Journal Club: A commonly used treatment for HCV, Interferon Beta, may largely act through microRNAs

While on vacation, an interesting study on the effect of interferon beta on microRNA levels was published in the journal Nature (Pedersen et al.: Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature doi:10.1038/nature06205).

In this study, Pedersen and colleagues were initially interested in whether interferons had the potential to modulate cellular microRNA levels. Not very surprisingly, this potent class of cytokines up- and downregulated a number of microRNAs. Strikingly, however, eight of the interferon beta-induced microRNAs had microRNA seed complementarities with an HCV genome. Moreover, miR-122, a microRNA that has now been shown by a number of laboratories now to facilitate HCV replication, was downregulated by interferon beta.

The link between HCV and interferon-regulated microRNAs is intriguing, since interferon beta is at the center of current HCV treatment regimens. In order to test whether the antiviral activity of interferon beta on HCV replication was indeed mediated by microRNA regulation, the authors asked whether interferon beta could still inhibit HCV replication in the presence of mimics of the upregulated and HCV matching microRNAs and an inhibitor of miR-122. In agreement with the notion that interferon-regulated microRNAs mediate a large part of interferon beta inhibition of HCV, such a mixture of small RNAs alleviated interferon beta inhibition of HCV replication from 90% to around 50% of untreated control in a tissue culture system.

HCV has a long-standing tradition in the RNAi Therapeutics field. As such, a number of drug candidates are expected to enter the clinic in the near future that directly target the HCV genome by RNAi. In addition, since HCV replication is supported by miR-122, it has become the focus of the first wave of microRNA-targeting therapeutic programs. Due to the ability of viruses to escape drug inhibition through mutation, a combination of these approaches appears promising. As much as no other current HCV antiviral alone can reliably get rid of HCV altogether, I do not expect any RNAi-related stand-alone therapy for HCV to be successful. However, when combined with potent agents such as Vertex Pharmaceutical’s late-stage protease inhibitor VX-950, RNAi may be able to further knock down HCV sufficiently so that it can be entirely cleared by the body. Moreover, many patients do not complete interferon therapy due to its severe side-effect profile, and alternatives are desirable. The strategy proposed in the paper may therefore lead to a treatment that works through the same antiviral pathway as interferon beta, but without the side-effects.

Lastly, I would like to briefly comment on the evolutionary aspects of the studies. It is very unlikely, given the rapid evolution of viruses alone, that the sequence of the implicated microRNAs was shaped due to selection based on HCV inhibition. Accordingly, the authors find that the sites complementary to the microRNA seeds are not all conserved in the different HCV genotypes (note: whether this is related to the varying efficacy of interferon beta on different genotypes in the clinic was not discussed). It is only through comparing the modulated microRNAs with a lot of viruses that they found the link with HCV. It is therefore fortuitous that interferon-modulated microRNAs should have anti-HCV activities. Of note, this is similar to a paper published 2 years ago in the journal Science (Lecellier et al.: A cellular microRNA mediates antiviral defense in human cells. Science 308: 557) which showed for the first time that a cellular microRNA may restrict the replication of a mammalian virus through good fortune.

The Race to Knocking Down Cardiovascular Disease

Given the burden of cardiovascular disease in the Western world representing a multibillion dollar drug market, finding a drug to complement statins in reducing complications due to high levels of bad cholesterol is naturally high on the priority list of many drug developers. The recent OTS Meeting and a Press Release by Alnylam emphasising their leadership by having obtained first-ever data on safely and effectively knocking down PCSK9 with RNAi in non-human primates, illustrate home the promise of RNA-based therapies for CVD. The interest is largely rooted in the fact that targets such as PCSK9, ApoB100, and potentially microRNA-122, well known determinants of blood cholesterol levels, but which have proven impossible to target by traditional small molecule approaches. Moreover, these targets are expressed in the liver, and it is clear by now that current systemic oligo delivery technologies allow them to be knocked down in vivo. Hence, the race is on to who will be first to develop a safe and efficacious oligonucleotide-based therapy for hypercholesterolemia and stand to reap the benefits of a potential blockbuster in the first phase of RNA-based drugs.

