Wednesday, July 8, 2009
The RNAi Therapeutics Blog is Back
After taking a break for almost a year to finish up my post-doctoral studies and to figure out what to do with my future, I have decided to pick up writing this blog again. In addition to what I hope will be a small contribution to explaining RNAi Therapeutics to the scientifically and financially interested public, it will also help me to better feel the pulse of RNAi Therapeutics through my interactions with you. So please feel free to comment. Initially, I will only comment when there is highly significant breaking news and will become more active again starting mid-September. There should be much to write about with first clinical data from the Alnylam and Tekmira SNALP-siRNA trials coming up which in my opinion could be a real game changer. Fingers crossed!
Alnylam 1500, GSK 800
In what was possibly greeted in investor circles with a slight yawn, GSK and Alnylam announced today that Alnylam would add 1500 issued or pending RNAi Therapeutics-related patents to GSK’s 800 patent filings into an IP pool designed to facilitate the development of drugs for neglected tropical diseases. So what was this all about?
“We are delighted that Alnylam will join GSK in this important programme by adding their unique RNAi technology [emphasis mine] to the patent pool.”
This indicates to me that Alnylam and GSK are getting along pretty well. Both managements should be familiar with each other following the GSK-Regulus deal for the development of microRNA therapeutics for inflammatory disease (Regulus is the microRNA joint venture of Alnylam and ISIS). A positive experience there with small RNAs may have given GSK extra incentive to join the ranks of Roche, Takeda, Novartis, and Pfizer in considering RNAi Therapeutics as a bona fide remedy, if not cure for Big Pharma’s stuttering innovation machine and oncoming wave of expiring blockbuster. And yes, Merck is also one of them, and it is likely that when they bought Sirna Therapeutics in 2006, it left GSK looking for a new RNAi Therapeutics partner. Just before that in 2006, GSK and Sirna signed a major RNAi alliance for respiratory disease, and since then Merck has not made the impression that it likes to share its mysterious RNAi know-how.
You know where this is going, and regardless of whether an Alnylam-GSK RNAi platform alliance will actually be announced this year or whether this is just another example of my RNAi delusion, it is worth speculating about the scope of such a potential deal of which we expect at least one this year from Alnylam. Similar to the 2007 Roche platform deal, my guess with regards to therapeutic areas would be oncology, respiratory diseases, metabolic diseases and certain liver diseases. Since these are all areas in which significant improvements have been made in siRNA delivery in the last 2 years, the terms may be even more favorable.
And yes, it would be nice for GSK and Alnylam to combine their expertise in malaria drug development and liposomal siRNA delivery to the liver, respectively, to translate promising pre-clinical results by Alnylam and collaborators into a much needed weapon for a disease that disrupts the lives of up to 500 million people a year mostly in impoverished countries. That RNAi is even considered for such purposes is also a sign that the eventual cost of goods for RNAi Therapeutics should be well below that of recombinant proteins, including monoclonal antibodies, which in turn may be more suitable for vaccination approaches.
Tuesday, September 23, 2008
The RNAi Therapeutics Blog Will Take a Break
While on vacation (and far away from the financial markets mess…), I had plenty of time to re-prioritize my research and investment efforts. To my pleasant surprise, the blog has turned out to be a very rich (not monetary) experience for me with many new contacts that allowed me to learn about the various aspects of biotech R&D and financing. Thank you for this!
Should my plans change or should my boss boot me out of the lab for under-performing, I might be back, and then it would probably be full-time. In the meantime, I hope everybody and RNAi Therapeutics in particular to get through the economic turmoil well.
Wednesday, September 3, 2008
RNAi Therapeutics and Innate Immunity- Keeping the Field Honest
As part of the RNAi Therapeutics review series in Human Gene Therapy earlier this year, former Protiva scientists Adam Judge and Ian MacLachlan (both now with Tekmira following the Protiva-Tekmira reunion) made some rather bold statements with regards to the interpretations of a number of pre-clinical RNAi Therapeutics validation papers (‘Overcoming the innate immune response to small interfering RNA’). As part of the same reviews series, in the risk section of “The Business of RNAi Therapeutics”, I also cautioned that some of the first RNAi Therapeutics candidates may show clinical efficacy, but not necessarily for all the right reasons.
