Sunday, December 30, 2007
Nevertheless, I feel that most systematic progress has been made in delivering RNAi to the liver with more progress likely to follow. There have now been a fair number of publications that clearly show target-specific RNAi knockdown in the liver, unlike maybe some of the lung studies where there were occasional data interpretation issues. Of course, in declaring 2008 ‘The Year of the Liver’, I should disclose that I am somewhat biased due to the hepatotropic focus of the laboratory that I work in.
For siRNA-mediated RNAi in the liver, I currently see three front-runners, although it is possible that among the considerable work that is ongoing and not yet published, others have reached a similar stage of technological maturity. These are the in-famous SNALPs, Mirus Bio’s “Dynamic PolyConjugates”, and MIT’s lipidoids. SNALPs, being worked on by Protiva, Tekmira, Alnylam, and Sirna/Merck, are probably 12-18 months ahead of the game and best characterized, whereas the data on PolyConjugates and lipidoids are very promising, but still somewhat spotty.
SNALPs, stable nucleic acid lipid particles, are set to enter the clinic in 2008 with programs in hypercholesterolemia and liver cancer (both Alnylam) and possibly a clinical candidate chosen by Tekmira. The hypercholesterolemia program is also an opportunity to get an early measure of therapeutic efficacy, possibly in ‘08. Protiva’s and Sirna/Merck’s intentions are less clear and I believe that Big Pharma often chooses to keep phase I programs secret for competitive reasons, so that it is theoretically possible that Sirna has already entered the clinic or is about to do so with the long-anticipated SNALP RNAi for Hepatitis C.
It is obvious that there were some delays in bringing SNALP RNAi to the clinic, and I believe that this is largely due to dosing and safety issues. Both of these concerns may come down to the propensity of SNALP liposomes to be taken up by immune cells such as Kupffer cells in the liver and plasmacytoid dendritic cells which a) function like a sink for the liposomes when they enter the liver so that SNALPs become available for entering the desired hepatocytes only after the sink is saturated, something that complicates dosing; and b) increases the risk of triggering unwanted cytokine responses. I am optimistic, however, that by varying the composition of SNALPs, safe and efficacious formulations may be found, particularly if the liposome uptake mechanism by the professional immune surveillance system were to be different from that by the hepatocytes, which I think is quite reasonable to assume. I therefore hope that the fact that so many scientists are working on SNALP RNAi is a sign of its promise rather than desperation. As such, the number of R&D staff at Tekmira has more than doubled from 17 to 39 in the year ending September 2006 largely due to work on SNALPs.
As I have written before, I am much taken by Mirus’ PolyConjugate work, although this is based on only a single paper published in the middle of ‘07 (see 24 July 07 Blog: “Mirus Scientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes”). The neat aspect of that work was that it showed that it should be possible to avoid the Kupffer cells in the liver and specifically target hepatocytes for knockdown with the help of carbohydrate ligands (I am curious whether similar targeting ligands would also work in the context of other formulations). As we know, this work is partnered with Pfizer, and I wonder whether the upcoming loss of exclusive marketing rights for their wonder-drug Lipitor will spur them into action here.
I am still waiting for more data on lipidoid-mediated RNAi which hopefully will become available soon as was indicated in the footnotes of an October 2007 paper on the effect of siRNA delivery on microRNA function. From both a business and scientific perspective, it will be interesting to see whether Alnylam may choose lipidoids over SNALPs for its first liver RNAi programs and the overlap of SNALP and lipidoid both in IP and manufacturing terms.
The liver may get further attention from the targeting of microRNA-122 for the treatment of hypercholesterolemia and Hepatitis C, and other programs on targeting certain microRNAs for the treatment of hepatocellular carcinoma. There are various programs by Santaris, Regulus and others that have progressed into larger animals and we may even see a first IND being filed for one of these applications by the end of 2008. Development in the antisense field, particularly the partnering of ISIS’ mipomersen, should also generate heightened awareness for RNA-based therapeutics of liver disease.
Other predictions for 2008:
1) Unpredicted delivery technologies
2) Pfizer finally makes its move
3) News on RNAi for wet AMD- good and/or bad?