Assuming that it is a safe bet that cholesterol levels can be reduced with oligo-based strategies, what will determine regulatory success? Given that low cholesterol is a life-long effort, any drug taken over a long period of time, even before disease onset, will have to be safe first of all. Risk can be largely grouped into four categories: target risk, risks inherent to the therapeutic platform, sequence risk, and risks associated with route of delivery and drug formulation. Arguably the target best validated on the grounds of human genetics is PCSK9, a protease that degrades LDL-receptors and therefore inhibits clearance of bad cholesterol from circulation. Research mostly from the University of Texas Southwestern has shown that mutations that increase the activity of PCSK9 increase cholesterol levels, whereas individuals with nonsense mutations in PCSK9 that reduce PCSK9 activity have lower cholesterol levels and, importantly, a much reduced risk for cardiovascular events. Moreover, the absence of any functional PCSK9 throughout life has no obvious adverse side-effect while retaining the health benefits of low cholesterol.

Before PCSK9 came to the fore, ApoB100, a protein required for the assembly of LDL-cholesterol, used to be the target of choice. Indeed, the development of PCSK9-based treatment strategies have extensively made use of ApoB100 as a marker protein for evaluating RNAi delivery and knockdown in the liver. Pioneering research mostly by ISIS Pharmaceuticals has shown that indeed ApoB100 knockdown has the ability to lower LDL-cholesterol. Although ISIS has not seen fatty liver in clinical trials and preclinical research of their lead antisense compound ISIS 301012 (currently in late phase II) to be a problem, various other groups have observed this side-effect following ApoB100 knockdown, which would not be that surprising given the role of ApoB100 in fat metabolism. However, even if fatty liver will be observed in larger phase III trials and post-approval, ISIS has made the right decision to test 301012 first for patient populations most at risk for CVD.

Similar to ApoB100 and PCSK9, inhibition of microRNA-122 by antisense technologies has been now shown numerous times to also have LDL-cholesterol lowering effects. Strangely, despite the fact that this is by far the most abundant microRNA in the liver, no obvious toxicities have been associated with miR-122 inhibition. Consequently, a number of groups such as Regulus and Santaris hope to develop this into a treatment for hypercholesterolemia.

Taken together, my bet is on PCSK9 knockdown to lead the way in oligo-based therapies for the long-term treatment of hypercholesterolemia. New targets, however, should emerge, partly as a result of now being able to apply RNAi itself for target identification, for example by transiently targeting essentially any gene of interest in the liver in vivo and the use of transgenic RNAi mice (Artemis), a combination of the two latest Nobel prize-winning technologies.

Next to target choice, the nature of the knockdown technology, antisense versus RNAi, itself will also have important safety implications. As I am quite fascinated about the prospect of RNAi for various reasons, please keep in mind that my natural inclination is to favour RNAi any time. In terms of potency, once equal amounts of oligos get delivered into the cell, RNAi has been shown frequently to be generally superior to antisense oligos (ASO), although antisense technologies can be quite diverse. Lower dosages will not only reduce cost of a treatment that has to be taken long-term, but, more importantly, allow for dosages that fall well within therapeutic windows. Moreover, in the case of RNAi, I feel quite comfortable with a technology where the risks such as immuno-stimulation, off-targeting, and potential interference with the endogenous microRNA pathway are reasonably well understood, intensely studied, bioinformatics- and chemistry-based solutions devised, and well taken into account in current RNAi-based drug development efforts. This in fact reflects a new awareness in RNA-targeted therapies, largely driven by the renewed interest generated by the discovery of RNAi. Accordingly, the therapeutic utility of any two RNAi compounds, or antisense compounds for that matter, may differ dramatically due to sequence-dependent toxities.
These toxicities may also be linked to route of delivery and related oligo formulation. A technically quite uncomplicated approach, as taken by 301012, is to simply administer relatively large amounts of unformulated oligos (200mg/week in the case of 301012) to make sure that enough of it ends up in the liver. By contrast, liver uptake of siRNAs is thought to require additional formulation. Indeed, liposomal formulations that are set to enter the clinic within the next year increase liver uptake of siRNAs from less than 1% of injected material to over 30%, allowing for lower dosages to be used. Some toxicities, however, were observed at relatively high dose levels with some of the cationic liposomes, and it remains to be seen whether lipidoids and other “not-so-cationic” liposomes will come to dominate the liver delivery field. Also, while most of the disclosed liposomal delivery vehicles efficiently enhance liver uptake, they are often not specific for uptake into the hepatocyte population in the liver, the cell type of interest. Particularly uptake into Kupffer cells, a type of immune cell in the liver, can lead to dosing and safety complications, and ultimately the path taken recently by scientists from Mirus, which by the way has an RNAi delivery collaboration with Pfizer, to specifically target formulated siRNAs to hepatocytes, but not other liver cell types, may substitute non-specific liposomes in the second wave of RNAi-based therapies for hypercholesterolemia. While delivery is often described as the Achilles Heel for RNAi therapeutics, the charge (ironically) and chemical similarity of siRNAs as a class makes them ideally suited to devise drug targeting strategies that can be broadly applied and should lead to safer therapies, something that is nearly impossible for say small molecules.

