Impressions from the 3rd Annual Meeting of the Oligonucleotide Therapeutics Society: Day 2

The second day of the Meeting was filled with cutting-edge science related to RNAi drug development with emphasis on RNAi mechanism, delivery, and cytokine responses triggered by various nucleic acid classes.

Tuschl kicked off the day by presenting data on the systemic identification of Argonaute interaction partners (mostly published data) and a new approach towards identifying microRNA targets. In contrast to microRNA target identification algorithms which heavily rely on sequence conservation, Tuschl and colleagues identified RNAs pulled down in Argonaute immuno-precipitations and then sequencing. Validating the approach, these RNAs were enriched for sequences with seed targets of the most abundantly expressed microRNAs in the tested cell line (1.8x enrichment over random). He finally reported on small molecule screens aimed at identifying inhibitors of microRNA maturation, however, with less success. It appears to me that he should stick to his guns and work at inhibiting microRNAs and their precursors instead by nucleic acids, such as antagomirs, which have proven to be much more potent and specific inhibitors of microRNA activity.

Ingo Roehl (Roche Gmbh, former Alnylam Europe) gave a nice presentation on progress in the development of analytical chemistries to support DMPK/PD (Drug Metabolism and Pharmacokinetics/dynamics) studies of RNAi Therapeutics. It was encouraging to see significant improvements that now allow siRNA and endogenous microRNA quantitations in the picomole-femtomole range in biological samples at high-throughput. In contrast to methods used by other groups, their coupled HPLC mass-spec setup allows for the discrimination between intact siRNAs and their degradation products. However, further improvements in sensitivity are desired as the potency of siRNAs means that biological activity can be observed long after siRNA levels drop below the level of quantitation.

Peter Linsley from Rosetta Inpharmatics (a subsidiary of Merck), which gained early “notoriety” for being the first group to point out the problem of off-targeting by siRNAs some years ago, highlighted the dilemma posed by the fact that the off-target signature of a given siRNA is extremely different in mouse and man. This means that mice will not function as a safety model for off-target toxicity studies. Furthermore, by limiting oneself to cross-species specific siRNAs for the sake confirming the treatment in the animal model, many siRNAs with better potencies and off-targeting signatures in humans may ultimately be missed. Also of interest is the fact that by leveraging its genomic expression profiling capabilities, Rosetta Inpharmatics has now established microRNA overexpression signatures for over 150 microRNAs, raising the question when Merck will officially announce its entry into the microRNA therapeutics arena.

Following on from his identification of SID-1 in being required for systemic RNAi in worms by acting as an siRNA channel, Craig Hunter (Harvard University) presented data to support a model in which siRNAs enter and exit cells via SID-1 by diffusion, whereas other proteins such as SID-2 are involved in binding RNAs so as to increase their concentration close to the siRNA channel. That this is relevant for the development of RNAi therapeutics is highlighted by the recent Nature Biotech paper by Dr. Stoffel from the ETH Zurich and Alnylam where data suggested a role for SID-1 in taking up siRNAs following docking of siRNA-studded lipoprotein particles to their cellular receptors. In addition to walking his audience the published data-heavy paper, Stoffel stated that he saw no reason why it should not be possible to create artificial lipoprotein particles containing siRNAs and targeting agents.

Instead of Dinah Sah or David Bumcrot presenting Alnylam’s progress on cancer RNAi therapeutics, Rachel Myers stood in to give a general overview of the approach Alnylam takes to RNAi-based drug development. Since little new primary data was presented, I thought it is noteworthy that both Ingo Roehl and Myers gave scientific credit for the development of the hotly contested SNALP development to Protiva Biotherapeutics and that the delivery technology used in last week's Nature paper on microRNA competition by siRNAs was called "Alnylam proprietary”. Also, it was a great relief for me to hear that Alnylam had reproduced all of Sailen Barik’s data on the RSV-RNAi treatment paradigm, including efficacy of siRNAs after RSV infection. Indeed, siRNAs reduce viral titers up to 3 days after infection in a mouse model where maximal viral burden is seen by day 4 of the infection. Moreover, illustrating that the upcoming proof-of-concept studies for RSV in the experimental infection model is not merely an academic RNAi therapeutics de-risking exercise, the patients that Alnylam expects to treat with ALN-RSV01 will have infection in the upper respiratory tract with little or no exposure in the lung. Consequently, ALN-RSV01 will be a combination of treating upper respiratory tract infection and prophylaxis for the lung. Everybody, of course, is quite restless now to learn about the outcome of the phase II experimental infection results which appear to be on track and from which will be presented in detail early next year without precluding a PR on the results in December.

