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.

Refocusing on the Fundamental Promise of RNAi Therapeutics

Certainly, the world’s economy has been going through a phase of heightened uncertainty and investors might be forgiven if they focused on the near-term rather than long-term endeavors such as the development of a new biotechnology. However, it would not be fair to blame the current apathy towards RNAi Therapeutics entirely on the economy and investor schizophrenia. Instead, the valid apprehensions about the platform with respect to delivery and innate immune stimulation need to be addressed in a scientifically rigorous manner, and then be followed up with a growing pipeline of highly innovative, targeted therapeutic candidates. Moreover, the focus of management and investor communication should be on the therapeutic opportunities rather than raising expectations about IP monetization and non-core business opportunities.

If this reminds you somewhat of Alnylam and appears to be an unfair characterization of other companies in the space, it is no coincident. Alnylam is widely regarded as the bellwether of RNAi Therapeutics and greatly influences the general perception of the technology. Personally, I’m always surprised how high the keyword ‘Alnylam’ ranks, usually among the top 2 search items, when it comes to visitors to this blog. Maybe I shouldn’t be, since Alnylam has certainly earned its place at the top through their vision of accumulating fundamental RNAi trigger IP early on and their ability to monetize on this through their platform partnerships. With close to $450M in the bank, however, and the scientific challenges that have come to the fore, the world would like to see more evidence that Alnylam has conviction that RNAi Therapeutics has real positive net present value.

Playing the devil’s/short’s advocate, what specifically makes one wonder whether Alnylam has lost its enthusiasm about RNAi Therapeutics? Besides the perceived focus on IP monetization over expanding the clinical pipeline, it is a lead program (ALN-RSV01) that targets an organ for which RNAi delivery does not appear to be as robust as e.g. in the liver and appears to be artificially kept alive to buy time until improved follow-on candidates or other programs fill the void; moreover, clinical results that indicated that ALN-RSV01 has an uphill battle in front coincided with the dark days when innate immune stimulation was thought to be a major class effect of RNAi Therapeutics and Alnylam was not seen to be sufficiently proactive; $20-25M of upfront and near-term payments to ISIS Pharmaceuticals for access to its single-strand RNAi Therapeutics-related IP, a technology that lags years behind dsRNAi Therapeutics- contrast this with its reluctance towards spending similar amounts on nearer-term dsRNA delivery opportunities; directing too much (public) focus on non-core business opportunities such as vaccines, stem cells, and even Regulus; no insider purchases in years, and real insider selling (not just option exercises) that makes you wonder about the personal commitment of management and whether they consider Alnylam a good investment at all. As a potential platform licensing partner I would ask myself that if IP monetization and non-core businesses are where Alnylam sees most value in, why should I invest $300M for a simple therapeutic platform license, particularly at a time when events surrounding the Tuschl and Kreutzer-Limmer patents appear to weaken Alnylam’s ability to exclude, and when $600M in payments to Alnylam has not been enough for a Big Pharma partner to enter a single program into the clinic?

The problem is that Alnylam has become the victim of its own IP success, diluting its efforts by trying to lead in all things RNAi Therapeutics, playing it safe by spreading its risks widely, maybe tempted by its enviable financial position. I am aware that there is a conflict between investing in the most valuable near-term opportunities and demonstrating the broad capabilities of a biotechnology platform. While the latter should not be neglected, and Alnylam is already doing a tremendous job by outsourcing much of it to academia and industry by providing siRNA reagents and playing the role of a facilitator, the scientifically limited capacity of a 150+ company could be focused on de-risking the technology further by showing unambiguous on-target RNAi knockdown in vivo, something that it is well placed to do due to its leading siRNA chemistry know-how and siRNA sequence design engine (a nice demonstration of this is the RNAi Roundtable on RNAi Lead Development). Such capability can also be harnessed for being an attractive platform licensing partner (besides having unambiguous freedom-to-operate for its RNAi triggers), as it is obvious that Big Pharma is looking for enablement first when evaluating RNAi Therapeutics partnerships.

