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.
Monday, October 8, 2007
Saturday, October 6, 2007
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.
,
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.
,
Friday, October 5, 2007
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.
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.
Friday, September 28, 2007
Nature Publishes Reassuring Study by Alnylam on the in vivo Delivery of siRNAs and their Effect on the Endogenous microRNA Pathway
Given that therapeutic RNAi takes advantage of an endogenous biological pathway, the introduction of very high levels of small RNAs certainly has the potential to interfere with related small RNA pathways, such as microRNA function.
Indeed, competition with the microRNA pathway in vivo which in some cases caused the death of mice, were first reported in a study by Grimm and colleagues in the journal Nature last year. Ironically, rather than a demonstrating a failure of the viral delivery system used in that study, it was the extreme efficiency with which small RNAs could be expressed with the double-stranded AAV vectors that allowed the competition to be observed. In what is an often overlooked aspect of these studies, compared to non-viral delivery methods, silencing efficiencies of over 95% can be easily achieved with these vectors at doses that have no adverse effects on either the microRNA pathway or the viability of mice.
In an ideal world, the Grimm et al. studies would have been embraced as an opportunity to study the dose-limiting steps of therapeutic RNAi to inform future RNAi therapeutics strategies. Instead, as the Press lives from feeding the public the simple messages, rather than reporting complicated truths, it decided to label the studies as yet another example of the dangers of gene therapy, and - somewhat understandably- caused some companies involved in developing RNAi Therapeutics to distance themselves from DNA-directed RNAi for political reasons.
As the trusted leader of RNAi Therapeutics, Alnylam was therefore given the platform to reassure the RNAi community this week in Nature that, unlike AAV-RNAi, liposomally delivered siRNAs had no obvious adverse effects on the endogenous microRNA pathway (John et al., 2007). The study further highlighted that liposomal siRNA delivery has advanced to a point where around 80% gene silencing in hepatocytes can routinely be achieved following systemic administration of therapeutically viable doses of siRNAs, including their repeat administration.
Although I welcome Nature’s decision to document progress in the important area of RNAi therapeutics, and understand Alnylam’s desire to publish in the highest profile journals, I would like to take this opportunity to address a few misconceptions about the studies. One important misconception is that delivering RNAi with AAV per se is more toxic. To make this point, a direct comparison of the intrahepatic levels of small RNA levels following both routes of administration would have been necessary. Given the >99% transduction efficiency of double-stranded AAV in mice and the consequently extremely high gene silencing efficiencies, it is quite likely that double-strand AAV vectors are currently the most potent delivery system to the liver in terms of small RNA delivery and gene knockdown.
I would therefore not be surprised at all to see similar competition with microRNA function following administration of very high siRNA dosages. This is supported by numerous studies that have shown competition for gene silencing when very high levels of two or more siRNAs were introduced simultaneously into tissue culture cells. However, given the ability of hundreds of microRNAs to function in a given cell at any time, such observations represent only extreme cases and suggest a wide therapeutic index. Unfortunately, the relatively small range of doses used in the John et al. studies (2mg/kg to 5mg/kg) did allow for a careful evaluation of related competition in vivo and concomitant dose-limiting toxicities.
I guess the purpose of this Blog really is my plea to the field of RNAi Therapeutics to keep learning from each other, instead of letting the Press and uninformed “analysts” play on the fears of investors, through their indiscriminate use of buzzwords, thereby polarising and separating what really belongs together. In this spirit, I would like to stress that this study is yet another proof-point of the viability of RNAi for therapy leading up to the possibly first proof-of-concept gene silencing results in Man to be revealed in the coming months- once again by Alnylam.
PS: Although a combination of liposomal delivery methods were used in these studies, the details were not disclosed. Apparently, another study on lipidoid-delivered siRNAs, a technology developed by the Langer and Anderson groups at the MIT, has been submitted to Nature Biotech and is about to be published. Lipidoids differ slightly from the Tekmira-owned SNALP technology, and looks likely to be the technology used for Alnylam’s first clinical systemic RNAi program (liver cancer or hypercholesterolemia), for which an IND is expected by the end of 2007. More than knockdown efficiency, we should be looking for the toxicity profile as I regard this to be the big unknown that will determine the success of this program, particularly if it turns out to be for hypercholesterolemia.
Indeed, competition with the microRNA pathway in vivo which in some cases caused the death of mice, were first reported in a study by Grimm and colleagues in the journal Nature last year. Ironically, rather than a demonstrating a failure of the viral delivery system used in that study, it was the extreme efficiency with which small RNAs could be expressed with the double-stranded AAV vectors that allowed the competition to be observed. In what is an often overlooked aspect of these studies, compared to non-viral delivery methods, silencing efficiencies of over 95% can be easily achieved with these vectors at doses that have no adverse effects on either the microRNA pathway or the viability of mice.
