, which is a non-enzymatically processed RNA molecule, wherein: (i) each RNA strand independently consists of 19-25 nucleotides in length, and (ii) at least one RNA strand forms a single-stranded 3'-overhang from 1 to 5 nucleotides, wherein said RNA molecule is capable of target-specific RNA interference.

The reason why such a comeback was possible can be traced back to the settlement over the Tuschl patents 'with Merck' in early 2011.  This allowed the Tuschl I and II prosecutions to be aligned such that the double-patenting issue for the 3' overhang claim could be overcome such that the broadness of the Tuschl I RNAi triggers could be rescued into the valuable human therapeutic uses. 

Among those without RNAi trigger licenses from Alnylam, Silence Therapeutics (and its partners, especially Quark) should be hit particularly hard by the latest development: 19bp RNAi triggers are no place to hide any more.   

There are thus two monsters of RNAi trigger patents that consequently co-exist in the commercially very important US market: Baulcombe for almost all types of therapeutically useful double-strand RNA lengths (20-24/30bp; see here) and Tuschl for the desirable 3' overhang feature of RNAi triggers.  It is unclear to me how two patents can essentially claim the same.  Either the patent office is aware of this and is satisfied with ultra-fine semantics distinguishing the two, or this is an issue worth reconsidering.

Another indication that the last word might not have been spoken may be the multiple requests by Alnylam (i.e. their law firm) to expunge certain materials that were rendered during the Tuschl patent prosecutions (possibly relating to timing and nature of the claimed invention), but are deemed trade secrets by Alnylam (can’t help but smile, here). Has Utah taken notes in time?

Bass versus Tuschl: Patentability of 3’ Overhangs Could Depend on Meaning of ‘Person of Ordinary Skill in the Art’

2000-2001 was one of the most exciting, fast-moving periods in RNAi history. All the evidence from the various fields converged to unravel the central mechanisms of RNAi resulting, amongst others, in the discovery of how RNAi can be triggered in human cells. Plant scientists discovered that small RNAs were involved, a group in Cold Spring Harbor found that the 3’ overhang-generating enzyme Dicer played a role, fly biochemists, led by Tom Tuschl, found that synthetic siRNAs could induce RNAi first in fly extracts, and then also human cells. Eureka!

The discovery of RNAi in human cells would not have been possible without the contributions by so many other bright minds. In addition to the research publications, the scientific discourse that happens at conferences and the hypotheses put forward in review articles are a vital part of the scientific process. Everybody stands on the shoulders of others, but it is also acknowledged that the peer recognition belongs to the person/group that publishes first. It is this peer recognition more so than monetary compensation that is the satisfaction of many a scientist. If I’m honest, yours truly probably wasn’t all that different.

Tom Tuschl clearly deserves the scientific recognition. Being the first to demonstrate in a robust manner RNAi in a range of human cells in such a competitive field is no minor feat, nor did it come out of nowhere. Tuschl, partly in collaboration with Zamore, Bartel, and Sharp, did a considerable part of the groundwork leading up to the breakthrough. One should not forget that Brenda Bass, who (or is it really the University of Utah) contests the intellectual ownership over the 3’ overhang feature of the ‘Tuschl siRNA’, was in fact commenting on a paper involving Tuschl during his time as a post-doc in Cambridge, Mass, when she speculated about the possibility of 3’ overhangs in the Cell Review. The other discussions that she claims to have had with the Tuschl siRNA inventors and Zamore were also likely strongly influenced by that research…although I cannot say that for sure. In that sense Tuschl stood on the shoulders of many RNAi giants, Bass on top of Tuschl, and then (maybe) Tuschl again on top of Bass. You get the picture.

Whether having earned the uncontested peer recognition will be followed by the issuance of strong patents, however, is an entirely different question, as I believe that there is merit to Brenda Bass’ contention that Tuschl was at least in part inspired by her work and ideas. On the other hand, I should repeat that because Bass chose to publicly disclose her ideas without filing for patent protection, her ideas became a free-for-all and part of the prior art so that trying to get her name on the patent would seem like inequitable conduct in broad daylight. Therefore, the consequences of the decision whether Bass was instrumental for Tuschl to choose the 3' overhangs in his siRNA design would seem one of patentability of 3’ overhangs, not ownership.

Why do I believe the University of Utah has a point with regard to Tuschl having been inspired by its very own Bass? Some evidence can be found in the commentary of the early papers themselves which I re-read with the question in mind of what gave Tuschl the actual impetus to choose the 3’ overhang feature.

Chapter 1: Long double-strand RNA is processed into small RNAs which direct RNAi in fly lysates (Zamore, Tuschl et al. Cell March 31, 2000)

In this paper, which is a central component of the Tuschl I patent application, Zamore and Tuschl studied the processing of long double-stranded RNAs in fly lysates and found that 21-23 nucleotide small RNAs were generated. They subsequently mapped the cleavage sites in the target mRNA and found that it was cleaved at 21-23 nucleotide intervals. Consequently, the authors suggest that the 21-23 nucleotide RNAs direct RNAi target cleavage- a hypothesis that would survive scientific scrutiny.

Of note, their proposed model of RNAi in Figure 7 shows siRNA without any evidence of 3’ overhangs.

Chapter 2: Bass proposes that small RNAs observed in Zamore et al. paper form 3’ overhangs (Cell April 28, 2000)

In this review in Cell, which shortly followed the Zamore et al. paper and which is a key exhibit in the Utah case, Bass proposes, for the first time, that since only RNase IIIs were known to be able to cleave double-stranded RNAs at specific sites, the small RNAs observed in the Zamore et al. studies ought to form 3’ overhangs as the typical feature of RNase III cleavage.

Importantly, she further speculates that the predicted instability of these 3’ single-stranded overhangs explains why Zamore et al. observed a mixture of small RNAs between 21 and 23 nucleotides, instead of small RNAs of a single size (i.e. 23 nucleotides) as one might have expected from the involvement of a single enzyme, since the single-stranded RNA 3’ overhangs of the initially generated siRNAs would be susceptible to degradation in the fly lysate. As we will see, this idea may have been picked up by Tuschl in his subsequent studies, providing additional support that he and his group indeed read this review very closely. Is Bass’ speculation in fact the reason why half the RNAi Therapeutics world and research/reagent providers still cling to the (actually largely useless, and sometimes indeed harmful) 3’ dTdT feature?

Chapter 3: First demonstration, by the Tuschl lab, that synthetic siRNAs, especially those with 3’ overhangs, can trigger RNAi (fly lysates; Elbashir et al. Genes and Development January 15, 2001)

Before the Tuschl group famously published in Nature on the finding that synthetic siRNAs with 3’ overhangs can trigger efficient RNAi in human cells, they published initial results with such siRNAs in fly lysates in Genes & Development. This paper is critical as it is the first use by the Tuschl group of siRNAs with 3’ overhangs. How would they explain the adoption of this feature for their RNAi trigger design?