The second area of (publicly declared) focus could be on identifying and validating a series of high-value gene targets expressed in organs for which delivery risk is currently lowest: liver, solid cancer, and the eye (the latter maybe even with a dual siRNA/ddRNAi approach). This effort includes hiring appropriate leading disease experts. While Elan Pharmaceuticals has too many management problems to even start elaborating in this blog, one thing that it has done well and what turned out to be a major attraction for investors and pharma partners is to build a world-class scientific team in neurology. In the end, 5-6 therapeutic target areas in which the company intends to develop its own therapeutics is more than enough, a number that many even much larger companies do not go beyond.

While it is possible that competition for therapeutic targets in the liver is intense and one would not like to be too open about specifics, the message right now appears to be that the liver is just one of the areas it views as equally promising, while the reality is that it is likely that after RSV01, the next 3 INDs will be for that organ (VSP02, TTR, PCSK9). One possible way to further catalyze the potential of RNAi Therapeutics for liver-related diseases would be by consistently raising awareness at a meeting like the upcoming American Association for the Study of Liver Disease annual meeting coming up in no other place than Boston, by maybe organizing/sponsoring a session dedicated to progress made thus far in RNAi knockdown in the liver. Emerging public health problems such as non-alcoholic steatohepatitis and HCV are just two examples of very important liver diseases that beg to be evaluated with RNAi. And just perusing the program makes me think ‘just how many more liver-related diseases could there be?’. Again, it may pay here to have a leading therapeutic focus area team in-house and/or dedicated liver disease SAB, lest Big Pharma snatches away the best targets. From an investor relation’s point of view, the message should be that this initial concentration on the liver is very consistent with the fundamental promise of the RNAi Therapeutics platform, namely that once you have identified suitable delivery for a particular organ, you can then rapidly roll out an entire suite of related programs. At the same time, other delivery technologies will mature and eventually start to produce equally numerous drug candidates, with one delivery wave after another feeding the overall RNAi Therapeutics pipeline. Certainly, the consequences of such a strategy if it does not work for the liver at all it won’t be pretty, but working on one problem intensely might get you a higher risk-adjusted probability of success than working on 3 different areas less so intensely.

RNAi delivery is a natural third area of investment focus. Much of the innovation will come from the outside, and Alnylam, as do the Roches and Mercks, needs to have a discerning eye about what could work or not. Of course, relative to other pure-play RNAi Therapeutics companies, having deep pockets and having probably seen close to a hundred if not many more collaboration requests and with its own scouting and experience with delivery, it is in a good position to do well in this game. Nevertheless, Big Pharma has even deeper pockets, although not necessarily the same deep know-how, and the $120M that Roche paid for Mirus’ early-stage DPC delivery technology demonstrates what a large company that cares about RNAi Therapeutics is willing to pay for a differentiated delivery platform that has shown potential in rodents. What may be more in the hands of the company is the study of basic siRNA uptake mechanisms (see the Max-Planck collaboration), and the development of more broadly applicable endosomal release technologies.

The good news is that the perception of RNAi Therapeutics could soon take a positive turn. In my mind, Tekmira’s JCI paper of achieving anti-cancer efficacy in mice through a rigorously demonstrated RNAi mechanism of action can be regarded as a turning point and is for everybody’s benefit. Before that, it almost started to look like non-specific class effects could prove to be a huge headache for RNAi Therapeutics not least from a regulatory point of view. The reason why you see me so excited about Tekmira at the moment is that they have anticipated the two main challenges of RNAi Therapeutics, namely delivery and avoiding innate immune stimulation, years ahead of the rest of the field, and frankly, without them it would be much darker in the lands of RNAi Therapeutics now. I will acknowledge, of course, that without Alnylam it would likely look much darker in some of Vancouver's neighborhoods, too. By achieving similar standards of target validation and being generally forward looking, Alnylam could again set themselves apart from its competitors and almost like a side-effect increase its partnership value. Further events that could soon contribute to this change in perception are the demonstration of clear knockdown efficacy with a reasonable safety profile, with Tekmira’s SNALP-ApoB results expected early next year and Alnylam’s TTR study results probably in late 2010. ALN-VSP02 safety data could add to the safety package. Equally important to me is to see a Big Pharma initiating an RNAi Therapeutics clinical program coming out from its own R&D. This is because RNAi Therapeutics-only companies are tempted to initiate clinical programs just to suggest their scientific prowess with often doubtful scientific rationale (we’ve seen a lot of this), while a technologically more diversified Big Pharma will think long and hard about whether RNAi Therapeutics is ripe for the clinic. Roche's intention for a SNALP IND in 2010 is therefore very encouraging.