4) Protiva-Tekmira dispute resolved (wishful thinking)
For a nice presentation on the powers of SNALPs by Protiva chief scientist Ian MacLachlan, please visit: http://mms.technologynetworks.net/RNAi06Presentations/maclachlan/Player.html
Monday, December 24, 2007
The Pardridge group has been working on so called Trojan Horses to get pharmaceutical agents across the blood-brain-barrier. This may be achieved by coupling the active drug ingredient to a monoclonal antibody that targets receptors on the vascular endothelium of the brain which can then ferry the entire complex through the endothelial cell layer and into the brain.
In this study, Xia and colleagues combine streptavidin-antibody fusion proteins to biotinylated siRNAs which form tight complexes mediated by the extremely high affinity of streptavidin for biotin. The antibodies recognize the transferrin and insulin receptors which are the most widely studied receptors in this apparently understudied area of research. After demonstrating that it is possible to generate reasonable amounts of pure Trojan Horses, the authors use them for knock down studies in tissue culture with 75-85% knockdown efficacy which was further dependent on the antibody. Importantly, the biotin-streptavidin interaction did not impair knockdown efficacy and both 5’ and 3’ biotinylation of the passenger siRNA strand was tolerated, in support of the flexibility of this technology.
They then move on to an in vivo tumour model where rat glioma cells expressing a reporter gene (luciferase) are implanted into the brains of rats and grown for 5 days. At this point, the animals were intravenously administered with the Trojan Horses incorporating an siRNA against the reporter gene. Remarkably, at a dosage of as little as 270ug/kg siRNA (this does not include the protein component of the formulation though), luciferase levels stabilized and were 4 fold less compared to animals injected with saline control alone, and no obvious toxicities were observed.
One critical control that I would have liked to see is a Trojan Horse with a control siRNA as the assay did not distinguish whether the relative decline in luciferase activity was the result of actual gene knockdown or due to some cytotoxic effect, so that one could rule out that binding of the monoclonal antibody to transferring receptors on the cancer cells itself was the cause for the reduction in luciferase activity. Another nice experiment would have been to target a cancer-related gene and look at survival and other therapeutic measures. In any case, it will be interesting to follow the progress of this technology which appears to be partnered with the Californian biotech company ArmaGen Technologies.
PS 1: In my 19 June 07 Blog: “New Breakthrough in the Systemic Delivery of RNAi for the Brain” I described a related study published in Nature where a rabies peptide facilitated the transfer of an siRNA across the BBB. In regards to that study, Xia et al. make the following cryptic remark: “The RVG peptide is hypothesized to cross the BBB via receptor-mediated transport on the brain capillary endothelial nicotinic acetylcholine receptor. However, activation of brain microvascular AChR causes BBB disruption.” Although temporarily disrupting the BBB may be a way to get drugs into the brain, it seems to raise safety concerns as the BBB serves to keep bad stuff, such as viruses out of the brain for a reason.
PS 2: Last week, Santaris signed a nice collaboration agreement with GSK for the development of LNA-based antisense drugs valued at up to $700M, further highlighting Big Pharma's interest in RNA-based therapies. While I have much to learn about the safety of LNAs, I have little doubt that they can be quite efficient in binding to their targets and are perhaps particularly attractive for antagonising microRNAs since the simple act of sequestering a microRNA would already be effective. Unfortunately for retail investors, the same week it announced another private funding round for M20 Euros. Santaris should find sufficient interest should it decide to go public.
Monday, December 17, 2007
Most shRNA approaches are based on DNA-directed expression of RNA hairpins in the nucleus that are then fed into the RNAi pathway. Somagenics’ approach is slightly different in that they are introducing the RNA directly into cells. Concentrating more on the science than IP issues here I only want to briefly remark that this probably overlaps quite a bit with the Hannon patents (licensed to both CytRx and Alnylam) on the use of shRNAs as RNAi triggers, both transcribed and synthetic.