ISIS’ ApoB100-targeting antisense 301012 has good chances of becoming the first oligo-based therapy for CVD, at least for people with familial hypercholesterolemia and for whom statins don’t work. Although only a fraction of the overall market, the sheer size of the cholesterol market makes this a lucrative goal nonetheless. I am somewhat surprised that, to my knowledge and despite potential target risk, there is little talk of other ApoB100-targeting therapies. It will be interesting to see what companies like Merck, which has clearly stated their admiration for 301012 at the last OTS Meeting, are willing to pay for rights to 301012. PCSK9-targeting therapies are in late preclinical development and therefore about 3 years behind 301012, but I believe these to be the safest bet for a widely applied oligo-based drug for hypercholesterolemia with a number of organisations ramping up their PCSK9 programs.

Alnylam appears to be leading this race with the recent announcement of first-ever non-human primate data of an RNAi compound that safely and effectively knocked down PCSK9 with concomitant reductions in total and LDL-cholesterol. An IND is planned for the end of this year, or early next year, and probably will depend on finding the delivery solution that most importantly is safe for long-term administration. Importantly, Alnylam enjoys a particularly strong IP position and know-how in targeting PCSK9 by RNAi, due to their own position in fundamental RNAi technology, and important collaborations on the biology of PCSK9 with UT Southwestern, which has been leading in the genetics of PCSK9, as well as in delivery with the Anderson/Langer lab at the MIT and exclusive access to Tekmira’s cationic liposomal delivery IP for RNAi. Sirna-Merck may want to dispute this with an patent on targeting the same PCSK9 by RNAi that issued recently and was filed in July 2006 as part of their brute-force approach to patenting genes for RNAi. Alnylam, however, presented their first PCSK9 RNAi data in mice at last year’s 2nd Annual OTS Meeting, and it is anybody’s guess when their or rather UT Southwestern’s first lab-book entry on PCSK9 RNAi occurred. Probably at a similar stage to Alnylam is the PCSK9-antisense collaboration of ISIS with Bristol-Myers Squibbs for which mouse data have been published earlier this year. Santaris’ antisense compounds for PCSK9/ApoB100 and miR-122 should also be heading soon towards the clinic.

New delivery technologies, including oral formulations, and targets should ensure that the oligo-CVD field will remain lively in the years to come. Also, since there have been a number of recent data demonstrating efficient targeting of RNAi to the endothelia of blood vessels, new RNAi strategies aimed directly at the atherosclerotic plaques may emerge.

It would not be the first time that several similar compounds, small molecule, antibody or recombinant protein, with essentially the same molecular targets, would co-exist in a market, a concept also very familiar to the hypercholesterolemia field. IP, careful clinical development involving the best scientists in both oligonucleotide technology, delivery and the biology of the drug targets, together with a bit of luck, will decide who will reap the largest benefits from the potentially first knockdown blockbuster.