In addition to mapping the RNAi cleavage sites in the target RNA, Elbashir and colleagues cloned the ~21-23nt small RNAs themselves. These were then analyzed for the modifications at the 5’ and 3’ ends, upon which they found that the small RNAs contained 5’ phosphates and 3’ hydroxyl groups. They then speculated that these termini indicate that they had been processed by RNase III as such ends are characteristic of cleavage by this enzyme. They then go on to show that synthetic siRNAs with 3’ overhangs were more efficient triggers of RNAi in fly lysates than the corresponding (blunt) RNAi triggers without them. The explanation for the choice of the 3’ overhangs was the RNase III speculation.

Importantly, Tuschl and colleagues acknowledge in their model at the end of the paper that Bass, in her 2000 Cell review, already proposed the involvement of an RNase III. I guess this is pretty good evidence that there was at least some inspiration, although they also suggested earlier in their description of the experimental results that this conclusion could also been drawn independently from the small RNA end modifications that they determined. Here, I would disagree with Tuschl and colleagues as there are other RNases that leave 5' phosphates and 3' hydroxyls, and it is really the understanding of a dsRNA expert like Bass that long double-stranded RNAs get processed by RNase III. That an RNase III was involved in RNAi was shown around the same time by the Hannon group in Cold Spring Harbor. But even if we assume that the conclusion of the RNase III involvement could have been drawn independently, the Bass review was part of the prior art at this point.

I was surprised that it was the RNase III speculation and not direct experimental evidence that led Tuschl and colleagues choose the 3' overhangs in this research. Without re-visiting this issue, I had been under the impression that it was the small RNA cloning results that allowed Elbashir et al. to reconstruct the 3' overhang siRNAs.

Final Chapter 4: Synthetic 3’ overhang siRNAs trigger RNAi in human cells (Elbashir et al. Nature May 24, 2001)

This is the famous paper where Elbashir and colleagues from the Tuschl lab showed for the first time RNAi gene silencing in human cells, using synthetic 3’ overhang siRNAs. Unlike the preceding Genes&Development paper, this one discloses the exact nature of the overhangs. With the exception of one siRNA, all of them contained a dTdT in the overhang (deoxy-ribose, not ribose). This immediate adoption of deoxy would seem strange initially, because the siRNAs that are generated from long dsRNAs would have ribonucleotides in their overhangs and why risk a loss of silencing activity by substituting them with deoxy? The cited reason was that it would reduce the cost of synthesis, and probably also stabilize them from degradation. The synthesis cost argument clearly is not a valid reason for using deoxy for a basic science study such as this one, so it makes sense that it was actually instability that was on their mind, an instability first suggested by Bass in her review.

In light of the evidence in the above publications alone, I believe that it will be very difficult to make the argument that Bass did not influence the 3’ overhang feature in Tuschl’s siRNA design. Nor do I see much evidence that Tuschl made an effort to hide this and quite fairly referenced the Bass review in the Genes & Development paper. I have seen much worse examples of groups deciding not to cite the work of others because it may take away from the novelty of their research. And furthermore, if the Bass review was prior art, why didn’t others jump on the idea and published before Tuschl? Was it because he had an unusual understanding of what was going on in the field and the Bass review, presentations, and discussions only gave an extraordinary person like Tuschl the right insights, or was it ‘just’ because he was already set up to do the experiments and that there would have been many others that were inspired by Bass and would have come up with the 3’ overhang siRNAs if Tuschl had gotten into a bad accident.

The decision whether a person in the art of ordinary skill would have been able to exploit Bass’ speculations to come up with the 3’ overhang siRNAs, similar to how it apparently influenced Tuschl and colleagues, is one of the critical questions challenging the patentability of the 3’ overhang feature, possibly the only RNAi trigger feature of relatively broad scope and value that Alnylam can still hope to control.

Brewed up in Seattle: Tekmira and Halo-Bio Seek to Capitalize on Multi-Targeting Potential of RNAi Therapeutics with Multi-Valent RNAi Triggers

It must have been over either coffee, the fuel of scientific discovery and frequent source of business development inspiration, or beer, a beverage that you may enjoy but not mix with business, that Seattlelites Mark Murray (CEO of Tekmira) and Todd Hauser (CEO/inventor of Halo-Bio) initially came up with the idea of joining forces to revolutionize one of the attractions of RNAi Therapeutics: Multi-Targeting, especially for the treatment of complex diseases, cancer and viral infections (see also previous blog entry on Merck’s hypercholesterolemia efforts; link to press release, here).

The technology to which Tekmira acquired a worldwide exclusive license is referred to by Halo-Bio as multi-valent RNAs (mv-RNAs). It is a molecule that in its basic manifestation consists of three separate double-stranded regions, forming a structure similar to a Mercedes star. The RNAi machinery is thought to use each strand as a guide for the targeting of the same or, more interestingly, separate and independent genes.

Data from this technology is yet to emerge, although I would imagine that Tekmira has carefully evaluated mv-RNAs and various other RNAi trigger options before signing this deal given its increasing hints that it is looking at non-Alnylam RNAi triggers. It has become apparent that RNA interference is a robust naturally occurring process that it can harness various RNA molecules as substrates for efficient gene silencing. Of course, not all of them are created equal. Also worth noting in this context is that efficient RNAi largely depends on the 5’ end of the guide strand so that the 3’ end can function to satisfy complementarity requirements such as those for the formation of mv-RNAs.

Besides potency, additional practical and scientific questions remain to be answered. For example, the use of 3 RNA strands to generate one RNAi trigger may increase cost and quality. On the other hand, for those that follow the Tuschl II patent prosecutions in more detail or have seen the somewhat boring, yet useful study by Roche on the purity of annealed siRNAs, it should have occurred to a number of folks in the industry that strand annealing may not be the only way to generate double-stranded RNAs. Furthermore, even when using three strands (one and two may also be possible), the actual complexity may actually decrease compared to using 3 traditional RNAi triggers with 6 individual strands for targeting the same number of genes. Another question would be whether and which mv-RNAs are loaded directly into the RNAi RISC complex, or whether they require prior processing by other nucleases.

mv-RNAs may also have particular utility in lipo- and polyplex settings, or facilitate formulation of SNALP liposomes, because of their increased negative charge.

Of course, this deal also has to be seen in the context of the Tekmira-Alnylam feud. First of all, it is worth noting that Tekmira stated that mv-RNAs are an attractive alternative for multi-targeting RNAi Therapeutics. It still has the more traditional RNAi trigger options from Alnylam which, not least due to their simplicity, appear preferable over mv-RNAs for single-targeting applications.

The transaction also increases Tekmira's business development freedom from Alnylam. Due to the structural differentiation of mv-RNAs from traditional RNAi triggers, it is possible that certain of Alnylam's exclusive rights to Tekmira technology do not apply. In addition, mv-RNAs may also be sub-licensed independent of Tekmira's delivery technology.

Alnylam faces RNAi trigger uphill battle- even 3’ overhangs not safe

The worth of Alnylam’s RNAi trigger IP has deterioriated raplidly over the last two years and Tekmira may not require Alnylam IP even when using Tuschl siRNAs.