Again, it is ironic that it is the success of Alnylam thus far that has facilitated a situation in which it is sometimes regarded as sluggish as a Big Pharma when compared to companies like Tekmira and even the New mdRNA both of which have looked into the abyss before and appear to be highly motivated and make efficient use of their capital. There is no more bullish signal to me when the Chief Scientific Officer of a company invests a significant chunk of his personal wealth into his company’s stock in the midst of the economic crisis and when the company is trading at half its cash. And sometimes I look back in nostalgia to the good old days when the Alnylam-Sirna rivalry kept both companies on their toes and increased investor interest.

Focus on platform partnerships was a necessary stage in the history of the company, but with $450M cash guiding for a platform deal is not essential and has only downside potential. It is now late October and we have yet to hear of the first of the two significant partnerships promised for this year and this is hurting share price. Unfortunately, this diverts attention from what this company is really about: applying the ability to down-regulate essentially any protein-coding gene towards the treating virtually any disease at its root and in a cost-efficient manner. If the excitement can be restored through clear scientific and clinical progress, share price will follow, partnerships will be regarded a pleasant surprise, all of which is good for investors and for speeding up the development of RNAi Therapeutics.

Aptamer-siRNAs: Another Shot at RNAi Therapeutics Delivery

There has been a trickle of papers lately describing the use of aptamers for the functional delivery of siRNAs such as for cancer and HIV. Aptamers are highly folded, 35-100 nucleotide long RNAs that can bind protein targets with relatively high affinities and specificities. One way of thinking about them is as the RNA equivalent of antibodies. Aptamers already are being tested as a therapeutic class of its own where they are typically designed to neutralize extracellular targets, with already one aptamer (Macugen) approved for wet AMD.

As such, aptamers should lend themselves for targeting associated therapeutic siRNAs to cells of interest, in a sense functioning like antibodies and small molecules that have likewise been recruited for targeted RNAi delivery. What distinguishes an aptamer-siRNA combination, however, is the promise of having to simply use only RNA synthesis to generate a pharmacologically viable siRNA therapeutic, obviating the need for complicated formulation technologies. Furthermore, when it comes to repeat-administration such a system may cause inherently little adaptive immunogenicity.

The reason why I have been somewhat skeptical on this technology is that like with so many siRNA targeting approaches, getting to the cell of interest is just a first step, and it is not obvious to me how after e.g. receptor-mediated endocytosis the rather large aptamer-siRNA conjugate would be able to cross the negatively charged lipid bilayer to get into the cytoplasm for incorporation into the RNAi-induced silencing complex (RiSC).

Nevertheless, a recent study in Nature Biotechnology (Dassie and colleagues: “Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors”) suggests that competitively low mg/kg dosages of intraperitoneally injected prostate-specific membrane antigen- (PSMA) targeted aptamer-siRNAs can efficiently knock down the popular cancer target PLK1 in a mouse xenograft model of prostate cancer. The study is a follow-up of a 2006 paper published in Nature Biotech by the same group from the University of Iowa (McNamara and colleagues: “Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras”) where intratumorally injected- i.e. not systemically administered- PSMA-targeted aptamer-siRNAs showed very efficient inhibition of tumor growth in the same model system.