The scope of the experiments were limited either to in vitro culture studies or its glorified in vivo counterpart, the hydrodynamic co-transfection experiment. Here, a large volume of RNA-containing liquid is injected into the tail vein of a mouse in a brief period of time, physically forcing the RNA into hepatocytes. It is of interest, that unlike the findings of the back-to-back papers of the Hannon and Rossi labs more than two years ago, hairpins (directed to the conserved HCV IRES) with minimal stems, 19 base-pairs, were generally more effective than those with extended 25 base-pair hairpins. While it has to be said that compared to the Hannon and Rossi papers which argued that the long stems that were processed by Dicer would increase RNAi efficacy, the sample size here was quite limited, it nevertheless shows that the processing of hairpins are often not as predictable as one would wish based on microRNA biology.
Johnston further reported that the shRNAs were at least as efficient as the corresponding siRNAs with some of the IC50s in the low picomolar range. I am curious how this will translate into in vivo uses for example because shRNAs, unlike classical siRNAs and Rossi-type two-stranded Dicer-substrates, contain the double-stranded RNA within one molecule which should make this structure thermodynamically quite stable. A potential disadvantage is that it is so much more expensive to synthetically make the ~42-55 nucleotide hairpin RNAs since cost and purity of manufacturing RNAs increases more than linearly after you reach a certain size, say 25 nucleotides. While these studies mostly used RNAs generated through biochemical synthesis in the test tube using recombinant phage RNA polymerases, for the clinic they are working together with Agilent to generate the “same” RNAs to scale. I say the “same” here since the phage polymerase leaves a triphosphate 5’ end while chemical synthesis does not. This is not a trivial issue here since 5’ modifications are known to influence RNAi processing. We will therefore have to wait how the transition from in vitro transcribed to synthetic shRNAs will affect the reproducibility of their data and potential concerns from the FDA. Similarly, some mention was made that shRNAs may tolerate RNA modifications less well than siRNAs, probably because they are subject to additional processing steps.
The use of more challenging animal models is also warranted before entering the clinic. It is therefore important for them to find suitable delivery solutions which they apparently have only started to. It was good to hear though hearing him mention Protiva/Tekmira’s SNALPs and Mirus Bio’s Dynamic PolyConjugates as probably the most advanced RNAi delivery platforms to the liver, reflecting my views on what’s out there in the literature. Overall, I like the fact that multiple RNAi approaches are in the pipeline to tackle HCV, namely siRNAs: Sirna/Merck; DNA-directed shRNAs: Tacere/Benitec and Nucleonics; and now synthetic shRNAs: SomaGenics. And maybe Tekmira/Protiva should harness the power of SNALP and officially nominate HCV as a clinical development program. I hope that the next 2 years should finally see programs moving into the clinic after HCV RNAi had been delayed in the wake of company-specific issues (Benitec; Merck/Sirna vs Protiva).
Thursday, December 13, 2007
Before initiating phase II studies in naturally infected adults, the company seeks to gather more information first on the pharmacodynamics of RSV-01 in an experimental infection model where the siRNA is administered by nasal administration (top-line data expected early 2008; company reported patient enrollement complete now), and second from the safety of an inhaled version of RSV-01, the eventual route of administration, in healthy adult volunteers. Results from the latter phase I trial were just reported at the 18th Annual Drug Delivery to the Lungs meeting in Edinburgh, Scotland.
This study included 109 subjects, 71 of whom received siRNA either in a single dose (0.1mg/kg to 3mg/kg) or multiple doses (0.01mg/kg to 0.6mg/kg once daily for 3 days). The safety data were encouraging as there were no serious adverse events reported. Nevertheless, the press release mentioned a “mild to moderate flu-like adverse event” at the higher dose group in the single-dose arm. While this is pure speculation, this reminds me of the fact that RSV-01 is an unmodified siRNA and should therefore be more prone to elicit cytokine responses, an area Alnylam by the way is taking quite seriously as they actively seek to recruit scientists working in the field of immunostimulatory nucleic acids, which by the way may be accelerated by the recent acquisition of Coley by Pfizer. Indeed, I would not be surprised if part of the remarkable antiviral efficacy of RSV-01 is based on the siRNA acting as an isiRNA, the term with which Gunther Hartmann from Bonn describes small interfering RNAs with immunostimulatory properties.