While Alnylam once had a decent shot at coming out as gate-keeping with Kreutzer-Limmer and Tuschl-I, failing in those patent prosecutions yet clinging on to ageing technology has meant that it failed to participate in RNAi trigger innovation, even if not gate-keeping. Anybody still remember Commodore or Atari or holds shares in Nokia and Research in Motion?

[correction 8/26/2011: I have re-read my entry and would like to clarify that Tuschl siRNAs are not 'out-dated' technology; it is more the underlying IP strategy that is failing].

Even as Max Planck, UMass, MIT, and Alnylam have just agreed to re-coordinate their Tuschl patent prosecution in the US, it is clear that the double-patenting issue was not the only issue preventing a strong Tuschl II from issuing. For example, by arguing that 3’ overhangs were implicit in T-I, yet T-I cannot claim this since it failed to recognize it, or by claiming 3' overhangs being obvious over other research on the discovery of Dicer in RNAi, the USPTO may have it both ways: deny a therapeutically useful T-I, yet reject T-II over obviousness involving T-I.

The T-II patents that have issued in the US are not that strong as they involve methods of synthesizing two RNA strands and annealing them to form double-stranded RNAs with 3’ overhangs. It is somewhat surprising that such a annealing method would have been granted a patent, and there are signs that the patent offices are starting to realize this.

Coffee or Beer? Have your say in whether you think the Tekmira-Halo deal is a hare-brained idea or stroke of genius by participating in the survey on the upper right hand corner of the blog.

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.

Clutching at Straws, Whitehead Attempts to Force Unwitting Scientist to Testify

According to recent court filings, the Whitehead is facing stiff opposition in getting a scientist to play a role in their defense in the Tuschl case.

The scientist in question is Brenda Bass from the University of Utah. In April 2000, she speculated in a review article that the small RNAs reported in the scientific publication underlying the Tuschl I patent application had been generated by an RNase III enzyme. Since RNase III enzymes are known to leave 3’ overhangs, the defense wants to use this speculative review article as proof that 3’ overhangs had been common wisdom in the gene silencing field at that time. This is designed to substantiate their claim that small RNAs 3’ overhangs were already implicit in Tuschl-I.

While I take my hat off to her remarkably prescient insights, the relevant remarks were clearly only speculations. Even if those speculations came from a well-respected scientist, most speculations in review articles do not turn out to be true to the same extent and for these reasons such speculations are generally not adopted as gospel in a scientific field, or by ‘those in the art’.

Maybe the Whitehead should start looking in the notebooks of Zamore, Bartel, and Sharp, or any other scientist for that matter, whether, since it was apparently so obvious, they were already in the know about the 3’ overhangs at the time (April 2000). This may be better than trying to annoy another person to become involved in a legal action. It essentially inconveniences her for being an insightful scientist.

If Whitehead is now alarmed that Dr. Bass does not really like to play according to their tune, then they really seem to be clutching at straws and continue to alienate the scientific community in their quest to lay claim on what belongs to the inventors of Tuschl II. And as a technicality, even if (unlikely) the judge could be convinced of this review article to have made 3' overhangs common wisdom, then given the filing dates it is still likely that Tuschl would be able to show that he first conceived of the overhangs before the April 2000 date.

Partly responsible for the alarm could be the fact that Max Planck and Alnylam are seeking, in addition to the permanent injunction, treble damages from the Whitehead and UMass for willfully conspiring to undermine the interests of Max Planck and Alnylam. If the $1.1B price tag for Sirna Therapeutics is any guide, it could get quite expensive. I just cannot get my head around Whitehead's eagerness to side with UMass, who along with their licensees Sirna/Merck and RXi seem to be the only financial beneficiaries should Tuschl I emerge as the fundamental RNAi trigger patent. In fact, Whitehead would even suffer financial harm by doing so, if it is true that Whitehead, MIT, and Max Planck would share equally in the combined Tuschl I-II royalties as per the Therapeutic Agreement. The only reason provided by them and Alnylam/Max Planck seem to be the political damage that siding with Max Planck instead of fellow UMass would entail. Clearly, there must be more to this than meets the eye.

Excerpt from the review:

Does PTGS by dsRNA Involve an RNase III–Like Enzyme?

Although the identity of the RNAi nuclease has not been determined, the characteristics of the short 21–25 nucleotide RNA pieces suggest they were generated by RNase III or a highly related enzyme (see [16] and [1], and references therein). RNase III is the only characterized nuclease known to cleave dsRNA at specific sites to generate dsRNA fragments of discrete sizes. For RNase III to stably bind a dsRNA, it must be at least two helical-turns in length, consistent with the observation that RNAi and transgene-induced silencing yield stable fragments of 22 base pairs. RNase III can produce fragments <22 class="apple-converted-space"> Figure 1, fragments less than 21–23 base pairs would not have been observed in the recent experiments because they would not remain stably bound to the enzyme and thus would be more accessible to degradation by other cellular nucleases.

Given the similarities between the cleavage products of RNase III and the RNAi nuclease, I have incorporated properties of the RNase III enzymes into the model of Figure 1. For example, RNase III makes staggered cuts that leave 3′ overhangs of two base pairs, as shown for the 23-mers of Figure 1. If RNAi involves an RNase III-like enzyme, it might explain why the small RNAs observed by Zamore and Tuschl range from 21–23 nucleotides. The initial cleavage might produce dsRNAs comprised of sense and antisense 23-mers, but the 3′ overhangs would be more accessible to single-strand–specific nucleases present in the extract, and trimmed to 21 and 22 nucleotide pieces. Zamore and Tuschl observe that cleavage of the dsRNA, unlike mRNA cleavage, does not absolutely require ATP. However, dsRNA cleavage is faster in the presence of ATP, and without ATP the pieces are predominantly the longer 23-mers. Certainly this is a clue to the role of ATP in this in vitro reaction, but at present its meaning is unclear.

Anti-miR122 Antagomir Successful in Fighting HCV in Chimpanzees

I just wanted to post this video by KENS 5 on the anti-miR122 studies in HCV-infected chimpanzees by Danish LNA company Santaris and researchers from the San Antonio Southwest Foundation for Biomedical Research. The study, just published online in Science, shows surprisingly potent reduction of HCV levels (2 1/2 logs) with an miR-122 antisense inhibitor and, at least equally important, without the emergence of viral escape mutants. While this study only involved four chimpanzees and human studies are still in the early phases (phase I, healthy volunteers), these results justify hopes that such a first-in-class agent could be a valuable component of future combination therapies with increased cure-rates, shortened treatment times, and maybe without the need for current standard-of-care component ribavirin.

Curiously in light of these promising results, it appears as if GSK let an option to the Santaris compound expire. While Big Pharma may have multiple reasons why it does not want to pursue a certain compound aside from the science (e.g. the fact that HDL-cholesterol was lowered as a result of miR-122 inhibition could warrant caution), for example product portfolio considerations, one possibility is that IP concerns and GSK's relationship with Regulus were important factors: Regulus has an exclusive license to the IP surrounding the fundamental work by Jopling and Sarnow on the role of miR-122 in HCV replication. While this may be good news for Regulus, I was made aware that the broad Esau and Tuschl patent applications on microRNAs as therapeutic targets and held by Regulus are being narrowed down considerably by the patent offices.