3rd Day of the OTS Annual Meeting: Novartis Demonstrates Anticancer Activity of Orally-administered siRNAs

The third and last day of the conference was another action-packed day with topics ranging from the basic science of RNAi to its clinical applications.

To better understand endogenous microRNA function and siRNA off-targeting, transcript analysis such as high-content microarrays has been widely used. This, however, captures only the changes in RNA levels, which is a problem since microRNAs and off-targeting have been thought to frequently act at the translational level with little changes in transcript abundance. Transcriptional analysis should therefore miss important microRNA targets and siRNA off-targets. Nikolaus Rajewski introduced an intriguing solution to this problem by harnessing a labelling technique coupled to mass-spectrometric analysis that allows changes in protein levels as a consequence of small RNA over-expression or inhibition to be measured in high-throughput. Preliminary results confirm that this technique indeed retrieves those targets that have been down-regulated both on the RNA and protein levels, but also discovers those genes where only the final protein output has been altered. If sufficiently robust, this technology should ultimately facilitate the development of, most importantly, safer, but also more potent RNAi medicines.

Arndt Borkhardt from Duesseldorf, one of the first to publish applications of RNAi in humans in 2002, presented an intriguing correlation between trisomy 21 (Down Syndrome)-related childhood leukaemia and over-expression of microRNAs encoded on chromosome 21. Importantly, only leukaemic cells, but not normal tissues over-express these microRNAs. Using zebrafish as a model system, he was able to show that the inhibition of these microRNAs indeed caused haematological abnormalities, corroborating a causal contribution of these microRNAs to leukaemia. It not strongly supports the rational of using microRNAs as cancer diagnostics, but also suggest these microRNAs to be valid targets for the treatment of childhood leukaemia. Continuing along the lines of microRNAs as therapeutic targets, Hermona Soreq from Israel made a compelling case that miR-132 and miR-182* (star) are important feedback regulators of the cholinergic anti-inflammatory response and may be targeted for modulating inflammatory responses in disease. Her geographical proximity to Rosetta Genomics makes me wonder whether this company will or has already become involved in thinking about clinical applications centered around miR-132 and 182*.

Jost Seibler represented Artemis, an Exelixis subsidiary specialising on generating mouse RNAi transgenic disease models. They have developed technologies that allows them to use constitutive and conditional (tet technology) RNAi transgenics in a matter of around 4 months, which far exceeds timelines for producing knockout animals. This makes these animal attractive target validation tools. Importantly, a number of examples demonstrated that essentially knockout phenotypes can thus be generated with U6 and H1 promoters driving shRNAs. It was also comforting to see that the shRNA hairpins worked in essentially all cell types and tissues with weaker knockdown only in B-cells and the spleen in general. It will be interesting to see whether other RNAi inducers work more efficiently in these cells and whether this is the consequence of some type of selective disadvantage on cells expressing high levels of small RNAs. 34 constitutive, and 48 conditional knockout models have already been successfully generated.

Volker Patzel definitely raised some eye-brows when he reported that specifically siRNA, and not other nucleic acid types, triggered gene silencing in a variety of prokaryotes. Complicating things was that the mechanism of action ranged from transient to persistent knockdown and even gene knockout effects, and that a variety of parameters such as cell transfection competency and host factors made the technique quite tricky. John Rossi certainly made a good point suggesting that bacterial genetics is most likely to uncover the mechanism of the observed silencing effects.