The reason for this should not come as a surprise to anybody in the oligonucleotide therapeutics field: long known from the experience with antisense and other oligonucleotide therapeutics classes, oligonucleotides such as siRNAs have the potential to induce innate immune responses which can have antiviral and anti-angiogenic activity independent of their gene knockdown capacity. In fact, there are significant efforts to harness this biological property for therapy in its own right, particularly the TLR responses. Furthermore, the potential for inducing innate immune responses by synthetic and DNA-directed RNAi has been well documented since 2003 and many of the pathways involved elucidated. Nevertheless, one should not ignore the fact that while RNAi Therapeutics may actually be able to take advantage of such activity as part of synergistically acting immunostimulatory RNAi Therapeutics, the risk is that the oligo-dependent immune responses are quite complex and therefore often difficult to predict and in the worst case may cause serious adverse events.
Since many of the early RNAi Therapeutics validation papers involved antiviral and anticancer applications, it was therefore reasonable to suspect that some of the studies misinterpreted therapeutic effects as the result of RNAi gene knockdown when, in fact, innate immune responses accounted for the majority of the activity. In support, the Tekmira researchers now report that almost all of the unmodified siRNAs reported in a sample of such papers were immunostimulatory whereas a single siRNA that, somewhat disturbingly so, was used as the control siRNA in many of the studies proved to be the exception having no such detectable activity. I should add, however, that the assay conditions were rather stringent (types of cells used and high siRNA concentrations) and just because an siRNA may induce immune responses under these conditions does not prove that these were actually responsible for the treatment effect seen in each of the cited studies. Also, if TLR therapeutics history is any guide, oligonucleotides that elicit immune responses in small animal models, do not necessarily do so in primates.
Given its potential as a whole new class of therapeutics, the scientific and clinical bar for RNAi Therapeutics is set particularly high and reports like the effect of TLR3 stimulation by siRNAs on preclinical models for wet AMD and the present paper by Tekmira tend to get quite a bit of press. While they remind us of the complexities involved in establishing a functional new drug discovery platform, they should also be regarded as promoting that process. In fact, the handful of bona fide RNAi Therapeutics groups, pure-plays and Big Pharma subsidiaries alike, are already taking oligo-induced innate immune responses very seriously and have taken advantage of the rapid progress in the field by applying best practices for identifying and correcting these responses (modification, siRNA structure) in developing the latest crop of RNAi Therapeutics candidates.
The acquisition of former TLR therapeutics company Coley Pharmaceuticals by Pfizer for example may be interpreted as Pfizer investing in solving siRNA-induced innate immune responses as one of the main challenges for RNAi Therapeutics they had identified. Similarly, Sirna Therapeutics and Protiva in their prominent 2005 Nature Biotech paper on RNAi delivery in a mouse model of hepatitis B recognized the potential of unmodified siRNAs to elicit non-specific viral suppression and solved the issue by appropriately modifying the siRNAs. Around the same time, Alnylam somewhat quietly generated IP related to double-strand RNA immune stimulation that it then exclusively licensed to Tekmira. Clearly, the main players in the field have not chosen to ignore the issue, but have invested considerable efforts with tangible results.
But what about the current RNAi Therapeutics clinical candidates that have already entered the clinic? There are one phase III (Opko Health) and two phase II (Sirna/Merck-Allergan and Quark-Pfizer) siRNA candidates for the treatment of wet AMD that obviously have naturally come under increased scrutiny. As far as I am aware, all three of these are ‘unformulated’, intravitreally injected siRNAs with one of them, Opko’s, being an unmodified siRNA. While it is not clear how well the mouse TLR3 studies translate into humans, they certainly raise the concern that non-specific responses might be responsible for any thus far clinically observed therapeutic effects, particularly since in the recent Nature study gene knockdown by this route was very limited at best (cholesterol-conjugated siRNAs, however, administered by the same route were shown to mediate functional gene silencing in the same study).
As is the case with Alnylam’s lead candidate ALN-RSV01 for the treatment of RSV infection which has raised similar concerns, it will be important to be forthcoming in the interactions with the regulatory agencies such that safe trials can be designed based on our best understanding of the mechanisms of action of the different siRNAs. While I haven’t read the documents, it certainly wouldn’t be the first time if such non-specific effects were noted as potentially contributing to treatment. In the future, it would not surprise me at all to see openly declared immunostimulatory siRNA drug candidates enter the clinic. If, however, these issues are not addressed upfront, and should adverse events occur as a result, this could easily backfire and future trials rendered much more onerous- something that should be in nobody’s interest. As for the prospects of the individual drug candidates in question, even if non-specific effects contributed to the therapeutic efficacy of these candidates, as long as they are safe and well tolerated they may very well be viable drugs.