In order to find the best therapeutic window, Alnylam has also been working on comparing the pre-clinical efficacy of single-dose versus multiple dose RSV-01. Gratifyingly, for the same amount of total siRNA administered, multiple dose administration was significantly more potent. This means that a good strategy of increasing antiviral efficacy while at the same time decreasing the risk of eliciting flu-like effects may be to choose multiple dose administration for the phase II studies of naturally infected patients.
Another interesting point mentioned in the press release is that siRNA delivery as evidenced by siRNA plasma levels was remarkably efficient compared to some pre-clinical data. Well, I guess had they not observed siRNA in the plasma, then the press release would have stated that avoiding systemic exposure was an additional safety feature of RSV01. This is how the phase I nasal data were interpreted. In any case, this is reminiscent of recent data for systemic siRNA administration to the liver where the efficacy and duration of RNA silencing in non-human primates was above expectation based on small animal experience and may have in fact contributed to the side-effects observed at the higher doses. But before actual plasma levels are reported, it is premature to speculate whether systemic siRNA administration by inhalation should be considered for other indications.
Overall, while the results indicate that the development of RSV-01 is not without risk, Alnylam continues to demonstrate that by conducting a wide-ranging scientific program supporting the compound, it will allow them to choose the most promising development path. The apparently efficient delivery efficacy via nebulizer makes me quite bullish about the antiviral efficacy of ALN-RSV01 so that safety should be the focus of future studies.
Sunday, December 9, 2007
The Wall Street Journal Reports that ‘Big Pharma Faces a Grim Prognosis’- In what Form will it take its RNAi Pill?
To go biotech, which is now producing many of the most innovative and high-margin drugs and which has so far largely avoided similar pressures and proven quite profitable if you invested in the right platforms such as monoclonal antibodies and recombinant proteins, appears to be one of the last options left for Big Pharma to survive. As the likes of Merck, Pfizer, and AstraZeneca jostle to become the leading biotech company of the future, RNAi as one of the few broad technology platforms with a unique mechanism of action has to be up very high on their priority list. So how will Big Pharma get into the game?
There are the early adopters such as Merck, Novartis, and Pfizer which have been quite public about their RNAi efforts. Merck initially played the nice guy that wanted to help companies like Alnylam translate the science of RNAi into drugs. However, with the acquisition of Sirna Therapeutics and later difficulties with Alnylam, it is clear that Merck had grander ambitions than just being a humble licensee. Pfizer on the other hand has been trying this and that as if they first wanted to confirm the clinical viability of RNAi and then make their move. Eventually, unless of course RNAi fails (which I tend not to believe), they will all have to, because even though you are now allowed to use patented technologies with the intent of developing commercial drugs, the moment you hit the market, you have to pay in one form or another, and it is common practice that the earlier you license the less it will cost you.
As I’ve been trying (in vain) to find out which Prior Art was cited in the EPO decision as a reason to restrict the scope of Kreutzer-Limmer to 15-21 base-pair double-strand RNA in 2006 (if somebody can help me here, please contact me), I have come across a webpage on the EPO site where some of the opposition history of K-L is documented. I found it quite interesting that initially, the opposition included the likes of ISIS Pharmaceuticals, now Alnylam’s modification partner for RNAi Therapeutics, and Novartis, the second Big Pharma after Merck to take a broad license from Alnylam. Both of them dropped their opposition and instead joined Alnylam. On the other side there are the likes of Sanofi-Aventis, AstraZeneca, and Atugen (now Silence Therapeutics), and we know that AstraZeneca eventually took a license from Silence, probably the cheaper option.