John Rossi himself was up next providing an update on the lentiviral RNA trials for HIV/AIDS that got under way earlier this year at the City of Hope (a collaboration with Benitec). This trial involves a lentivirus expressing 3 types of RNA therapeutics, an RNAi shRNA hairpin against an HIV gene, an HIV-TAR decoy, and a ribozyme directed against the host factor CCR5. It is a particularly important trial since this is the first time in gene therapy that not only a lentivirus is used as a vector, but also DNA-directed shRNAs are employed. Preclinical parameters such as safety and efficacy as measured by vector persistence, expression, HIV knockdown, and the ability of the pluripotent precursor blood cells to normally differentiate after lentiviral transduction were very encouraging. Unfortunately, his hopes to present data at this meeting from treating the first of three enrolled phase I HIV leukaemia patients were dashed due to 65% cell viability after a thawing step in the protocol, when at least 70% was the target. The FDA would have still allowed treatment to continue, but the clinical investigators felt they should not move the goal posts, and it is hoped that the 2nd and 3rd patient cells will fare better, so that they can be re-infused with the HIV-immunised blood progenitor cells. In the second half of his talk, Rossi detailed experiments inspired by a recent report from Bruce Sullenger’s group on successfully using RNA aptamers for targeting siRNAs in vivo. This is an interesting targeting approach since both the targeting agent as well as the therapeutic moiety derive from the same chemical class. As an alternative to the above lentiviral strategy, he would now like to evaluate such aptamer-siRNA RNA hybrids to target anti-HIV siRNAs systemically to infected cells with an gp120 aptamer developed by William James from Oxford University.

Arguably the highlight of the day was a presentation by Francois Watt from Novartis demonstrating for the first time sequence-specific anticancer activity of ORALLY administered siRNAs. These experiments were motivated by preceding mouse cancer model results using local and introperitoneal administration of siRNAs targeting the angiogenesis factors VEGF-R2 and Tie-2. These siRNAs were stabilised with an undisclosed modification at the 3’end for exonucleolytic stabilisation of the siRNA, which was confirmed by incubating them in gastric acids and intestinal lavage as a prelude to the oral delivery experiments. I wonder whether the 3’ end modification also facilitated the presumable uptake of the siRNAs. Clearly, it will be important to quantify the bioavailability of the otherwise unformulated siRNAs. Relatively large amounts of siRNAs had to be administered (100mg/kg), but I expect this to come down with more experience on oral delivery. Since Novartis had been involved in antisense development before their interest in RNAi, it can be speculated that their 3’modification had been informed by previous antisense results and may be covered by relevant modification IP. For all you Alnylam watchers, such positive data augurs well for Novartis exercising the Alnylam adoption license. Overall, my impression is that while companies can be very secretive with their corporate strategies, scientific data are often more believable since the pressure for them to publish glowing results in leading academic journals is less than for academic researchers. Also, they should have little motivation to further pour money into corporate partnerships if the science did not support it.

Francois Watt was a difficult act to follow, but Jeremy Heidel from Calando did a good job by detailing their development of cyclodextrin-based nanoparticles for delivery of siRNAs in cancer applications. Much of the data had already been published, but it was still a useful summary of their preclinical efficacy and tox studies for their first clinical program targeting RRM2 with transferrin-decorated nanoparticles. While transferrin did not affect the biodistribution of these particles, it enhanced siRNA uptake presumably simply by bringing the siRNA into proximity of the target cell which may then take up the siRNAs by transferring-independent mechanisms, a theme similar to the findings by Stoffel and Alnylam (recent Nature Biotech paper) and others. Importantly, these nanoparticles are bioactive at low mg/kg concentrations and can be safely repeat administered up to 27mg/kg. The only immune response observed was directed against the human transferrin component of the nanoparticles in mice, and importantly not the PEG component. This response against the increasingly popular transferrin should therefore not be observed in humans. Everything appears to be now in place for Calando to start their first systemic RNAi therapeutics trial.

Of course, one has to be aware that in the past many promising preclinical results in mouse models of cancer were not held up in human studies. This, however, is more a problem of choosing appropriate model systems and marrying them to the right targets, less a problem of the therapeutic platform per se. I am hopeful that, once efficient targeting is achieved, RNAi therapeutics will have a higher success rate in cancer than other treatment modalities since the choice of the target is not limited by the molecular structure of the protein.