Before the session on RNAi/oligonucleotide delivery, a number of speakers spoke about recent findings on the immunogenic properties of various forms of RNAs, including siRNAs. The importance of considering the expression pattern of the main oligonucleotide receptors (the endosomal TLR-3,7,8,9 and the cytosolic RIG-I and MDA-5), their localization, and their exact ligands became evident and will inform modification strategies aimed at avoiding the induction of unwanted cytokine responses following siRNA administration which is something that Rachel Myers noted Alnylam is still trying to get a better handle on (see the delay in the pandemic flu program). Other groups, however, such as Gunther Hartmann’s group from Bonn, embrace the immunogenic properties of some, particularly unmodified siRNAs and would like to combine it with their silencing activity for treating cancer and viral infections. Some encouraging data in that regard were presented.

Song Li (Pittsburgh) presented the use of neutral lipids for delivering a variety of oligonucleotides to the pulmonary circulation. Like others at the meeting, this approach illustrates a trend away from using cationic liposomes which have been associated with interacting with components of the blood and may trigger certain toxicities. Delivery to endothelial cells of larger vessels and capillaries was particularly efficient, and his group is currently collaborating with ISIS on targeting endothelin-1 for treating hypertension. Encouragingly, he was able to report on good gene knockdowns and quite impressive in vivo efficacy data, such as the reduction of hypoxia-induced right ventricular hypertrophy.

Similarly impressive in vivo efficacy data were then reported by Klaus Giese from Silence Therapeutics, mostly located in the host city of Berlin. Without showing all the controls, tumor burden, metastasis, cell proliferation were all strongly reduced following systemic delivery of siRNAs in a number of mouse tumor models. Although I cannot agree with his repeated claims on the uniqueness of their Atu-siRNAi design and related IP claims, the Atuplex delivery technology certainly deserves more credit. Unlike other lipid-based delivery methods, siRNAs here associate with the liposomes externally thus allowing liposomal charge to be modulated from cationic, via neutral, to anionic. Of practical importance, these particles can be lyophylised for storage and shipping and then resuspended without loss of silencing activity. Progress has also been made on lung delivery and a number of pre-clinical programs are being pushed forward into the clinic.

The founder and CEO of the nucleic acid delivery company Novosom, Steffen Panzner, continued the string of impressive in vivo efficacy data in a mouse model of RA (inflammation of the paws) following delivery of their proprietary siRNA-loaded Smarticles, a charge-reversible liposomal delivery technology. One problem of using the more desirable, in terms of safety, neutral and anionic lipids, is their reduced siRNA binding affinity. Novosome’s amphoteric liposomes circumvent this problem by binding siRNAs at low pHs, at which point they are positively charged, and rapidly shifting them to higher pHs for closing the liposomes. During this process, most of externally bound siRNAs are shaved off, with the additional benefit of consequently minimising endosomal exposure of the siRNAs which may trigger TLR7-mediated cytokine responses.

The scientific day was closed by a talk from Alan Sachs of Merck who set out to emphasise that despite a dearth of recent information on Sirna’s/Merck’s preclinical and clinical RNAi therapeutics pipeline, that they were more than ever committed at developing RNAi therapeutics. Unfortunately, this was not followed by a presentation of primary scientific data and specific examples, but rather how Merck thinks about RNAi drug development in general and repeated appeals of please partnering with Merck, particularly in the area of targeted siRNA delivery. It appears to me that Merck would like to rival Alnylam in accessing the best minds in the RNAi drug delivery field and is offering to send out their siRNAs for free so that others can formulate them and report back on their findings without any strings attached as to the use of these results. Sachs feels that Merck, through their expertise on the genomics and genetics of gene expression through Rosetta Inpharmatics, that it knows best which targets are most suitable to go after for drug development. Since siRNAs allow essentially any gene to be targeted, he like other feels that this is the natural drug development platform to be harnessed and thinks about 21 month development timelines from gene identification to preclinical proof-of-concept given the availability of suitable biomarkers. While searching for more targeted delivery solutions and collaborators, Merck is meanwhile establishing a platform for siRNA delivery to the liver and, encouraged by ISIS' 301012 results, is clearly motivated in targeting ApoB100 with siRNAs for the treatment of hypercholesterolemia. I would not be surprised at all, to see them join the PCSK9 frenzy as well. Being second in a $50-60B should not be that bad.
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Quo Vadis, Merck?