It therefore appears that in the not-so-distant future we will see Big Pharma split into two camps- those with Alnylam, and those against Alnylam. The rationale for the latter either being the belief that they can find a way around Alnylam’s IP estate, or at least avoid some of the royalties by trying blunt-end dsRNA of longer than 21 base-pairs should K-L’s scope not stand. Of course, the higher the going rate for the license fees, the more the temptation to go that route, and maybe Alnylam bravely does not even mind it that way too much, since a sense of exclusivity probably makes the terms of the licenses more attractive for them. Nevertheless, for somebody with the balance sheet of many in Big Pharma, this would not only appear to be scientifically quite risky, but also penny-wise and pound-foolish. As the WSJ documented so well, the times have probably come to realize that a lot of the innovation has happened outside their research labs and no matter the correlation between wallet size and ego, being humble at the right time may help you survive the next 10 years. The same EPO website also says that Janssen Pharmaceutica, a subsidiary of Johnson and Johnson, has dropped their opposition- what do you conclude?
Saturday, December 1, 2007
The Confusing World of AtuRNAi, Stealth siRNAs and mdRNAs (III and Final Part): The RNAi Therapeutics Fashion Show
Vick’s views obviously were not echoed by Alnylam’s CEO, John Maraganore, also at an investor presentation, who emphasized that no single patent will give you the right to work on commercializing RNAi Therapeutics. For freedom-to-operate, it takes a whole range of fundamental patents, such as IP covering the use of double-stranded RNAs for gene silencing in humans (Kreutzer-Limmer, Tuschl, Kay), nucleic acid modifications to make them drug-like (Crooke), and even the use of an dsRNAse-mediated mechanism itself (Crooke). It is the view of many, including myself, that it was Alnylam that has understood it very early on to gain exclusive and non-exclusive access to all of the early patents and applications that could even remotely impinge on the use of RNAi in the clinic. What this means is that only Alnylam has a blocking IP estate and that no matter which fundamental (e.g. Fire-Mello) or fringe patents you may have access to, you will still have to pay your dues in one form or another at the Alnylam toll gate. It is worth remembering that at least in the US, a patent does not give you the right to do anything, but the right to block somebody else from using the underlying technology.
So now let’s turn our attention to the RNAi Therapeutics catwalk, looking at the IP and technology strengths and weaknesses of a couple of better known RNAi Therapeutics companies (excluding Big Pharma with RNAi operations):
1) Alnylam Pharmaceuticals: The leader in the translation of the science of RNAi into drugs. Has virtually freedom-to-operate and blocking IP estate, now validated by a number of high-profile partnerships. Ability to gain access to the most promising delivery platforms desirable; IP protection may be sought for knocking down certain genetically validated genes for certain diseases, like they have done for VEGF before. Sorry- “Not-For-Sale”, want to become a top-tier biopharmaceutical themselves.
2) RXi (RNAi subsidiary of CytRx): Has assembled a motley array of patents including access to Tuschl I via UMass and Hannon (both co-exclusive with Alnylam), although many have not issued. These patents may lessen the pain when paying the Alnylam toll. Credible and engaged scientific advisory team including Nobel laureate Craig Mello (UMass) and Greg Hannon (Cold Spring Harbor). Puzzlingly, this company has not made much public progress in moving RNA therapeutics into the clinic. What are they waiting for?
3) Silence Therapeutics: Proven RNAi Therapeutics know-how with already two licensed compounds in the clinic and more to come soon. I am pleased by their progress on siRNA delivery to the vasculature, boding well for their anticipated cancer trials. Their 23 base-pair Atu-RNAi was granted IP protection in Europe and they hope to obtain same in the US soon. This allowed them to close a number of partnerships with Big Pharma, although the upfront payments were not enormous. They are, however, moving on very thin ice if they believe that with this one patent they have freedom-to-operate. It is mostly for their know-how that I believe that they may be one of the next RNAi acquisition targets by Big Pharma. Such a takeover would be “friendly” given Silence’s frequent statements that they would not mind being bought out.