Since systemic administration is deemed to be necessary for an siRNA therapeutic against prostate cancer, the investigators reasoned that they could achieve such delivery by increasing the potency of the aptamer-siRNA by primarily improving siRNA potency through siRNA design (changing an initially blunt siRNA into a Tuschl-type 3’ overhang type) and then attaching the ubiquitous PEG to increase circulation times so that the aptamer-siRNA would have an increased chance of finding its target. As hoped for, both strategies substantially improved in vivo performance. Impressively, PEG addition increased the half-life of the molecule from less than 35 minutes to over 30 hours (!) and this was accompanied by improved silencing and tumor inhibition. The 3’ overhang siRNA (actually it was a Dicer substrate- more on this later) was also much better than the original blunt-ended version. While most aptamer-siRNAs are bi-molecular which reduces the maximum length of RNA to be synthesized, a unimolecular precursor microRNA mimic performed best. This could due to increased stability of an intramolecular duplex and/or a more “natural” appearance to the RNAi machinery. Practically, however, bimolecular conjugates may be preferable as RNA synthesis becomes exponentially less efficient with size and is also for this reason that the authors further reduced the length of the aptamer from the earlier study.

Overall, all of the many controls that they were probably asked for by the reviewers confirmed the specificity of the results: the therapeutic effect correlated very well with the degree of knockdown, both in vitro and in vivo; binding and silencing was only observed in PSMA-bearing cells; no innate immunostimulation that might explain the anti-cancer effect was detected; 5’ RACE showed that there was in vivo RNAi activity. Finally, only ~21nt siRNAs were detected following administration of the Dicer-substrate RNAi triggers which suggests highly efficient Dicer processing. Generally, it has to be said that while the shorter, traditional siRNAs have many advantages in terms of specificity and immunity, Dicer-substrates may be ideally suited for conjugate approaches such as this, as Dicer-processing would liberate and thereby activate the functional siRNA whereas Argonaute loading, in theory, should be diminished by a direct conjugate to the siRNA (however, strategies such as reversible disulfide bonds might work for such a configuration).

As an aside, the studies are further validation of PLK1 as a very good target for RNAi Therapeutics in oncology. PLK1 is one of the most highly over-expressed genes in cancer, and knockdown studies have shown that cancer cells are very sensitive to the reduction in PLK1 levels while normal/healthy cells, even if transfected with PLK1 siRNA are unaffected. PLK1 is also the target for a SNALP cancer therapeutic candidate developed by Tekmira for solid cancers that is slated for IND next year (Alnylam with a 50:50 opt-in right until start of phase II).

In a sign that there is also commercial interest in aptamer-siRNAs, the leading aptamer company Archemix and Dicer-substrate company Dicerna recently agreed to collaborate on aptamer-siRNA delivery. Archemix, which shares a building with Alnylam, similarly chose to collaborate with heart- and muscle-focussed miRagen on the delivery of microRNA therapeutics. Archemix’ sudden move into small RNA therapeutics is also quite interesting given their failed IPO attempt and speculations of a reverse takeover of Silence Therapeutics.

So where do I think aptamer-siRNA delivery technology stands? I’m still somewhat skeptical and would like to see more of these studies from various laboratories. An important question that was posed by an accompanying News and Views article from Alnylam scientists (which btw makes it very likely that the paper was reviewed by them) is whether the surprising cytosolic uptake of the RNA is a peculiarity of the PSMA antigen or could be a more widely mechanism for presumably endosomal escape that could be exploited. Studies into the precise molecular mechanism of the uptake, as with all RNAi delivery systems, are needed. One could also imagine that to enhance uptake, membrane-active agents may be added to the PEG-aptamer-siRNA, although this would be contrary to the initial concept of a simple design. In summary, the more varied approaches being explored, the better for RNAi Therapeutics. For now, aptamer-siRNAs are just one of those to be watched.

MicroRNA-26 yet another Small RNA in the Fight against Liver Cancer

When you combine the fact that current systemic small RNA delivery technologies should work best for the liver and solid cancers, that RNAi opens up many of the well validated, but hitherto un-druggable cancer targets, and that endogenous small RNA regulatory pathways (microRNAs in particular) turn out to play central roles in cancer biology, then it should not come as a surprise that various Small RNA Therapeutics approaches are poised to greatly advance the care of liver cancer patients. Surgical resection is the most common treatment, while the pleiotropic small molecule inhibitor Sorafenib that has been shown to increase median survival from 7.9 to 10.7 months is the most advanced drug for this treatment. Clearly, in light of this and the over 600,000 deaths from primary hepatocellular carcinoma (HCC) alone each year, many of them in East Asia (HBV-related), and many more deaths from the metastatic spread of other cancers to the liver, the unmet medical need is significant.