Note: For a more detailed discussion of this paper, please read the review by Mark Kay and myself in Molecular Therapy (click here).

Argonaute Gymnastics May Guide siRNA Design (Journal Club)

Argonautes are at the heart of all RNAi-related small RNA-regulated processes. A structural understanding of how they load, activate, and utilize siRNAs to seek out and repress target mRNAs would therefore prove invaluable for the rational design of potentially ever more potent and specific siRNAs. The high-impact journal Nature appears to agree, as it has been publishing at an astonishing frequency collaborative work from the Patel and Tuschl labs at Rockefeller on beautiful structures of Argonautes at the various stages of their catalytic cycle.

In the latest publication in this series, they catch Argonaute with the activated guide strand bound to first a short (12 nucleotides), and then an elongated target RNA (15 nucleotides and longer). What is remarkable is that the enzyme undergoes a substantial structural rearrangement when binding to the longer target, most notably accompanied by the popping out of the guide strand 3’ end from the PAZ pocket where it usually sits in the single-stranded form. This is thought to reflect a 2-state mode of binding fully complementary target RNAs and may differ in terms of structural re-arrangement to what happens in the setting of microRNA target recognition which is the cause for siRNA off-targeting. What is always neat to see in those structures is how they rationalize a lot of preceding molecular biology and bioinformatics work. In this case, this includes the importance of the ‘seed’ nucleotides 2-8, the sensitivity of RNAi target cleavage to mismatches and modifications, and the relative unimportance of the 3’ terminal guide bases 17-21.

The structure may help to expand on work that shows that it is possible to design small RNAs for which target cleavage and translational repression can be functionally separated. Previous work by Dharmacon (now part of Thermo Fischer) for example showed that methylation at position 2 of the guide strand abrogates its ability to translationally repress targets, while leaving cleavage intact. While those siRNAs should be highly specific, the somewhat slow adoption of this strategy indicates to me that more work needs to be done so that similar strategies are more generalizable. Based on the two-state model and present structure, such modification-induced distortions may critically affect seed-binding energy on which translational repression is so much dependent, while the conformational shift that accompanies the recognition of fully complementary on-targets, may lock the Argonaute-guide-target complex into a stable cleavage-competent conformation. The structure may help define universally applicable modification strategies that are targeted towards the 5’ end of the guide strand.

The work also demonstrates how target cleavage is sensitive to modifications around the cleavage site, and finds certain positions better tolerate them than others. This may prove to be important for aiding siRNA activation which is thought to primarily function via cleavage of the passenger strand. Finally, the series of Argonaute structures with bound guide strand provided some nice snapshots of how the 5’ end is nestled in the so-called MID-domain of the enzyme. It is not far-fetched to believe that by optimizing this interaction (e.g. 5’ end modification, nature of the base itself etc), the capture and longevity of the guide strand in the Argonaute protein can be increased with possibly increased duration of activity of the activated enzyme complex, and in the case of single-stranded RNAi, improved loading, too. Finally, this type of structural work is also highly relevant for research into how to reconcile the need for modifications for increasing siRNA stability and avoiding innate immune responses with RNAi activity.

The next major structural insight that should guide design strategies for therapeutic RNAi will be from the elucidation of mammalian Argonaute structures (humans have 4 Argonautes), as the present work focused on archaebacterial Argonautes due to the relative ease of working with them. These, however, will differ in several important aspects from mammalian Argonautes, for example in that they utilize DNA as guides and maybe even as their natural targets. Nevertheless, with such healthy progress on the molecular understanding of RNAi, the efficiency of the RNAi Therapeutics drug development platform will only increase, and it is fun to imagine what it could look like in 10 years, at which point we may be able to watch a movie on the life of an Argonaute protein by the Patel lab.

The Importance of 3’ Overhangs in RNAi Therapeutics

[Note: some of what follows, has already been discussed in the entry ‘On the Importance of Being Tuschl’]

There has been a flurry of activity surrounding the Tuschl patents. It appears that at stake is no less than the Tuschl II patent which claims the use of 3’ overhangs in siRNAs. The uncertainty comes from some of the owners of the Tuschl patent estates prosecuting the Tuschl I patent series such that it uses scientific data underlying the Tuschl II patents. Although I cannot see that 3’ overhangs are explicitly claimed in Tuschl I, the presence of such overhang data in Tuschl I could invalidate the claims in Tuschl II as their prior use would suggest that overhangs were already practiced in the art and/or trigger double-patenting/interference issues.

It has always been a surprise to me that virtually out of nowhere, Tuschl II data, generated at the Max Planck in Gottingen after his time at the MIT, would appear in Tuschl I, which is based on work at the Whitehead/MIT and UMass. The work underlying Tuschl I identified that short siRNAs are generated from longer dsRNAs (in Drosophila extracts) and that the short siRNAs are the likely mediators of RNAi. No mention of overhangs, and no reason to anticipate that overhangs would confer an advantage. The work by Elbashir, Lendeckel, and Tuschl that underlies Tuschl II characterized the siRNAs processed in Drosophila extracts and noted that they contained 3’ overhangs. Synthetic versions of these siRNAs were then found to mediate RNAi in Drosophila extracts and, in the famous Nature paper, human cells. The 3’ overhangs were not just functionally irrelevant consequences of RNase III processing, but found to confer a distinct advantage in RNAi silencing efficacy. This advantage has been borne out in many studies since by labs throughout the world, and the reason seems to be that the overhangs allow for efficient Argonaute loading of the guide strand. For a while, 3’ overhangs appeared to have an additional advantage over blunt-end siRNAs in that they would avoid some of the innate immune responses, although this is an area in which the jury is still out. Another finding that has been borne out by numerous studies is that 19-21 bp lengths work better than smaller or larger siRNAs (not discussing here Dicer-substrates though).

Tuschl I claims double-stranded RNAs of 21 to 23 nucleotides. Note that it says nucleotides, and not base pairs, so in theory it could be dsRNAs that do not have to be blunt ended. But again, at the time of the invention, nobody in the field would have been able to anticipate the presence, let alone an advantage of the overhangs. Tuschl II claims dsRNAs with individual strands of 19-23nt in length, with at least one of the ends having a 3’ overhang.

I do not want to comment here too much on the specifics of the case, and Doug Macron from ‘RNAi News’ has done a great job in following the story- except to say that it is clear that Max Planck (and Alnylam) would never have agreed to such use of Tuschl II data, and even if there had been some procedural issues (e.g. Tuschl ‘swore’ etc, when in reality most academic scientists trust that their patent agents would act in their best interest, and therefore just sign off patent documents that come across their desk/bench) I believe that at some point fiduciary duty has been violated by those hired and paid, also by Max Planck and Alnylam, to prosecute the Tuschl patents.