4) Nastech: As RNAi was slowly getting some attention, Nastech quickly assembled a rather large team of experienced nucleic acid scientists that has been busy churning out patent application after patent application with the aim of working around Alnylam’s blocking IP position. Like Silence Therapeutics (and Dicerna) they hope that the scope of Tuschl II and Kreutzer-Limmer will ultimately not cover “long” small interfering RNAs. The ice may be thicker here than for Silence though. Apparently over 150 patent applications, more than Alnylam, according to CEO Steven Quay. Looked up one of their more recent ones on cross-linked peptide-siRNA particles for RNAi delivery. If I understood that one data slide correctly, they only achieved a 15% knockdown (???). Their other applications rather be better in terms of enablement, otherwise I would get the impression that the 150 applications are part of an overblown balloon. Nevertheless, should be considered a takeover candidate for Big Pharma that wants to hit the ground running in RNAi. However, following the P&G disaster, their position in partnership and fund-raising negotiations is quite hurt.
5) Benitec: Australian DNA-directed RNAi company, beaten a hasty retreat from the US after management and dubious patent issues caused them to run out of money. Still, a Benitec-sponsored HIV trial is well and underway at the City of Hope and it can only be hoped that they will remain a force in realizing the promise of DNA-directed RNAi. Only small in-house scientific team, strongly relying on their clinical partners for technology know-how. Major issue is their ability to attract funding when there is a lot of uncertainty surrounding the quality of their IP claims. Nucleonics, which has a phase I DNA-directed RNAi trial for HBV ongoing and is out to raise more funds, considered their arch-rival, but a number of other gene therapy companies, including Targeted Genetics and Introgen, also likely to extend their work in RNAi. DNA-directed RNAi efforts are somewhat less affected by Alnylam’s dominant IP estate, but overlaps exist.
6) Calando (subsidiary of Arrowhead Research Corp.): One of the increasing number of RNAi companies with a focus on delivery, in this case based on cationic cyclodextrin polymers that bind siRNAs for systemic, and potentially targeted delivery. Tox/efficacy data from non-human primate work indicates that this formulation may have promise for cancer applications. Need to license core siRNA patents. Although they may not be a prime acquisition target at this stage, clinical proof-of-concept studies may make them interesting for Big Pharma and Alnylam in 3-5 years.
7) Protiva-Tekmira: Although arch-rivals, I mention them together, because they belong together, both working on cationic liposomal delivery of RNAi. Right now, cationic liposomes are the most advanced systemic RNAi delivery method and both companies consequently had no lack of partnering interest. Unfortunately, IP (Tekmira) and related enabling know-how (Protiva) appear to be split between the companies, and maximal value creation should be achieved through collaboration, rather than wasting their time in the courts. One would hope that both parties realize soon that the whole is so much more valuable than the sum of the parts. If they would face the economic realities they would get back to business and either re-unify or cross-license (I will never get tired of making that plea, in case you have not noticed).
8) Innumerable nanotech delivery efforts, some of which are set to rise to more prominence. One such company is Intradigm that has done some interesting work on RNAi delivery a couple of years ago. Essentially all of them aim to partner their technologies sooner or later. A niche player is Cequent Pharmaceuticals, based on “trans-kingdom RNAi” where orally administered bacteria that express short hairpin RNAs are used to deliver RNAi to the small and large intestine for the treatment of related diseases such as inflammatory bowel disease and cancer. While their Nature Biotech paper showed promise, due to the out-of-the-box nature of this invention and many open mechanistic questions, more data is needed to make me feel at ease with this technology. Nevertheless, the $9M funding round this summer, including the Novartis Option Fund, should be taken as a vote of confidence.
While Alnylam is not-for-sale, many of the other pure play RNAi Therapeutics companies were obviously established and are managed with the intention of being sold off to larger corporations. Sirna Therapeutics was the first one to go and more are likely to follow. Accordingly, John Rossi stated in an interview about the Dicer-substrate start-up Dicerna that this company was basically established with the intention of selling it off to an innovation-starved, RNAi-challenged Big Pharma later on. Consequently, I believe that the RNAi Therapeutics landscape in 20 years will likely largely consist of Alnylam, Alnylam-licensed Big Pharma companies, some of which will have bought in nucleic-acid know-how in the form of small pure-plays biotechs. Those that did not find any suitor will have a difficult time of surviving, and may only survive by hitting the clinical jackpot early on in the game.
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