Finally, it is curious as to what exactly motivated Tekmira to re-test an entire battery of published siRNAs for their potential of inducing non-specific effects. It is possible that Tekmira has evaluated siRNA therapeutics for a number of the same applications like flu and wet AMD and were frustrated to see publications come out that according to their experience should have been artefacts (scientists tend to measure themselves by the number of publications and their impact factors and don’t like to see their own published work de-valued this way). Another part of the answer may also have been to keep the field honest at this early stage of RNAi Therapeutics drug development before long-term damage is caused: “However, surprisingly few of the reported studies have adequately tested, or controlled, for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.” By setting a rigorous new standard, Tekmira also signals their expertise not only in RNAi delivery, but also in siRNA chemistry and safety (like Coley, Tekmira has a long-standing interest in the use of immunostimulatory oligonucleotides for therapy). Supporting their claim, Tekmira/Protiva’s publications on abrogating TLR7/8 responses and SNALP RNAi delivery have proven to be extremely reproducible in many different laboratories.
The road to RNAi Therapeutics reality won’t be smooth. As much as it is important to tackle the scientific hurdles head-on, investors and the press should also make an effort to discriminate between ‘good’ and ‘bad’ science.
The reason for this should not come as a surprise to anybody in the oligonucleotide therapeutics field: long known from the experience with antisense and other oligonucleotide therapeutics classes, oligonucleotides such as siRNAs have the potential to induce innate immune responses which can have antiviral and anti-angiogenic activity independent of their gene knockdown capacity. In fact, there are significant efforts to harness this biological property for therapy in its own right, particularly the TLR responses. Furthermore, the potential for inducing innate immune responses by synthetic and DNA-directed RNAi has been well documented since 2003 and many of the pathways involved elucidated. Nevertheless, one should not ignore the fact that while RNAi Therapeutics may actually be able to take advantage of such activity as part of synergistically acting immunostimulatory RNAi Therapeutics, the risk is that the oligo-dependent immune responses are quite complex and therefore often difficult to predict and in the worst case may cause serious adverse events.
Since many of the early RNAi Therapeutics validation papers involved antiviral and anticancer applications, it was therefore reasonable to suspect that some of the studies misinterpreted therapeutic effects as the result of RNAi gene knockdown when, in fact, innate immune responses accounted for the majority of the activity. In support, the Tekmira researchers now report that almost all of the unmodified siRNAs reported in a sample of such papers were immunostimulatory whereas a single siRNA that, somewhat disturbingly so, was used as the control siRNA in many of the studies proved to be the exception having no such detectable activity. I should add, however, that the assay conditions were rather stringent (types of cells used and high siRNA concentrations) and just because an siRNA may induce immune responses under these conditions does not prove that these were actually responsible for the treatment effect seen in each of the cited studies. Also, if TLR therapeutics history is any guide, oligonucleotides that elicit immune responses in small animal models, do not necessarily do so in primates.
Given its potential as a whole new class of therapeutics, the scientific and clinical bar for RNAi Therapeutics is set particularly high and reports like the effect of TLR3 stimulation by siRNAs on preclinical models for wet AMD and the present paper by Tekmira tend to get quite a bit of press. While they remind us of the complexities involved in establishing a functional new drug discovery platform, they should also be regarded as promoting that process. In fact, the handful of bona fide RNAi Therapeutics groups, pure-plays and Big Pharma subsidiaries alike, are already taking oligo-induced innate immune responses very seriously and have taken advantage of the rapid progress in the field by applying best practices for identifying and correcting these responses (modification, siRNA structure) in developing the latest crop of RNAi Therapeutics candidates.
The acquisition of former TLR therapeutics company Coley Pharmaceuticals by Pfizer for example may be interpreted as Pfizer investing in solving siRNA-induced innate immune responses as one of the main challenges for RNAi Therapeutics they had identified. Similarly, Sirna Therapeutics and Protiva in their prominent 2005 Nature Biotech paper on RNAi delivery in a mouse model of hepatitis B recognized the potential of unmodified siRNAs to elicit non-specific viral suppression and solved the issue by appropriately modifying the siRNAs. Around the same time, Alnylam somewhat quietly generated IP related to double-strand RNA immune stimulation that it then exclusively licensed to Tekmira. Clearly, the main players in the field have not chosen to ignore the issue, but have invested considerable efforts with tangible results.
But what about the current RNAi Therapeutics clinical candidates that have already entered the clinic? There are one phase III (Opko Health) and two phase II (Sirna/Merck-Allergan and Quark-Pfizer) siRNA candidates for the treatment of wet AMD that obviously have naturally come under increased scrutiny. As far as I am aware, all three of these are ‘unformulated’, intravitreally injected siRNAs with one of them, Opko’s, being an unmodified siRNA. While it is not clear how well the mouse TLR3 studies translate into humans, they certainly raise the concern that non-specific responses might be responsible for any thus far clinically observed therapeutic effects, particularly since in the recent Nature study gene knockdown by this route was very limited at best (cholesterol-conjugated siRNAs, however, administered by the same route were shown to mediate functional gene silencing in the same study).