While I consider it possible that some fancy workarounds of the Alnylam IP estate may give certain freedom-to-operate to some companies, scientifically the sweet spot of siRNAs are those 19-21bp in length with at least one 3’ overhang end and the drug development economics would dictate these to be used and licensed. This area is very well covered by Alnylam through Kreutzer-Limmer, Tuschl I and II, although an unexpected outcome of the Tuschl proceedings could change the picture. On the other hand, one cannot dismiss the possibility that some non-Tuschl-like structure in combination with a certain modification pattern similar to what Silence Therapeutics claims e.g. actually works and is advantageous contrary to expectation, that is non-obvious, and I'd be happy to look at data that systematically demonstrates this.

Did Alnylam Patent News Send CytRx Shares Down 14%?

It should not come as a surprise that Alnylam today announced that the previously allowed Tuschl II covering siRNAs was granted in Europe. The fact, however, that another year has passed and Tuschl II keeps on sailing through the worldwide patent offices while the Tuschl I patent application is being delayed, further cements the impression that it is Tuschl II that is the key patent for siRNAs, not the related Tuschl I.

Tuschl I, co-exclusive to CytRx, Sirna/Merck, and Alnylam, is thought to have been a major motivation for Merck to buy Sirna in late 2006. Like Tuschl II, it covers gene silencing in mammalian cells, but interestingly the classical siRNA features, 19-21 base-pair double-stranded RNAs with 3’ overhangs, are claimed only in Tuschl II, whereas Tuschl I generally claims ~21-23 nucleotide silencing RNAs and their precursors, thus somewhat landing it in between antisense and RNAi.

Needless to say, the perceived value and potential of CytRx, or its RNAi spin-out RXi for that matter, as a Sirna-like takeover candidate by Big Pharma much depends on the fate of Tuschl I. So today’s 14% fall in CytRx shares on expanded volume, while those of Alnylam gained 2%, may be directly linked to today's patent announcement. Further delays in the registration of RXi as a publicly listed company and the recent shelf offering should have contributed to the sinking feeling CytRx shareholders must have felt today.

It then looks like 2008 could also be a defining year in terms of who owns the IP in RNAi, as the number of core RNAi Therapeutics companies is about to peak and funding gets directed to those with strong IP positions as investors become more discerning in their RNAi investment decisions. CytRx, Nastech, Benitec, and Silence Therapeutics will be under particular pressure to prove themselves as they return to the markets for funding.

Tuschl I patent application:
http://www.wipo.int/pctdb/en/wo.jsp?wo=2001075164&IA=WO2001075164&DISPLAY=STATUS

Tuschl II patent:
http://www.wipo.int/pctdb/en/wo.jsp?WO=2002%2F044321&IA=WO2002%2F044321&DISPLAY=STATUS

The Confusing World of AtuRNAi, Stealth siRNAs and mdRNAs (Part II)

(RNAi IP discussion continued from previous entry)

If long dsRNA had worked exactly in humans as it did in the worm and plants, then you would have expected an immediate flood of publications reporting the same. Long dsRNA for gene silencing, however, were impractical for most vertebrate cell applications due to the induction of non-specific cytokine responses that essentially shuts down most gene expression and therefore does not allow for targeted gene silencing. An exception may be embryonal cells which lack an interferon response and for which long dsRNA was reported to induce specific gene silencing first in zebrafish in 1999 (Wargelius et al. Biochem Biophys Res Commun. 263:156) and then in mice (a mammal) in late 2001 by the Filipowicz group (Basel, Switzerland).

These latter findings, however, were overshadowed earlier in 2001 by a publication from the Tuschl group in Germany, representing the culmination of a body of work he first started as a post-doc in the Bartel/Sharp labs during his time at the MIT, and then as an independent investigator at the Max-Planck Institute in Goettingen. While small RNAs were then known to derive from long dsRNAs, their molecular role in guiding the recognition and destruction of target mRNAs was only hypothesized and their structure mostly unknown. A breakthrough towards this understanding came by establishing a biochemical system in Drosophila (fly) lysates that recapitulated RNAi in the test tube (1999, MIT). One year later, they reported that during this reaction both the long dsRNA as well as the target mRNA is cut at 21-23 nucleotide intervals (MIT, 2000), thereby providing a link between dsRNA processing and mRNA targeting. In early 2001, then at the MPI, Elashir and colleagues in Tuschl’s lab further delineated the relationship between dsRNA processing and target mRNA cleavage in the Drosophila system, including the observation that the mRNA is cut around 10 nucleotides from the 5’ end of a 29 base-pair dsRNA (kind of Dicer-substrate). Importantly, by sequencing the 21-23 nucleotide RNAs by borrowing a cloning technique developed for the discovery of microRNAs around the same time, they found that the small RNAs were clustered consistent with long dsRNA processing into 21-23 base-pair DUPLEX RNAs. Moreover, the small RNAs were found to contain 5’ monophosphates and 3’ hydroxyl groups all consistent with the notion that long dsRNA was processed by an RNase III enzyme into 21-23 base-pair duplexes (reported to be the Dicer enzyme by Hannon in Cold Spring Harbor in the same month).

This led them to test whether small duplex RNAs were crucial functional intermediates between long dsRNA and mRNA cleavage, by synthesizing duplex RNAs and adding them to the Drosophila system. I quote: “Perhaps the 21-nt RNAs are present in double-stranded form in the endonuclease complex, but only one of the strands can be used for target RNA recognition and cleavage”. Indeed, this prediction turned out to be correct and synthetic duplex RNAs could silence mRNAs in this system, and duplexes with 2-3 nucleotide 3’ overhangs, the hallmark of the hypothesized RNase III-type processing, worked best. These data then formed the basis for the Tuschl I patent series to which Alnylam, RXi, and Sirna Therapeutics obtained co-exclusive licenses. My guess is that Alnylam actually would not mind if this patent wasn’t issued after all, since for some obscure reason UMass, unlike the Whitehead Institute, MIT, and MPI decided to grant RXi and Sirna co-exclusive licenses. Equally curious is the fact that while in the January 2001 paper the duplex RNAs were shown to work only in fly lysate, in the Tuschl I series, out of the blue, human cell studies are described. I could well imagine that the ultimately issued Tuschl I patent will be solely focused on the fly data, so that the first human siRNA description would be exclusive to the Tuschl II series (I would encourage you to read my 27 May, 2007 Blog “2007RNAi Therapeutics IP: The Importance of Being Tuschl” on this issue).

Clearly, work in human cells was ongoing at the time in Tuschl’s lab, and they conclude the fly paper in Genes and Development with the ominous statement: “The siRNAs may be effective in mammalian systems, where long dsRNAs can not be used because they activate the dsRNA-dependent protein kinase (PKR) response (Clemens 1997). As such, the siRNA duplexes may represent a new alternative to antisense or ribozyme therapeutics.” The compositions, methods, and uses of synthetic siRNAs in human cells are described in excruciating detail in the Tuschl II patent series, much of which has issued in the EU and US and is exclusively licensed to Alnylam.