This potential has not been lost on the RNAi Therapeutics industry. There are now a number of RNAi Therapeutics liver cancer programs already in or approaching the clinic, most prominently Alnylam’s VSP-02, but also others such as one by mdRNA. At the same time, there is increased validation for microRNA-based therapeutics for liver cancer both of the antagonist type (e.g. Rosetta-Regulus collaboration) and the agonist type. In the case of the latter, two recent high-profile papers in Cell (Kota et al., 2009: Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model) and the New England Journal of Medicine (Ji et al., 2009: MicroRNA Expression, Survival, and Response to Interferon in Liver Cancer) suggest that microRNA-26 mimicry is a serious contender.

Kota and colleagues got started after making the observation that miR-26a is strongly down-regulated in a mouse model of liver cancer driven by the myc oncogene. Importantly, miR-26a levels were subsequently found to be also reduced by about 50% in liver cancer samples compared to the matched healthy liver tissue. While global down-regulation of microRNAs is a well known general property of cancer, what made miR-26a a particularly attractive candidate for microRNA replacement is that a) it is a broadly expressed microRNA and over-expression in non-target cells therefore should be well tolerated; and b) its over-expression in a liver cancer cell line decreased cell proliferation which was attributed to miR-26a directly targeting the cell cycle regulators Cyclin D2 and E2.

They then chose AAV technology to deliver a microRNA mimic to the livers of the same mouse model. AAV delivery to normal liver can be extraordinarily efficient, with essentially 100% transduction using so-called self-complementary vectors in mice. The track record for HCC has been more mixed, but using a self-complementary vector of the AAV8 serotype the authors achieved more than 90% transduction. To their great satisfaction, tumor growth was greatly retarded following systemic administration of the mimic compared to an expression vector in which the microRNA cassette had been deleted (60% tumor burden to 20% tumor burden). Importantly, whereas there was wide-spread apoptosis in the liver cancer, the surrounding normal tissues and more distant tissues with a high proliferative index (e.g. testis) appeared to be untouched. This was the first demonstration of efficacy for a systemically administered microRNA mimic that was not targeted against the cancer-initiating oncogene itself.

Further support that miR-26a is a good candidate for mimicry in liver cancer comes from a bioinformatic study just published in the New England Journal of Medicine which confirmed in a population of HBV-related HCC in China the down-regulation of miR-26 in liver cancer. Moreover, as predicted by the Kota et al. study, liver cancer patients with the least amounts of miR-26 had a worse prognosis. On the other hand, it is the same miR-26 lo patients that benefited most from interferon-alpha therapy. Such a finding may have its clinical use since for a drug with borderline efficacy like interferon in liver cancer. Being able to exclude those patients that will not benefit from a given drug may give them an opportunity to try other drugs or at least be spared of the side-effects. When used during drug development as a companion diagnostic, of course, such a test would moreover have major benefits for increasing the success rate of developing a drug candidate. Companies in the microRNA Rx/Dx space like Rosetta Genomics, Asuragen, and Regulus may want to take a look at being part of a companion Dx-Rx development program where patients are selected for a miR-26 mimicry liver cancer trial based on their miR-26a status. More generally, I shall look forward to seeing a wider adoption of microRNAs as companion Dx and, looking out from my soapbox, even a tighter integration of microRNA Dx/Rx capabilities.

In summary, miR-26a mimicry is yet another promising small RNA approach to treating a disease with a high unmet medical need, not just sometime in a distant future, but very tangible. Again, the lesson here is that once a given organ can be addressed by small RNA delivery, a flood of therapeutic targets immediately become available to keep Small RNA Therapeutics busy for some time on this one organ alone.