As is the case with Alnylam’s lead candidate ALN-RSV01 for the treatment of RSV infection which has raised similar concerns, it will be important to be forthcoming in the interactions with the regulatory agencies such that safe trials can be designed based on our best understanding of the mechanisms of action of the different siRNAs. While I haven’t read the documents, it certainly wouldn’t be the first time if such non-specific effects were noted as potentially contributing to treatment. In the future, it would not surprise me at all to see openly declared immunostimulatory siRNA drug candidates enter the clinic. If, however, these issues are not addressed upfront, and should adverse events occur as a result, this could easily backfire and future trials rendered much more onerous- something that should be in nobody’s interest. As for the prospects of the individual drug candidates in question, even if non-specific effects contributed to the therapeutic efficacy of these candidates, as long as they are safe and well tolerated they may very well be viable drugs.
Finally, it is curious as to what exactly motivated Tekmira to re-test an entire battery of published siRNAs for their potential of inducing non-specific effects. It is possible that Tekmira has evaluated siRNA therapeutics for a number of the same applications like flu and wet AMD and were frustrated to see publications come out that according to their experience should have been artefacts (scientists tend to measure themselves by the number of publications and their impact factors and don’t like to see their own published work de-valued this way). Another part of the answer may also have been to keep the field honest at this early stage of RNAi Therapeutics drug development before long-term damage is caused: “However, surprisingly few of the reported studies have adequately tested, or controlled, for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.” By setting a rigorous new standard, Tekmira also signals their expertise not only in RNAi delivery, but also in siRNA chemistry and safety (like Coley, Tekmira has a long-standing interest in the use of immunostimulatory oligonucleotides for therapy). Supporting their claim, Tekmira/Protiva’s publications on abrogating TLR7/8 responses and SNALP RNAi delivery have proven to be extremely reproducible in many different laboratories.
The road to RNAi Therapeutics reality won’t be smooth. As much as it is important to tackle the scientific hurdles head-on, investors and the press should also make an effort to discriminate between ‘good’ and ‘bad’ science.
Thursday, August 28, 2008
Johnson & Johnson Likely Tekmira’s Next Official SNALP RNAi Delivery Partner
Since I know that many readers of this blog share a keen interest in Tekmira and the fate of SNALP delivery, one if not the leading systemic delivery technology for RNAi gene knockdown in the liver, and likely with use for solid cancers and immune-related diseases as well, you may like what I dug out today from the long-neglected junk mail pile on my desk: An abstract for the 6th Annual CHI conference on ‘RNAi for Therapeutics’ (Boston 22-23 October) by Pieter Peeters from the Johnson&Johnson company entitled ‘LIVER-SPECIFIC KNOCKDOWN USING SNALP-FORMULATED siRNAs TO STUDY PATHWAYS IN LIPOPROTEIN SECRETION AND STEATOSIS’.
The abstract body, which can also be found online here, as follows:
“As the application of RNA interference in vivo further develops, we are pursuing several promising technologies for systemic delivery. The goal of the current study was to evaluate the SNALP (“Stable Nucleic Acid Particles”) systemic RNAi delivery platform to knockdown genes encoding for key enzymes in triglyceride synthesis. SNALP-Formulated siRNAs were found to effectively knockdown mRNA levels by >90% in liver compared to a SNALP-Delivered scrambled siRNA control. The effect knock-down in liver was further investigated following a 3-week high-fat feeding challenge. The results demonstrated the efficient in vivo SNALP-mediated delivery of siRNA by systemic route and the utility of targeting novel targets to reduce fat storage in liver and improve hepatic steatosis.”
It is thus apparent that J&J is one of the as yet undisclosed SNALP evaluation partners of Tekmira, and has extensively used the delivery technology for target discovery/validation purposes as well as with a view of using it as a therapeutic modality itself. Given the obvious success of their efforts (note that J&J chose to present SNALP from among the other 'promising' delivery technologies it has evaluated), I would not be surprised for J&J to soon join the ranks of Alnylam, Merck, Takeda, Bristol-Myers Squibbs, and Roche, as the next major Tekmira SNALP RNAi delivery partner.
While I do not say that SNALP is the end-all, be-all for gene knockdown of the liver with new technologies likely to emerge over time, the extensive successful use of the technology by third-parties is great validation of its reproducibility and clinical relevance for advancement into the clinic at this time, and a testament to not just the liposomal patent position of Tekmira, but also the RNAi-related know-how of its scientists (see also Tekmira’s publication on the immunostimulatory potential of siRNAs and how to avoid it here).