A lot of the claims by other companies such as Silence Therapeutics and Invitrogen’s Stealth siRNAs center around the fact that Tuschl II emphasizes the 3’ overhangs of siRNAs, and that blunt-end siRNAs are therefore not subject to Alnylam’s IP estate, but completely ignore the fact that Tuschl, both in his fly and human work indeed tested blunt-end siRNAs, just that they did not perform as well as the overhang siRNAs. I speculate that the reason why Alnylam has not come out and spelt out this fact is because they may think that their equally exclusively licensed Kreutzer-Limmer patent series (use of short dsRNAs for gene silencing in mammals) provides even better coverage for the use of blunt-end siRNAs. Alnylam’s competitors, including Merck, have therefore focused their efforts of fighting Alnylam’s IP dominance on narrowing the scope of Kreutzer-Limmer, particularly in Europe, and have succeeded in doing so last summer to reduce the covered length to 15-21 nucleotides. However, a so called divisional patent application based on the Kreutzer-Limmer patent was granted in Europe in 2005 and has even broader claims than the original patent (15 to 49 base-pair duplexes). It is further ironical that a weakening of Kreutzer-Limmer would only strengthen Tuschl II’s scope. In addition, Tuschl II further covers modifications and conjugations to siRNAs, a claim which Alnylam has cemented by obtaining an exclusive license to the Crooke modification patent estate from ISIS.

While RXi may have marketed their recent StealthTM siRNA license from Invitrogen for therapeutic purposes, in my mind “StealthTM” siRNAs are nothing more than a marketing gimmick disguising the fact that these are 25 base-pair, blunt-end siRNAs with a supposedly magical pattern of base modifications. I would therefore not be surprised if Stealth failed to fulfill the non-obviousness criteria, in addition to the fact that I have not seen any evidence that Stealth, per se, performs any better, if not worse than the classical Tuschl siRNA design. What RXi probably won’t tell you is that Invitrogen has deemed it necessary to gain access to the Kreutzer-Limmer patents through a licensing agreement with Alnylam for the use of Invitrogen’s siRNAs for research applications only.

With regards to Dicer-substrate, licensed by both Nastech and Dicerna, I see practical value in that Dicer-substrates may be beneficial for RNAi delivery purposes in that they provide increased flexibility in covalently conjugating the Dicer-substrate to the delivery carrier, while siRNAs have to be reversibly conjugated, e.g. via disulfide linkages, to achieve the same. However, this does not guarantee the uniqueness of Dicer-substrate since a lot of the duplex length and the conjugation idea is subject to the pre-dating Kreutzer-Limmer and Tuschl patent series. Moreover, in his fly experiments with 29 base-pair duplexes, Tuschl already demonstrated “RNase III-substrate”. Hannon should also have relevance for Dicer substrate in that he was the first to describe Dicer to be the enzyme that mediates dsRNA processing in flies, and likely humans.

Hannon continued his work on the practical implication of dsRNA processing and was one of the first to explicitly describe the use of Dicer-substrates in humans in the form of DNA-directed hairpin expression cassettes driven by a Pol III promoter. While this 2002 paper in Genes and Development was as much inspired by the newly emerging knowledge on microRNA processing as much as by Tuschl’s 2001 findings, “Tuschl and colleagues first showed that short RNA duplexes, designed to mimic the products of the Dicer enzyme, could trigger RNA interference in vitro in Drosophila embryo extracts”), the European group (Brummelkamp et al.) that published in Science on the same subject the same month were mostly inspired by Tuschl: “We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells.” In any case, both Alnylam and RXi have gained access to the Hannon patents which touch on both DNA-directed RNAi and Dicer-substrate (synthetic or DNA-directed).

In a confusing turn of events, however, Hannon reported in 2005 that hairpins, in this case synthetic versions though, with longer duplex regions often worked better (= more potent and reliably) than the classical 19 base-pair hairpin design. This could be explained by the observation that the efficiency of RNAi should be enhanced by requiring a Dicer processing step since this is coupled to the RiSC-mediated gene silencing step. The fact that the original 19 base-pair hairpins, first thought to be processed by Dicer, were found to be inferior could be explained by their inefficient processing into siRNAs by some RNases not normally related to RNAi. Essentially the same conclusion was reached by a paper in the same issue of Nature Biotechnology from John Rossi’s group at the City of Hope, this time, however, by employing synthetic 25-30 base-pair duplexes (licensed to Nastech and Dicerna) instead of synthetic hairpins. For a discussion of Dicer-substrate science and IP, please refer to my October 31, 2007 Blog: “A new player in RNAi Therapeutics: Dicerna Pharmaceuticals”.

Besides RNA polymerase III-driven small hairpins, DNA-directed RNAi can also be initiated through more microRNA-like constructs. This was enabled by the elucidation of the microRNA silencing pathway and the trick basically is to design DNA vector constructs that will mimic one of the RNA intermediates during microRNA processing. These methods have the advantage that RNA polymerase II promoters can be employed with potential tissue-specific or other regulation. This should allow for potentially safer DNA-directed RNAi, although RNA polymerase III constructs have extreme knockdown potencies. Brian Cullen’s (Duke) and particularly Narry Kim’s (Seoul, Korea) groups have spear-headed these efforts, but I have not heard from companies yet specializing on the use of such RNAi constructs for therapeutic purposes. It is further likely that the original DNA-directed RNAi patents will be quite important for the commercialization of these later methods.

In addition to employing RNAi triggers that funnel into the RNAi pathway upstream of siRNAs, it is also theoretically possible to make use of at least two more intermediates functioning downstream of siRNA generation: single-stranded guide RNA that recognizes target mRNA within RiSC and a 3-stranded intermediate in which the passenger (=non-targeting) strand is interrupted based on findings from a number of groups, again almost simultaneously about 2 years ago, that the passenger strand was cut prior to guide RNA RiSC loading, analogously to how target mRNAs are cleaved.

The single-strand siRNA method is mostly investigated by ISIS for commercial purposes, probably because it feels that their IP position on single-stranded antisense RNAs would make them the dominant player in single-stranded RNAi. It should be kept in mind, however, that it was again the Tuschl group, known to be close to Alnylam, that first reported on single-stranded RNAi inducers (Martinez et al., 2002) and patents have been filed. Moreover, evidence so far suggests that single-stranded RNAs are only very inefficiently recognized by the endogenous RNAi machinery and it is doubtful that any potential advantages of single-stranded RNAs versus duplex RNAs would ever make up for the inferior potency. Patents covering 3-stranded siRNAs have been filed for by Nastech, although they have not been associated with any of the initial reports on siRNA passenger strand cleavage. It will therefore be interesting to determine the priority dates of the various discoveries, and probably more importantly, data as to the efficiency of these “meroduplex RNAs” (same initials as Nastech’s planned RNAi spin-out mdRNA) compared to other RNAi inducers. Such 3-stranded siRNAs may offer certain advantages with regard to conjugation chemistries and the fact that short RNA strands are cheaper to synthesize than larger ones, but until this is proven it will look just like another thinly disguised patent work-around attempt.