In an ideal world, the Grimm et al. studies would have been embraced as an opportunity to study the dose-limiting steps of therapeutic RNAi to inform future RNAi therapeutics strategies. Instead, as the Press lives from feeding the public the simple messages, rather than reporting complicated truths, it decided to label the studies as yet another example of the dangers of gene therapy, and - somewhat understandably- caused some companies involved in developing RNAi Therapeutics to distance themselves from DNA-directed RNAi for political reasons.
As the trusted leader of RNAi Therapeutics, Alnylam was therefore given the platform to reassure the RNAi community this week in Nature that, unlike AAV-RNAi, liposomally delivered siRNAs had no obvious adverse effects on the endogenous microRNA pathway (John et al., 2007). The study further highlighted that liposomal siRNA delivery has advanced to a point where around 80% gene silencing in hepatocytes can routinely be achieved following systemic administration of therapeutically viable doses of siRNAs, including their repeat administration.
Although I welcome Nature’s decision to document progress in the important area of RNAi therapeutics, and understand Alnylam’s desire to publish in the highest profile journals, I would like to take this opportunity to address a few misconceptions about the studies. One important misconception is that delivering RNAi with AAV per se is more toxic. To make this point, a direct comparison of the intrahepatic levels of small RNA levels following both routes of administration would have been necessary. Given the >99% transduction efficiency of double-stranded AAV in mice and the consequently extremely high gene silencing efficiencies, it is quite likely that double-strand AAV vectors are currently the most potent delivery system to the liver in terms of small RNA delivery and gene knockdown.
I would therefore not be surprised at all to see similar competition with microRNA function following administration of very high siRNA dosages. This is supported by numerous studies that have shown competition for gene silencing when very high levels of two or more siRNAs were introduced simultaneously into tissue culture cells. However, given the ability of hundreds of microRNAs to function in a given cell at any time, such observations represent only extreme cases and suggest a wide therapeutic index. Unfortunately, the relatively small range of doses used in the John et al. studies (2mg/kg to 5mg/kg) did allow for a careful evaluation of related competition in vivo and concomitant dose-limiting toxicities.
I guess the purpose of this Blog really is my plea to the field of RNAi Therapeutics to keep learning from each other, instead of letting the Press and uninformed “analysts” play on the fears of investors, through their indiscriminate use of buzzwords, thereby polarising and separating what really belongs together. In this spirit, I would like to stress that this study is yet another proof-point of the viability of RNAi for therapy leading up to the possibly first proof-of-concept gene silencing results in Man to be revealed in the coming months- once again by Alnylam.
PS: Although a combination of liposomal delivery methods were used in these studies, the details were not disclosed. Apparently, another study on lipidoid-delivered siRNAs, a technology developed by the Langer and Anderson groups at the MIT, has been submitted to Nature Biotech and is about to be published. Lipidoids differ slightly from the Tekmira-owned SNALP technology, and looks likely to be the technology used for Alnylam’s first clinical systemic RNAi program (liver cancer or hypercholesterolemia), for which an IND is expected by the end of 2007. More than knockdown efficiency, we should be looking for the toxicity profile as I regard this to be the big unknown that will determine the success of this program, particularly if it turns out to be for hypercholesterolemia.
Monday, September 24, 2007
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.
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.
Wednesday, September 19, 2007
Journal Club: Alnylam and Collaborators Make Progress in Understanding and Optimising siRNA Uptake In Vivo
In yet another elegant paper, Alnylam and collaborators from the ETH in Zurich and the Rockefeller report this week in the journal Nature Biotech on the mechanism of siRNA uptake in vivo (Wolfrum et al.). This is significant, since a systematic approach to understanding siRNA delivery in vivo should most adequately address the delivery challenge that is considered by many to be the main barrier to the broad application of RNAi as a therapy. It is also a sign of the maturity of the RNAi field in general that no only are there now a variety of innovative delivery systems evaluated almost by the day, systems that show promise serve as leads for a detailed investigation of the underlying biology.
The study by Wolfrum and colleagues follows another high-profile publication 3 years ago (Soutschek et al.) where Alnylam scientists demonstrated gene silencing in mice following systemic administration of cholesterol-conjugated siRNAs. That study showed that although such siRNAs could silence genes particularly in the liver and gut, quite high amounts of siRNAs were needed (50mg/kg). By studying the uptake of the siRNA conjugates in these tissues, the authors not only hoped to understand why they functioned at all, but also to optimise their potency.