Beyond the use of SNALP-RNAi in target validation, where will J&J likely be interested in applying the technology clinically? My first bet certainly would be on the treatment of hepatitis C infection. Not long ago, we learnt from a report in RNAiNews that J&J was about to contribute to a $25M fund-raising round for the DNA-directed RNAi company Nucleonics before the deal was called off on questions about scientific misconduct. I honestly could not believe that J&J would even consider collaborating with a company that has entered an RNAi phase I clinical trial for hepatitis B that was so patently destined to fail because of the known inefficiency of liposomal plasmid (not siRNA) delivery.
Developing an RNAi Therapeutic for HepC has also to be seen within the context of building upon J&J’s imminent franchise in this hard-fought market. Together with marketing and development partner Vertex Pharmaceuticals it is currently conducting phase III studies for telaprevir, a protease inhibitor that has all the looks of becoming the next big breakthrough drug in HepC (my other favorite biotech drug story is Tysabri, in case you wanted to know). With predicted cure rates of 60-70% for type I after the introduction of protease inhibitors, RNAi antivirals should be able to enhance cure rates due to their complementary mechanism of action.
Two years ago, J&J awarded RNAi discoverer and Nobel Laureate Craig Mello the Dr. Paul Janssen Award for Biomedical Research. This alone leads me to believe that J&J should have broad ambitions in RNAi as a platform technology. As the number of conference abstracts related to RNAi in the liver (especially fibrosis, metabolic disease) suggests, the liver alone should yield sufficient targets to keep SNALP scientists busy for now.
PS: Since I know that this blog is also read by some larger investment companies, despite the apparently low volume of Tekmira on the Toronto Stock Exchange, it should be possible to acquire shares in size without moving the price too much as the overhang of Protiva stock following the merger is noticeable. End of advertisement.
The abstract body, which can also be found online here, as follows:
“As the application of RNA interference in vivo further develops, we are pursuing several promising technologies for systemic delivery. The goal of the current study was to evaluate the SNALP (“Stable Nucleic Acid Particles”) systemic RNAi delivery platform to knockdown genes encoding for key enzymes in triglyceride synthesis. SNALP-Formulated siRNAs were found to effectively knockdown mRNA levels by >90% in liver compared to a SNALP-Delivered scrambled siRNA control. The effect knock-down in liver was further investigated following a 3-week high-fat feeding challenge. The results demonstrated the efficient in vivo SNALP-mediated delivery of siRNA by systemic route and the utility of targeting novel targets to reduce fat storage in liver and improve hepatic steatosis.”
It is thus apparent that J&J is one of the as yet undisclosed SNALP evaluation partners of Tekmira, and has extensively used the delivery technology for target discovery/validation purposes as well as with a view of using it as a therapeutic modality itself. Given the obvious success of their efforts (note that J&J chose to present SNALP from among the other 'promising' delivery technologies it has evaluated), I would not be surprised for J&J to soon join the ranks of Alnylam, Merck, Takeda, Bristol-Myers Squibbs, and Roche, as the next major Tekmira SNALP RNAi delivery partner.
While I do not say that SNALP is the end-all, be-all for gene knockdown of the liver with new technologies likely to emerge over time, the extensive successful use of the technology by third-parties is great validation of its reproducibility and clinical relevance for advancement into the clinic at this time, and a testament to not just the liposomal patent position of Tekmira, but also the RNAi-related know-how of its scientists (see also Tekmira’s publication on the immunostimulatory potential of siRNAs and how to avoid it here).
Beyond the use of SNALP-RNAi in target validation, where will J&J likely be interested in applying the technology clinically? My first bet certainly would be on the treatment of hepatitis C infection. Not long ago, we learnt from a report in RNAiNews that J&J was about to contribute to a $25M fund-raising round for the DNA-directed RNAi company Nucleonics before the deal was called off on questions about scientific misconduct. I honestly could not believe that J&J would even consider collaborating with a company that has entered an RNAi phase I clinical trial for hepatitis B that was so patently destined to fail because of the known inefficiency of liposomal plasmid (not siRNA) delivery.
Developing an RNAi Therapeutic for HepC has also to be seen within the context of building upon J&J’s imminent franchise in this hard-fought market. Together with marketing and development partner Vertex Pharmaceuticals it is currently conducting phase III studies for telaprevir, a protease inhibitor that has all the looks of becoming the next big breakthrough drug in HepC (my other favorite biotech drug story is Tysabri, in case you wanted to know). With predicted cure rates of 60-70% for type I after the introduction of protease inhibitors, RNAi antivirals should be able to enhance cure rates due to their complementary mechanism of action.