In summary, it is clear that it was Fire and Mello’s discovery on long dsRNAs as RNAi inducers in worms and Tuschl’s extensive body of work leading to the delineation of the classical duplex siRNA that opened up RNAi for therapeutic use. Many of the other developments are directly derived from both of these fundamental discoveries and require appropriate IP licenses. The combination of Tuschl II, Kreutzer-Limmer, and all the other patents it has either exclusive or non-exclusive access to, makes Alnylam the gate-keeper of RNAi Therapeutics. The exact terms of companies wishing to commercialize RNAi Therapeutics will vary depending on their co- or non-exclusive access to some of these fundamental patents, how far removed their exact siRNA derivatives are from the classical siRNA design as well as the ability to prove their utility. While this dominant IP position by Alnylam may make them unpopular and almost look like a bully, one should not forget that concentration and clarity of IP encourages investments particularly in the risky business of drug development and therefore will increase the likelihood of maximizing the therapeutic potential of the technology. It should also be said that Alnylam has been pretty good in de-risking RNAi technology, thereby benefitting the whole field of RNAi Therapeutics, in addition to granting access to RNAi technology through their licensing policy, although the terms will increase the longer you wait. Outside this core RNAi IP, other IP, particularly relating to delivery, but also access to validated targets will prove valuable, albeit much more fragmented.

In my last blog on this RNAi IP series, I would like to briefly discuss individual companies according to technology strength and IP position. Alnylam’s view on this issue will be presented in a special IP-focussed investor presentation on November 28 at the 19th Annual Piper Jaffray Health Care Conference and can be followed live or recorded by webcast on the company’s website.

Erratum: Please note that in my discussion of Dicer-substrate in the October 31, 2007 Blog: “A new player in RNAi Therapeutics: Dicerna Pharmaceuticals”, I mistakenly stated that Hannon’s long hairpin RNAs were DNA-directed, when they actually studied synthetic versions of these hairpins.

The Confusing World of AtuRNAi, Stealth siRNAs and mdRNAs (Part I)

“Next-generation” RNAi is all the rage in the world of the ambitious RNAi start-up. The definition (note that everything is allowed under the guise of the satire): “An RNAi-inducing molecule derived from the classical Tuschl siRNA design, however with a magical pattern of modifications and variations in the exact length of the RNA duplex, sometimes an NA duplex, with overhang or not. As important as the chemistry that may sound impressively inventive to the lay (investing) public is that a proper name is chosen to further accentuate its apparent uniqueness. This is intended to suggest freedom-to-operate with the ultimate aim of attracting investments from people hoping the company will eventually catch up to the market cap of Alnylam (why would you invest in any of their direct rivals otherwise?).”

Considering that it has become commonplace to hear CEOs talk about their RNAi being so unique and advanced that they are now operating in parallel universes, the staid Tuschl siRNA must have really lost its relevance for the development of RNAi Therapeutics. While I think that some select siRNA derivatives given names such as StealthTM RNAi or Dicer-substrate definitely warrant further investigation, as it is yet unclear how well they will perform relative to the simple, but fundamental siRNA design, what I would like to do is to cut through the marketing fog and provide a brief overview of the types of RNAi inducers currently being used at the bench or in the clinic and how I think they relate to each other in terms of IP. In this post I will lay the foundation by giving a summary account of the history of RNAi as a tool, including some of the fundamental patents (and applications), before dissecting some of the Next-generation siRNA designs in a follow-up posting.

Studies on RNAi-related gene silencing really started in the early 90’s in plants with the observation of co-suppression whereby genes that share sequence similarity inhibited each others’ expression. Usually, this was triggered by the inappropriate processing of one of the gene products, typically from an introduced designer gene that is recognized as aberrant and therefore as a threat by the plant RNAi surveillance system. While the mechanism by which this occurs is a scientifically very interesting question, it cannot be used for gene silencing in humans and therefore has little or no relevance to RNAi Therapeutics IP. Parallel work on gene silencing in worms by Fire and Mello, of course, discovered that it was long double-stranded RNA (dsRNA) that was central to inducing RNAi and patents were filed covering dsRNAs longer than 25 base-pairs for gene silencing. This patent can be licensed non-exclusively by almost anybody that wants it, and despite it being based on work in worms and the long dsRNA nature in the stated claims, it is nevertheless considered to be a license that you should add to your IP portfolio anyway, I guess just because it has proven so fundamental to the understanding of RNAi in general and nobody would want to argue that. I also think this highlights the fact that real fundamental scientific insight will be credited by the patent courts even if the exact length of the duplex or modification pattern was not spelt out in the claims letter by letter.

Shortly after Fire and Mello published their research, Kreutzer and Limmer from the University of Bayreuth in Germany reasoned that short dsRNAs may have similar gene silencing effects in mammalian cells. This prediction, as we know, turned out to be true and now forms the basis of the Kreutzer-Limmer patents claiming short dsRNA of around 15-49 base-pairs for the induction of gene silencing in mammalian cells, although the exact length is the subject of patent challenges, including Merck’s opposition in Europe. This early work was considered important enough by Alnylam for them to acquire Ribopharma AG, the company founded on the Kreutzer-Limmer patents. Although I consider Tuschl’s subsequent work to be quite a bit more fundamental to the use of RNAi in mammals, Alnylam understood that it was important to remove any uncertainty as to the dominance of their RNAi IP position given the relative timing and overlapping content of Kreutzer-Limmer and Tuschl.

Around the same time, Hamilton and Baulcombe discovered that small RNAs were generated during plant RNAi. While they were prescient in predicting that these may mediate RNAi, they did not formally prove it and the structure of the siRNA that was detected in those experiments remained unknown. A world away, in Australia, DNA-directed hairpin vectors for reliably inducing RNAi were being described by Waterhouse and colleagues from the CSIRO. Based on the utility and impact of these vectors on plant research, the patents derived from these studies should give the CSIRO a strong position in the agricultural uses of RNAi. In many ways, the commercial development of plant RNAi is more progressed than therapeutic RNAi as traits can now be altered relatively quickly without having to resort to lengthy breeding and selection. I guess the most important question will be how uniform these knockdown phenotypes will be across a field of crops. The CSIRO patents also form partly the basis for Benitec’s claims to the therapeutic uses of DNA-directed hairpin RNAs. The Graham patents form the other pillar of Benitec’s contested patent estate describing the use of DNA cassettes driving the expression of various forms of dsRNAs, although I find these patents to be quite theoretical in nature and wonder whether most of the described non-Pol III expression cassettes would actually work for gene silencing in most mammalian cell types (to be continued…).


Two noteworthy developments last week that I would briefly like to comment on:

1) ISIS released further phase II data for their ApoB-targeting antisense compound mipomersen. The 200mg/week dose reduced by about half the level of bad cholesterol in patients already on stable statin therapy. This looks quite impressive and if no safety issues come up in the larger phase III trials, then this has the potential to become a blockbuster. I’ve been quite critical about mipomersen in the past, particularly due concerns about fatty liver which many scientists in the field would have expected to observe following ApoB knockdown. Safety data for the 200mg dose, based on liver enzyme measurements, however do not indicate this to be a problem. Ultimately, the proof is in the pudding and I would be happy to ultimately have to admit to have been wrong on this issue. ISIS explains the absence of fatty liver due to transcriptional compensatory changes in fat metabolism. Overall, these data augur well for the development of all RNA-targeting platform technologies, including RNAi Therapeutics, as it suggests that minor off-targeting should be well tolerated in many cases.