It is not often that an early-stage biotech company decides to terminate a collaboration agreement with Big Pharma and the investment community cheers it. But this is exactly what happened last week, after a brief bout of confusion, when Alnylam announced the termination of their partnership with Merck & Co. Inc. for the joint development of RNAi therapeutics, citing moves by Merck they deemed incompatible with Alnylam’s dominant IP in RNAi therapeutics.

Merck was an early entrant into the emerging RNAi therapeutics field, and acknowledged Alnylam’s fundamental technology and IP leadership when they signed a broad co-development deal in 2003. It was clear, however, that Merck’s ambition has always been to play a more prominent role in RNAi therapeutics, and has been instrumental in energising the effort through a number of initiatives such as their support for this year’s RNAi Therapeutics meeting in Keystone, an RNAi delivery challenge, and bringing leading bioinformatics to bear on addressing off-targeting through their Rosetta Inpharmatics subsidiary.

One year ago, Merck woke up the investment community by announcing the acquisition of Alnylam’s chief rival, Sirna Therapeutics, for a then whopping $1.1B. Following the millennium technology bubble and stalled progress with developing ribozyme therapeutics, Sirna transformed itself into an RNAi company by leveraging its existing RNA capabilities to rapidly screen many genes with numerous siRNAs and claim them in their patent applications as exclusive RNAi therapeutics targets. Based on this achievement, Sirna Therapeutics promoted itself as the leader in RNAi Therapeutics, although it has always been questionable whether such a brute-force approach would eventually stand up to the novel and non-obvious criteria required of a patent.

So why did Merck end up buying Sirna, not Alnylam which they chose to partner with in the first place? One can almost be certain that Merck approached Alnylam. However, unlike management at Sirna Therapeutics, schooled in the art of turning around flagging businesses and probably a little bit focussed on maximising short-term shareholder value, Alnylam always had full confidence in the strength of their IP position and the prospect for RNAi therapeutics and consequently put in place mechanisms that would make an unsolicited takeover almost impossible

Ironically, the events that followed the Sirna acquisition by Merck further underlined Alnylam’s dominant RNAi therapeutics IP position. It seems odd that for a company that touted itself as the leader, many of its former staff have now left the company, and it appears that except that Sirna’s proprietary development pipeline, except for its AMD program that it had licensed to Allergan, had been completely overhauled.

From Alnylam’s perspective, the advantage of terminating their partnership with Merck appears clear. As an early deal that largely benefited Alnylam in terms of credibility rather than financially, it was far less attractive compared to the more recent deals with Roche and Novartis (up to $120M compared billions in future milestones plus royalties), so that the freed up time, resources, and RNAi therapeutics targets (nine following an amended 2006 agreement in addition to Nogo for spinal cord injury) can now be focussed on more rewarding partnerships and in-house development programs. It also means that Alnylam would not be fostering a Merck increasingly positioning itself as a rival rather than partner by denying them access to their considerable know-how.

It also becomes more and more apparent that Merck is suffering from the consequences of having inherited Sirna’s aggressive business practices. As part of an ongoing dispute with the Canadian biotech company Protiva Biotherapeutics which accused Sirna of having misappropriated trade-secrets relating to liposomal RNAi delivery, a recent court order is likely to have adversely impacted Sirna’s systemic RNAi delivery efforts with potentially further financial liabilities down the road. In Europe, Sirna was also engaged in opposing Alnylam’s Kreutzer-Limmer patents relating to the use of short double-stranded RNAs for gene silencing in mammalian cells, the very same IP to which Merck gained access through their Alnylam collaboration. After the Sirna takeover, however, Merck appears to have made the crucial mistake of continuing to oppose those patents. I suspect, although I am not familiar with the details of the Merck-Alnylam agreement, that this alone should have given Alnylam the right to unilaterally terminate the type of relationship that Alnylam, in the wake of the Alnylam-Roche alliance, repeatedly said it would never pursue again.

To be sure, the value of Sirna may have been higher to Merck than to other pharmaceuticals since Sirna’s technical RNA capabilities should have nicely complemented Merck’s existing RNAi therapeutics development and bioinformatics initiatives. However, given the strength of Alnylam’s patent portfolio, the Tuschl and Kreutzer-Limmer series to name just two, it is difficult to imagine that Merck is really convinced that it has the freedom to bring RNAi therapeutics to market without a license from Alnylam. It will therefore be interesting to follow whether Merck will now pursue a late, but more costly licensing strategy, or whether it will respond to the new situation in unusual ways. In any case, I hope that the new rivalry between Alnylam and Merck will be a friendly albeit competitive one for the benefit of the entire field of RNAi therapeutics.