Two years ago, J&J awarded RNAi discoverer and Nobel Laureate Craig Mello the Dr. Paul Janssen Award for Biomedical Research. This alone leads me to believe that J&J should have broad ambitions in RNAi as a platform technology. As the number of conference abstracts related to RNAi in the liver (especially fibrosis, metabolic disease) suggests, the liver alone should yield sufficient targets to keep SNALP scientists busy for now.
PS: Since I know that this blog is also read by some larger investment companies, despite the apparently low volume of Tekmira on the Toronto Stock Exchange, it should be possible to acquire shares in size without moving the price too much as the overhang of Protiva stock following the merger is noticeable. End of advertisement.
Wednesday, August 27, 2008
Journal Club: mRNA Levels are a Good Indicator of Off-targeting
Next to delivery and immune stimulation, sequence-specific off-targeting is considered one of the fundamental challenges in the development of RNAi Therapeutics. Such off-targeting largely occurs by microRNA-like gene silencing following recognition of off-target RNAs that have limited sequence complementarity to the guide strand of the siRNA. Since microRNA gene silencing had long been believed to inhibit translation without affecting mRNA levels, there has been concern that the true extent of RNAi off-targeting may be larger than originally reported in studies assessing changes in global RNA levels. By achieving the feat of simultaneously measuring microRNA-induced changes in thousands of proteins, two high-quality papers now find that, surprisingly, mRNA levels are indeed a very good indicator of off-targeting, validating the use of established high-throughput RNA assays during the siRNA selection stage of RNAi Therapeutics development.
Key to the success of the Bartel lab at MIT (Baek et al.: The impact of microRNAs on protein output) and the Rajewski lab in Berlin (Selbach et al.: Wide-spread changes in protein synthesis induced by microRNAs), was their use of the SILAC technique (Stable Isotope Labeling with Amino acids in Cell culture). In this procedure, newly synthesized proteins are labeled with amino acids containing different isotopes. To determine the impact of microRNAs on global protein synthesis (~5000 proteins were identified in the studies), the samples with and without a given small duplex microRNA were labeled with different isotopes. This results in peptides from the same protein exhibiting slightly shifted peaks during protein mass-spectrometry, and the ratio of the peak intensities belonging to a pair of peptides is taken as a measure of microRNA-mediated gene silencing (1= no change). Changes at the protein level thus determined were then compared to changes on the mRNA level by microarray analysis.
As expected, the authors found that among the down-regulated proteins were highly enriched those that contained microRNA seed target sites in their 3’ untranslated regions (UTRs). Consistent with our notion of microRNAs as regulators of a group of genes rather than individual genes, typically 200-500 proteins were changed by more than 30% following the introduction or deletion of a microRNA, with about half of them likely the consequence of a direct interaction with the microRNA. Remarkably, despite the introduction of rather large amounts of microRNAs into the tissue culture cells (25-100nM using lipofection), most of these changes were rather subtle, and very rarely were changes of more than 3-fold detected. While it is always possible that wide-spread subtle changes may lead to a directed cellular response or cause toxicity in the case of RNAi Therapeutics, this observation is in agreement with microRNAs often acting as rheostats to maintain cellular homeostasis and suggests that the introduction of random short duplex RNAs should not necessarily lead to gene regulatory chaos and distinct phenotypes.
The surprise came when it was found that changes in protein levels could for the most part be accounted by changes in mRNA levels. Only very few proteins were reduced without concomitant mRNA changes. Without going into the details, the studies nevertheless support a model in which translational repression is the primary mechanism of microRNA-mediated gene silencing and mRNA destabilization a consequence thereof. Consistent with this hypothesis,
in the rare instances where 2 microRNAs were located close to each other, silencing was synergistic, yet the mRNA level was not further reduced. This is quite interesting as it may explain how the early use of microRNA reporter systems in which multiple microRNA target sites were closely spaced and which consequently caused strong gene suppression could have led to the notion of translational silencing only. The 20-30% reduction in mRNA could have easily been dismissed as experimental noise.
For practical purposes, however, measuring mRNA levels are highly predictive of proteomic changes and this should allow for the selection of RNAi Therapeutics candidates that have an increased likelihood of not causing toxic changes in gene expression. Merck has long been a proponent of this approach and partly for this reason have closely aligned the systems biology expertise of subsidiary Rosetta Inpharmatics with their RNAi incubator Sirna Therapeutics, in addition to employing systems biology for identifying the right gene targets.