Efficient in vivo drug delivery requires favourable pharmacokinetics. Particularly, a drug has to be present in the blood for sufficient length of time so that it has a chance to accumulate in its target tissue. One reason for example why many experimental drugs fail is because they are rapidly excreted through the kidneys. This may often be prevented if the drug could interact with components of the blood such as the abundant lipoprotein particles.
Indeed, the authors find that siRNAs conjugated to cholesterol or other lipophilic molecules associated with the similarly greasy HDL and LDL lipoprotein particles. These would ferry them around in circulation and bring them into the proximity of cells that carry on their surface receptors for either HDL and/or LDL. Strikingly, pre-assembling the siRNA with purified HDL and LDL particles quite significantly increased the potency of the siRNAs. Furthermore, mice lacking either of the receptors for the lipoproteins were much less prone to gene silencing by the same pre-formulated siRNA particles.
In a further interesting twist, it was shown that siRNAs were not taken up by the cells as part of internalising lipoproteins, but that the siRNAs would take advantage of their proximity to the cell membrane during the docking, release, and re-docking process of their lipoprotein carriers with their receptors. Amazingly, through a combination of gene knockdown experimentation and blockage by antibodies, at least one of the actual entry routes for the siRNA was inferred to be the human homologue of the SID-1 gene that had earlier been shown to mediate systemic RNAi in the worm C. elegans.
Systemic RNAi describes the spread of an siRNA from one cell to another cell in the same or even different tissues. Systemic RNAi in worms and plants is associated with the amplification of RNAi, and both systemic RNAi as well as RNAi amplification were thought to have been lost during human evolution. It is therefore a surprise that SID-1 would still function in siRNA uptake, with demonstrated selectivity for siRNAs relative to other types of nucleic acids. This also raises the intriguing possibility that some sort of natural siRNA uptake should occur in humans.
Of more immediate importance, the present paper opens the door for the systematic screening of new lipophile-siRNA conjugates with improved association kinetics with lipoprotein particles, or even pre-formulation of such conjugates with lipoproteins or other natural or synthetic carriers of the blood. I look forward to what this line of investigation will yield next.
The study by Wolfrum and colleagues follows another high-profile publication 3 years ago (Soutschek et al.) where Alnylam scientists demonstrated gene silencing in mice following systemic administration of cholesterol-conjugated siRNAs. That study showed that although such siRNAs could silence genes particularly in the liver and gut, quite high amounts of siRNAs were needed (50mg/kg). By studying the uptake of the siRNA conjugates in these tissues, the authors not only hoped to understand why they functioned at all, but also to optimise their potency.
Efficient in vivo drug delivery requires favourable pharmacokinetics. Particularly, a drug has to be present in the blood for sufficient length of time so that it has a chance to accumulate in its target tissue. One reason for example why many experimental drugs fail is because they are rapidly excreted through the kidneys. This may often be prevented if the drug could interact with components of the blood such as the abundant lipoprotein particles.
Indeed, the authors find that siRNAs conjugated to cholesterol or other lipophilic molecules associated with the similarly greasy HDL and LDL lipoprotein particles. These would ferry them around in circulation and bring them into the proximity of cells that carry on their surface receptors for either HDL and/or LDL. Strikingly, pre-assembling the siRNA with purified HDL and LDL particles quite significantly increased the potency of the siRNAs. Furthermore, mice lacking either of the receptors for the lipoproteins were much less prone to gene silencing by the same pre-formulated siRNA particles.
In a further interesting twist, it was shown that siRNAs were not taken up by the cells as part of internalising lipoproteins, but that the siRNAs would take advantage of their proximity to the cell membrane during the docking, release, and re-docking process of their lipoprotein carriers with their receptors. Amazingly, through a combination of gene knockdown experimentation and blockage by antibodies, at least one of the actual entry routes for the siRNA was inferred to be the human homologue of the SID-1 gene that had earlier been shown to mediate systemic RNAi in the worm C. elegans.
Systemic RNAi describes the spread of an siRNA from one cell to another cell in the same or even different tissues. Systemic RNAi in worms and plants is associated with the amplification of RNAi, and both systemic RNAi as well as RNAi amplification were thought to have been lost during human evolution. It is therefore a surprise that SID-1 would still function in siRNA uptake, with demonstrated selectivity for siRNAs relative to other types of nucleic acids. This also raises the intriguing possibility that some sort of natural siRNA uptake should occur in humans.
Of more immediate importance, the present paper opens the door for the systematic screening of new lipophile-siRNA conjugates with improved association kinetics with lipoprotein particles, or even pre-formulation of such conjugates with lipoproteins or other natural or synthetic carriers of the blood. I look forward to what this line of investigation will yield next.