Posters- an interesting poster on flu (influenza A) RNAi was presented by the infectious disease group at the MPI in Berlin showing that LNA-stabilised, but not unmodified siRNAs, when formulated with PEI caused potent knockdown of flu viral titers in a mouse model following intravenous administration. Santaris, the LNA antisense company had a poster dedicated to their ApoB100 antisense program, showing that 12mer LNA gapmers are better at suppressing ApoB100 than longer gapmers. However, I was puzzled by their findings that not only was LDL potently reduced, but that HDL was also suppressed.

Lastly, I wanted to find out more about the rationale for BMS to partner with ISIS for targeting PCSK9 by antisense, when their poster presentation on targeting PCSK9 by RNAi was so positive. I was taught that Big Pharma may use different techniques for their target validation and that the ultimate choice of the targeting technology was determined by other factors. Apparently, the greater experience with systemically administered antisense compounds in the clinic compared to RNAi and the need for formulating siRNAs for efficient delivery to the liver, trumped the need for considerably higher doses of antisense oligos to achieve equivalent knockdown potencies. Ultimately, I came away with the impression that while BMS saw the promise of PCSK9 in the treatment of hypercholesterolemia, Alnylam was just not willing to partner their PCSK9 program at this early stage. In any case, I wish BMS and ISIS well on developing PCSK9-targeted therapies and it would not be unprecedented if two different technologies targeting the same gene will ultimately come to market. I expect, however, that the therapy with the better safety profile will ultimately dominate in the market. While much is being made about the off-targeting potential of siRNAs, it is remarkable that 3 days of observing RNAi next to other nucleic acid-based therapies, demonstrated that while the RNAi field tackles all sorts of potential safety considerations head-on, off-target profiling and the study on the induction of cytokine responses were essentially missing in the presentations on other RNA-targeting technologies.

The 3rd Annual Meeting of the Oligonucleotide Society was a full success. Many thanks to the organisers and presenters for this!!! This is a time when the right ingredients to making oligonculeotide therapies are clearly coming together, and this should become even more evident at the 4th Meeting to be held next year in Boston.

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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Impressions from the 3rd Annual Meeting of the Oligonucleotide Therapeutics Society: Day 1

The 3rd Annual Meeting of the Oligonucleotide Society (OTS) got under way in the stylish Langenbeck-Virchow Auditorium situated in the centre of a culturally vibrant Berlin. The OTS brings together the many disciplines in the rapidly expanding field of oligonucleotide-based drug development, ranging from aptamers, ribozymes, antisense, immunostimulatory nucleic acids to RNAi. Reports from clinical studies of these treatment modalities and oligonucleotide chemistries were at the centre of today’s lecture sessions.

Brett Monia from ISIS Pharmaceuticals chaired the first session and set the stage by reporting that while there are only four approved oligo-based therapies, more than 50 are currently in clinical development. Many of these are designed for cancer applications, but infectious, metabolic, and inflammatory disease applications are emerging as major new focus areas. This may not only be due to considerable unmet medical needs, but also because these may offer better success rates for clinical development. A few talks on cancer therapies with ASOs (antisense oligos) documented the challenges of designing smooth clinical trials for cancer therapies. One problem is the heterogeneity of cancers and matching the gene target with the right cancer. Early trials on a given compound typically involve small patient numbers with a wide spectrum of cancer manifestations. Although safety trials, almost anecdotal evidence of biological activity in a few patients then often guide the design of later phases of clinical development. Clearly, for cancer, there is a need for the development of more reliable biomarkers as early indicators of therapeutic efficacy and for patient stratification, which may be something that Rosetta Genomics’ microRNA diagnostics may be able to facilitate.

Focusing on diseases like viral infection and metabolic disease where efficacy can be routinely evaluated in early stages of clinical development (viral titers, LDL-cholesterol, glucose) should therefore be lower hanging fruits for the next wave of oligonucleotide-based therapeutics. This transformation in the industry is illustrated by ISIS’ new focus on metabolic disease and its out-licensing of the more challenging ASO applications. Rosanne Crooke recounted the clinical experience with 301012, ISIS’ lead compound for the treatment of hypercholesterolemia. As I wrote earlier, one major concern with targeting ApoB100 is the risk of steatosis of the liver due to inhibition of a protein required for the export of triglycerides and which has indeed been observed by a number of groups targeting ApoB100 by RNAi. Possibly motivated by the existence of individuals with null mutations for PCSK9, the new target of choice for hypercholesterolemia, with no evidence for adverse effects due to lack of the LDL-receptor processing protein, a conference participants asked about the existence of ApoB100 knockout mice. Interestingly, no such mice exist, because ApoB100 would have additional roles during development.