It is very comforting to know that the extent and severity of off-targeting on the protein level is not significantly greater than what we have observed at the RNA level. Moreover, the ability to use high-throughput RNA expression technologies for assessing off-targeting and chemical modification techniques which have proven to significantly further reduce off-targeting potential of siRNAs should minimize the risk of RNAi Therapeutics candidates to fail due to sequence-specific off-targeting. A good siRNA at high concentrations in tissue culture reduces about 5-10 off-target RNAs by more than 2-fold (and as we know now proteins to a similar degree, not more), while the target is the most suppressed gene. By acting at the RNA level, RNAi Therapeutics has the distinct safety advantage in that it allows for off-targeting to be assessed in such a comprehensive manner. Before investing $1 billion dollars in the development of a drug, it appears to be well worth taking advantage of this by trying to understand these changes.
Key to the success of the Bartel lab at MIT (Baek et al.: The impact of microRNAs on protein output) and the Rajewski lab in Berlin (Selbach et al.: Wide-spread changes in protein synthesis induced by microRNAs), was their use of the SILAC technique (Stable Isotope Labeling with Amino acids in Cell culture). In this procedure, newly synthesized proteins are labeled with amino acids containing different isotopes. To determine the impact of microRNAs on global protein synthesis (~5000 proteins were identified in the studies), the samples with and without a given small duplex microRNA were labeled with different isotopes. This results in peptides from the same protein exhibiting slightly shifted peaks during protein mass-spectrometry, and the ratio of the peak intensities belonging to a pair of peptides is taken as a measure of microRNA-mediated gene silencing (1= no change). Changes at the protein level thus determined were then compared to changes on the mRNA level by microarray analysis.
As expected, the authors found that among the down-regulated proteins were highly enriched those that contained microRNA seed target sites in their 3’ untranslated regions (UTRs). Consistent with our notion of microRNAs as regulators of a group of genes rather than individual genes, typically 200-500 proteins were changed by more than 30% following the introduction or deletion of a microRNA, with about half of them likely the consequence of a direct interaction with the microRNA. Remarkably, despite the introduction of rather large amounts of microRNAs into the tissue culture cells (25-100nM using lipofection), most of these changes were rather subtle, and very rarely were changes of more than 3-fold detected. While it is always possible that wide-spread subtle changes may lead to a directed cellular response or cause toxicity in the case of RNAi Therapeutics, this observation is in agreement with microRNAs often acting as rheostats to maintain cellular homeostasis and suggests that the introduction of random short duplex RNAs should not necessarily lead to gene regulatory chaos and distinct phenotypes.
The surprise came when it was found that changes in protein levels could for the most part be accounted by changes in mRNA levels. Only very few proteins were reduced without concomitant mRNA changes. Without going into the details, the studies nevertheless support a model in which translational repression is the primary mechanism of microRNA-mediated gene silencing and mRNA destabilization a consequence thereof. Consistent with this hypothesis,
in the rare instances where 2 microRNAs were located close to each other, silencing was synergistic, yet the mRNA level was not further reduced. This is quite interesting as it may explain how the early use of microRNA reporter systems in which multiple microRNA target sites were closely spaced and which consequently caused strong gene suppression could have led to the notion of translational silencing only. The 20-30% reduction in mRNA could have easily been dismissed as experimental noise.
For practical purposes, however, measuring mRNA levels are highly predictive of proteomic changes and this should allow for the selection of RNAi Therapeutics candidates that have an increased likelihood of not causing toxic changes in gene expression. Merck has long been a proponent of this approach and partly for this reason have closely aligned the systems biology expertise of subsidiary Rosetta Inpharmatics with their RNAi incubator Sirna Therapeutics, in addition to employing systems biology for identifying the right gene targets.
It is very comforting to know that the extent and severity of off-targeting on the protein level is not significantly greater than what we have observed at the RNA level. Moreover, the ability to use high-throughput RNA expression technologies for assessing off-targeting and chemical modification techniques which have proven to significantly further reduce off-targeting potential of siRNAs should minimize the risk of RNAi Therapeutics candidates to fail due to sequence-specific off-targeting. A good siRNA at high concentrations in tissue culture reduces about 5-10 off-target RNAs by more than 2-fold (and as we know now proteins to a similar degree, not more), while the target is the most suppressed gene. By acting at the RNA level, RNAi Therapeutics has the distinct safety advantage in that it allows for off-targeting to be assessed in such a comprehensive manner. Before investing $1 billion dollars in the development of a drug, it appears to be well worth taking advantage of this by trying to understand these changes.