Monday, September 17, 2007
Has Rosetta Found the Cornerstone to Corporate Success?
Last week’s formation of Regulus Therapeutics should give a boost to the whole field of microRNA-based therapeutics as it lends credibility to microRNAs as a new drug development and diagnostics platform. Part of this boost should come in the form of funding from larger pharmaceutical and diagnostics entities for companies with a credible and well-developed microRNA IP estate.
I would therefore like to take a closer look now at Rosetta Genomics, next to Regulus arguably the only other major pure-play microRNA-focussed company. Rosetta has pleasantly surprised me by assembling a strong IP portfolio, which it has then followed up with a series of well-designed corporate and academic partnerships. This is complemented by a growing tool-box allowing for clinically-relevant extraction, detection and measurement of microRNAs. Like other players in this field, Rosetta believes that given the emerging importance of microRNAs in gene regulation, these molecules would also be involved in human disease so that they could be both harnessed for clinical diagnostics and therapeutics.
Rosetta is an Israel-based company, founded on the discovery and patenting of human microRNAs using high-throughput computing and bio-technologies (2005 Nature Genetics study). In the wake of the Human Genome and other sequencing projects, the founders of Rosetta hypothesised that the key to human complexity was not due to an increased number of genes, but at least partly due to the emergence of primate- and even human-specific microRNAs, and their search for new microRNAs consequently accommodated that notion. This was against the mainstream of most microRNA discovery efforts then which heavily relied on the notion of biological conservation, and Rosetta would be able to detect a number of microRNAs that had been missed.
Indeed, their hypothesis was supported by their 2005 Nature Genetics paper, almost doubling the number of sequenced human microRNAs at that time (adding 89 microRNAs), a number of them not conserved beyond primates. Based on partly theoretical considerations, predictions as to the total number of microRNAs were also revised upwards from initial estimates in the field of around 250 to well over 800. These efforts have resulted in patent applications exceeding 500,000 pages, probably using the same computing power used for predicting microRNAs.
I should add, however, that most of these non-conserved microRNAs were restricted to 2 clusters in the genome and should therefore be of less diagnostic value as would be expected for an equal number of more randomly distributed microRNAs. Furthermore, most of the previously cloned microRNA, particularly those by Thomas Tuschl, licensed exclusively for therapeutics use to the parent companies of Regulus, Alnylam and ISIS, should be amongst the biologically most important microRNAs simply based on their higher expression levels (the reason why they were detected by cloning in the first place).
At that point, I thought just another publication based on bioinformatics that was showing that the complexity of microRNAs may be higher than initially thought. Also, their theoretical approach and computer-driven technologies made me wonder whether this would ever develop into a meaningful hands-on biotechnology operation.
Rosetta took a number of steps to change this perception. First, it has gained access, at least for diagnostic use, to the large majority of human microRNAs through licensing agreements, most importantly with the Max-Planck Institutes and Rockefeller. Next, similar to what Alnylam has done, they have come out with a number of high-quality, peer-reviewed publications, ranging from microRNA detection technologies to the functional elucidation of certain disease-associated microRNAs. Partly, this was done through academic collaborations which allows them to stay product focussed and capitalise on opportunities should they arise from discoveries in microRNA research. Other collaborations with corporate and clinical partners have given them access to relevant technologies such as one with ISIS for the therapeutic targeting of microRNAs using antisense technology, and clinical specimens from hospitals which will be used to test their diagnostics.
How they were able to orchestrate this transformation is not clear to me and quite impressive, but looking at the line-up of illustrious early investors and SAB (scientific advisory board), populated with Nobel Laureates and the likes of Robert Langer (also on Alnylam’s SAB), suggests that they have enough influence to get the attention of key audiences. The expansion of their activities in the US should further nurture current and future partnerships and attract new investors.
These investors may be attracted by Rosetta’s first issuances of microRNA patents and its strategy to use early revenues from their more mature microRNA diagnostics efforts to fund the potentially more lucrative area of microRNA-based therapeutics on quite attractive financial terms. It is their aggressive goal to have 3 microRNA diagnostics products on the markets by the end of next year, with their most advanced program being for the classification of Cancer of Unknown Primary (CUP) where the goal is to identify the original tissue from which a cancer has spread. A recent presentation at the AACR cancer meeting suggests that this can be achieved with 85% accuracy by profiling 19 microRNAs. Their initial therapeutic pipeline, meanwhile, focuses, similar to Regulus, on diseases of the liver, such as liver cancer and HCV infection. This is done in collaboration with ISIS Pharmaceuticals, and it will be interesting to see how the recent formation of Regulus will affect this relationship.