2) Pfizer announced the acquisition of Coley Pharmaceuticals for almost triple of Coley’s market cap before the offer. Coley Pharmaceuticals is an oligonucleotide therapeutics company that exploits the immunostimulatory properties of oligonucleotides for applications such as boosting vaccines or in the fight against cancer. Actually, I’ve been quite impressed by their OTS presentation in Berlin, particularly their vaccine program. This comes only days after a blog this month where I asked the question when Pfizer will make its big move in RNAi Therapeutics (11 Nov 07 Blog: “When Will Pfizer Finally Make its Big Move in RNAi Therapeutics?”). It is notable that Coley is a Massachussetts company and I would like to think that the proximity to Alnylam will not be an impediment to Pfizer’s new biotech initiative that also appears to more and more focus on oligonucleotide therapeutics. With the new oligo expertise in-house (note that Alnylam does not have another subsidiary to throw into the next deal) and plans to add more staff to a research facility in Cambridge (the headquarters of Alnylam) in addition to a possible biotech incubator near Boston, the plot thickens.

Regulus Rx, Where One and One Equals Five

One has to be impressed by the way John Maraganore, chief astronomer and CEO of Alnylam, and his team are maximising the value of their company’s scientific know-how and intellectual property. Last week’s launch of Regulus Therapeutics, a joint venture with ISIS Pharmaceuticals focussed on the development of microRNA-based therapeutics, is the latest in a string of strategic decisions that positions Alnylam to lead and participate in the very promising field of RNAi-related therapies while wisely managing the company’s resources and respecting the scientific challenges ahead. The scientific and financial results speak for themselves and are starting to catch the attention and respect of Wall Street and beyond.

Forming this joint venture not only consolidates and complements much of the IP in microRNA-based therapeutics, it also allows the respective companies to focus on their core operations in RNAi and antisense while giving the new company a distinct identity and independence that should incentivise their employees and help attract 3rd party funding, particularly in the form of alliances with larger partners. The joint venture also acknowledges the fact that microRNA therapeutics sets a new paradigm for treating disease with its unique set of challenges and risks.

Based on pioneering work by Regulus SAB member David Bartel and his group at the MIT, it is now believed that the approximately 1000 human microRNAs regulate around one third of our genes, “that is one third of our genes” (Stanley Crooke). It follows that each microRNA targets multiple genes. Targeting a microRNA for therapy is therefore based on the premise that the functions of these genes are linked to a common biological process hence limiting the potential for unwanted side-effects by disturbing unrelated pathways. In this sense, microRNA- and RNAi-based therapies are opposite philosophies where siRNAs seek to surgically target single disease-associated genes, whereas microRNAs are aimed at whole networks. Genetic research supports both approaches, and a number of microRNAs have been shown in animal models and human cells to specifically affect discrete regulatory pathways, a fact that is arguably best understood by two key SAB members of Regulus Rx, namely David Bartel and David Baltimore. A recent catalogue of microRNAs established by yet another Regulus SAB member, Thomas Tuschl from the Rockefeller, however, suggests that many microRNAs are constitutively expressed in a number of tissues, thereby slightly questioning whether this is the case for most microRNAs. Although a truly exploding field of research, compared to RNAi Therapeutics where there are already an abundance of well-defined targets, the development of Regulus Rx will heavily depend on progress made in elucidating the exact role of specific microRNAs.

Regulus Rx’s first development program, targeting miR-122 for the treatment of Hepatitis C Virus (HCV) infection is a particularly interesting case. This program has been licensed from Stanford University where Catherine Jopling from Peter Sarnow’s group has shown that miR-122 specifically interacts with part of the HCV genome and thereby stimulates HCV replication. MiR-122 is by far the most abundant microRNA in the liver, and it is surprising that no obvious toxicities have been associated with its inhibition in vitro or in vivo, as demonstrated by work from both Alnylam and ISIS scientists. It is even thought that inhibiting it may have applications in managing cardiovascular disease. Earlier this year, Alnylam and ISIS were issued a patent covering miR-122 as a therapeutic target. I am quite curious to learn whether last week’s announcement of HCV miR-122 as the first development program of Regulus is also based on results targeting HCV replication by miR-122 inhibition in vivo. In this regard, it is notable that a leading group in HCV biology, the Rice group at Rockefeller only recently confirmed miR-122’s importance in HCV replication in a paper jointly published with Thomas Tuschl from the same institute.

Regulus Rx will be based in Carlsbad, CA, not only because of the better weather, but also because this is where ISIS Pharmaceuticals is located. This acknowledges the fact that antisense technology, pioneered and long dominated by ISIS, is the key enabling technology for antagonising mature microRNAs many of which are processed out of introns of RNA Polymerase II transcripts and therefore not a target for RNAi. This underscores the importance Alnylam has attributed to ISIS’ patent estate, also demonstrated by their exclusive licensing ISIS’ oligonucleotides modification patents for the use in double-stranded RNAi Therapeutics. It also sends out the subtle message that as Alnylam respects other parties’ IP it expects anybody interested in RNAi Therapeutics to follow this example by licensing from Alnylam.

The 50:50 joint venture also speaks volumes to the negotiating leverage Alnylam has gained in recent years, made possible in large part by its solid financial position. Only 3 months ago, the financial strength of Alnylam has allowed it to revise a partnership agreement with Medtronic that has given Alnylam a larger stake in the financial success of any drugs coming out of that collaboration in return for increased funding by Alnylam. Unlike ISIS which not long ago had to repeatedly issue debt to fund their operations and is biting its nails to gain financial freedom by partnering its ApoB100 antisense program, Alnylam could easily pay the $10M to equal the companies’ stake in the new venture. In addition to its financial muscle and Tuschl III which gives Regulus exclusive access to some of the most important microRNA targets, Alnylam further brings to the table its invaluable access to leading scientists in academia and Big Pharma alike.

Finally, it is notable that the otherwise wide-ranging technology access licenses that Alnylam has granted Novartis and Roche did not include microRNAs. It would therefore be appropriate that following last week’s development the newco be put on a solid financial footing through a significant partnership. But let’s give them some time for this as although the day may never end on Alnylam, on earth their day still has only 24 hours.

(For more on the scientific rationale for microRNA therapeutics, please read my Blog from 26 May, 2007: “MicroRNAs as Therapeutic Targets”)


PS: Once the dust has settled, we should learn more about the exact scope of Regulus Rx. Specifically, it is not clear to me yet whether in addition to antagonising microRNAs by antisense, the scope of Regulus will encompass the microRNA agonist approach as well where microRNAs mimics are introduced for therapeutic purposes. While this would strengthen Regulus’ portfolio, it would be a significant contribution of Alnylam IP since such mimics would essentially be based on siRNA technology.