Despite reports of steatosis due to knockdown of ApoB100 with RNAi, Sirna Therapeutics is yet another biotech company interested in targeting it for lowering cholesterol. In a talk about siRNA delivery to the liver, Barry Polisky, CSO of Sirna Therapeutics/Merck also noted that in Sirna’s experience, there are genes that can be knocked down extremely well (e.g. ApoB100) and some that are relatively refractory to knockdown by RNAi. Although anecdotal, given the extensive experience of Sirna Therapeutics with screening for effective siRNAs by tiling them across entire genes, and similar findings by Qiagen and others, it will be interesting to determine what makes a gene a good target for RNAi, particularly whether it involves its normal regulation by microRNAs.

Polisky further focused on the need for siRNA modifications to make RNAi therapeutics a reality. Currently, Sirna’s standard siRNA design seems to involve si(R)NAs that lack essentially all 2’OH groups, except for the three 5’ nucleotides of the guide strand, while the passenger strand has 5’ and 3’ modifications that avoid it to be loaded into RiSC to reduce off-targeting. Other speakers and posters also noted that since most nucleotide modifications had been developed with antisense therapies in mind, new modifications may exist for optimal RNAi efficacy, particularly in vivo. Indeed, new synthesis strategies are being developed that will allow this to be tested, such as the 6-membered carbohydrate substitutions of ribose reported by Piet Herdewijn from the University of Leuven, Belgium.

Polisky further characterized siRNA delivery to the liver as efficient, albeit not optimal. Progress is clearly illustrated by the fact that >30% of siRNA formulated into liposomal nanoparticles now end up in the liver with first-pass kinetics with no evidence for toxicity in the 1-9mg/kg range (mostly rodent studies), whereas >99% of unformulated siRNAs are rapidly filtered out by the kidney.

The liver is also the organ where the first microRNA-based therapies are likely to be targeted. Like Regulus and Rosetta, I was amazed, or maybe not, at how many groups are currently working at abrogating miR-122 function for treating either hypercholesterolemia or HCV infection. LNAs, owned by the privately held Danish company Santaris, appear to be a particularly promising oligonucleotide class for achieving potent microRNA down-regulation in vivo. Similarly, antisense strategies based on LNA appear to rival siRNA potency in a number of settings, and it will be interesting to learn more about the toxicologies of these compounds, which would certainly be facilitated if Santaris decided to enable to field by allowing better access to their technology. It also highlights the need for specialized chemistries for therapeutic targeting of microRNAs which in my mind is something that Regulus ought to consider.

With regard to delivery outside the liver following systemic administration, PEI-formulated siRNAs (see recent PolyPlus-Alnylam deal) are now being used successfully by a number of groups for lung delivery. Other interesting delivery data was presented by a Portuguese group in a poster on RNAi in the brain. By non-covalently adding transferrin protein to cationic liposome-siRNA particles, they were able to efficiently target neuronal cells both in vitro and in vivo which, similar to cancer cells (see Calando’s cancer cell targeting with transferrin), are studded with transferrin receptors.

PS: A nuisance to most scientists in the field, immunostimulation by oligonucleotides is exploited by Dynavax as an adjuvant for vaccines. Poor adjuvants activity lead to insufficient immune responses and the need for multiple booster injections affecting compliance. HBV vaccination is a case in point, and as a consequence around 50% of patients in the US fail to successfully complete vaccination. Very high HBsAg antibody titers after only two injections of recombinant HBsAg with an immunostimulatory oligo from Dynavax as adjuvant, instead of alum, suggests that they may be able to greatly improve on those numbers.