After a difficult IPO and little attention from Wall Street, the time is ripe for Rosetta and Regulus to lead the charge in translating the important biology of microRNAs into medical use.
I would therefore like to take a closer look now at Rosetta Genomics, next to Regulus arguably the only other major pure-play microRNA-focussed company. Rosetta has pleasantly surprised me by assembling a strong IP portfolio, which it has then followed up with a series of well-designed corporate and academic partnerships. This is complemented by a growing tool-box allowing for clinically-relevant extraction, detection and measurement of microRNAs. Like other players in this field, Rosetta believes that given the emerging importance of microRNAs in gene regulation, these molecules would also be involved in human disease so that they could be both harnessed for clinical diagnostics and therapeutics.
Rosetta is an Israel-based company, founded on the discovery and patenting of human microRNAs using high-throughput computing and bio-technologies (2005 Nature Genetics study). In the wake of the Human Genome and other sequencing projects, the founders of Rosetta hypothesised that the key to human complexity was not due to an increased number of genes, but at least partly due to the emergence of primate- and even human-specific microRNAs, and their search for new microRNAs consequently accommodated that notion. This was against the mainstream of most microRNA discovery efforts then which heavily relied on the notion of biological conservation, and Rosetta would be able to detect a number of microRNAs that had been missed.
Indeed, their hypothesis was supported by their 2005 Nature Genetics paper, almost doubling the number of sequenced human microRNAs at that time (adding 89 microRNAs), a number of them not conserved beyond primates. Based on partly theoretical considerations, predictions as to the total number of microRNAs were also revised upwards from initial estimates in the field of around 250 to well over 800. These efforts have resulted in patent applications exceeding 500,000 pages, probably using the same computing power used for predicting microRNAs.
I should add, however, that most of these non-conserved microRNAs were restricted to 2 clusters in the genome and should therefore be of less diagnostic value as would be expected for an equal number of more randomly distributed microRNAs. Furthermore, most of the previously cloned microRNA, particularly those by Thomas Tuschl, licensed exclusively for therapeutics use to the parent companies of Regulus, Alnylam and ISIS, should be amongst the biologically most important microRNAs simply based on their higher expression levels (the reason why they were detected by cloning in the first place).
At that point, I thought just another publication based on bioinformatics that was showing that the complexity of microRNAs may be higher than initially thought. Also, their theoretical approach and computer-driven technologies made me wonder whether this would ever develop into a meaningful hands-on biotechnology operation.
Rosetta took a number of steps to change this perception. First, it has gained access, at least for diagnostic use, to the large majority of human microRNAs through licensing agreements, most importantly with the Max-Planck Institutes and Rockefeller. Next, similar to what Alnylam has done, they have come out with a number of high-quality, peer-reviewed publications, ranging from microRNA detection technologies to the functional elucidation of certain disease-associated microRNAs. Partly, this was done through academic collaborations which allows them to stay product focussed and capitalise on opportunities should they arise from discoveries in microRNA research. Other collaborations with corporate and clinical partners have given them access to relevant technologies such as one with ISIS for the therapeutic targeting of microRNAs using antisense technology, and clinical specimens from hospitals which will be used to test their diagnostics.
How they were able to orchestrate this transformation is not clear to me and quite impressive, but looking at the line-up of illustrious early investors and SAB (scientific advisory board), populated with Nobel Laureates and the likes of Robert Langer (also on Alnylam’s SAB), suggests that they have enough influence to get the attention of key audiences. The expansion of their activities in the US should further nurture current and future partnerships and attract new investors.
These investors may be attracted by Rosetta’s first issuances of microRNA patents and its strategy to use early revenues from their more mature microRNA diagnostics efforts to fund the potentially more lucrative area of microRNA-based therapeutics on quite attractive financial terms. It is their aggressive goal to have 3 microRNA diagnostics products on the markets by the end of next year, with their most advanced program being for the classification of Cancer of Unknown Primary (CUP) where the goal is to identify the original tissue from which a cancer has spread. A recent presentation at the AACR cancer meeting suggests that this can be achieved with 85% accuracy by profiling 19 microRNAs. Their initial therapeutic pipeline, meanwhile, focuses, similar to Regulus, on diseases of the liver, such as liver cancer and HCV infection. This is done in collaboration with ISIS Pharmaceuticals, and it will be interesting to see how the recent formation of Regulus will affect this relationship.
After a difficult IPO and little attention from Wall Street, the time is ripe for Rosetta and Regulus to lead the charge in translating the important biology of microRNAs into medical use.
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