Saturday, August 23, 2008
Disingenuous Leerink Swann Report on Value of RNAi Therapeutics
What a difference 2 years can make…
• "RNA interference could be the next breakthrough technology and we believe RNAi is well positioned at forefront of development of novel nucleic acid based therapeutics." – Leerink, 2006
• "We believe RNAi's therapeutic technology platform could yield a robust source of pipeline candidates…" – Leerink, 2006
• "Alnylam is narrowly focused upon the development of a Nobel Prize winning technology called RNA interference (RNAi) as a drug development tool that we feel is more suited for research purposes than the development of therapeutics." – Leerink, 2008
Originally, I intended this blog to be positive and reflect my enthusiasm for RNAi Therapeutics. While my enthusiasm remains intact, I also learned that as the financial stakes in the industry have increased rapidly, it has not only attracted supporters hoping that one day RNAi will improve healthcare, but also an ever-growing list of detractors hoping to capitalize on the financial misfortunes and medical failures of what would appear to be a benign biotechnology. As the drug development and approval process is as political as it is about science and medicine, and requires significant investments, I see it within the scope of this blog to defend RNAi Therapeutics from some of these misconceptions that are being planted.
This week’s report by Leerink Swann analyst Jonas Alsenas, initiating coverage of Alnylam with a ‘Sell’ rating (the only one by Leerink Swann at that!) and a 2009 price target of $14-16, is probably the most amateurish, yet apparently effective attempt to sabotage the development of RNAi Therapeutics. This report which essentially states that nucleic acid therapeutics have never been a success, and therefore never will be, and that the recent interest in RNAi is merely a reflection of the poor state of Big Pharma in general (but what about all the other technologies that do not get similar interest in this climate?), is littered with so many factual mistakes that one has to come to the conclusion that a) either is the author utterly unqualified to comment on biotechnology and RNAi and should look for another job soon, or b) that it was driven by an agenda to hurt the share price of Alnylam for the benefit of the massive short interest in the company, or maybe c) just the a revenge of somebody having missed the boat. BTW, 2008 is a bit late for a “healthcare investment bank” to initiate coverage of the leading company in the RNAi space.
But now to the most important part of today’s blog entry. In what follows, a good friend of mine, scientist, and long-time follower of the RNAi Therapeutics space has volunteered to dissect the Leerink Swann report to expose the mistakes and intentions behind it. Not only is he very well qualified to do so, he also has a uniquely refreshing writing-style that cuts through the clutter which I’m sure you are about to enjoy as much as I do.
• "RNA interference could be the next breakthrough technology and we believe RNAi is well positioned at forefront of development of novel nucleic acid based therapeutics." – Leerink, 2006
• "We believe RNAi's therapeutic technology platform could yield a robust source of pipeline candidates…" – Leerink, 2006
• "Alnylam is narrowly focused upon the development of a Nobel Prize winning technology called RNA interference (RNAi) as a drug development tool that we feel is more suited for research purposes than the development of therapeutics." – Leerink, 2008
Originally, I intended this blog to be positive and reflect my enthusiasm for RNAi Therapeutics. While my enthusiasm remains intact, I also learned that as the financial stakes in the industry have increased rapidly, it has not only attracted supporters hoping that one day RNAi will improve healthcare, but also an ever-growing list of detractors hoping to capitalize on the financial misfortunes and medical failures of what would appear to be a benign biotechnology. As the drug development and approval process is as political as it is about science and medicine, and requires significant investments, I see it within the scope of this blog to defend RNAi Therapeutics from some of these misconceptions that are being planted.
This week’s report by Leerink Swann analyst Jonas Alsenas, initiating coverage of Alnylam with a ‘Sell’ rating (the only one by Leerink Swann at that!) and a 2009 price target of $14-16, is probably the most amateurish, yet apparently effective attempt to sabotage the development of RNAi Therapeutics. This report which essentially states that nucleic acid therapeutics have never been a success, and therefore never will be, and that the recent interest in RNAi is merely a reflection of the poor state of Big Pharma in general (but what about all the other technologies that do not get similar interest in this climate?), is littered with so many factual mistakes that one has to come to the conclusion that a) either is the author utterly unqualified to comment on biotechnology and RNAi and should look for another job soon, or b) that it was driven by an agenda to hurt the share price of Alnylam for the benefit of the massive short interest in the company, or maybe c) just the a revenge of somebody having missed the boat. BTW, 2008 is a bit late for a “healthcare investment bank” to initiate coverage of the leading company in the RNAi space.
But now to the most important part of today’s blog entry. In what follows, a good friend of mine, scientist, and long-time follower of the RNAi Therapeutics space has volunteered to dissect the Leerink Swann report to expose the mistakes and intentions behind it. Not only is he very well qualified to do so, he also has a uniquely refreshing writing-style that cuts through the clutter which I’m sure you are about to enjoy as much as I do.
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