Sunday, December 30, 2007

2008: The Year of the Liver

It is true that next year could bring the first human proof-of-concept for an RNAi therapeutic. But since results from the experimental infection studies for ALN-RSV01 were originally expected in late 2007 and the results would be an overhang of work done in ’07, I find it more appropriate to pick a fresh candidate. It is also true that the lung may rival the liver in the number of programs moving into the clinic in ‘08 given recent data that suggest delivery of siRNAs to the lung by nebulizer to be fairly innocuous, and the fact that AstraZeneca and GSK should be busy working in that area following their agreements with Silence and Sirna Therapeutics, respectively.

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:

Monday, December 24, 2007

Journal Club: Trojan Horses that Harbor RNAi to Dupe the Blood-Brain-Barrier

An interesting paper relating to the delivery of RNAi Therapeutics to the brain via systemic administration was recently published by the Pardridge group at UCLA in the journal Pharmaceutical Research (Xia et al.: “Intravenous siRNA of Brain Cancer with Receptor Targeting and Avidin-Biotin Technology.”).

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

SomaGenics Reports on the Use of shRNAs for the Treatment of HCV

At the end of a day packed full with fun RNA science from the San Francisco Bay Area, Brian Johnston, President and CEO of SomaGenics presented some of his company’s data on the use of shRNAs for the treatment of HCV, historically one of the early therapeutic targets for RNAi.

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

Breaking News: Alnylam Reports on the Safety of Inhaled ALN-RSV01

The eyes of the RNAi Therapeutics world are on Alnylam as they are approaching critical clinical milestones for their lead development program ALN-RSV01 for the treatment of respiratory syncytial virus infection.

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?

As I am procrastinating about what to write in a review about the ‘Business of RNAi’, I have tried to get more into the heads of Big Pharma rather than seeing the world from the RNAi company’s point of view as I used to. In a nice front-page article of the Wall Street Journal this week (, Barbara Martinez and Jacob Goldstein vividly painted a dark portrait about looming patent expirations and generic competition, declining drug approval rates, and a research enterprise that has just gotten too bureaucratic to respond to the new pressures.

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

Shortly after Alnylam announced issuance of patent protection for the Kreutzer-Limmer series covering double-stranded RNAs between 15 and 49 base-pairs in Germany this week, the CEO of Silence Therapeutics, Jeffery Vick, shot back at an investor conference presentation reassuring their investors that this development would not affect Silence’s freedom-to-operate, including its all-important ability to close lucrative partnerships based on their proprietary (?) Atu-siRNA technology. Vick’s confidence stems from the observation that the first Kreutzer-Limmer patent had been opposed in Europe before and was consequently reduced to cover dsRNA of 15 to 21 base-pairs in 2006 (see previous Blog entry), and therefore would cover Silence’s blunt, 23 base-pair Atu-siRNAs. He vowed to fight the new patent, which is now likely to be issued throughout Europe in due course, to restrict its scope. My impression is that the patent attorneys will have a feast in the years to come, and just wait until Alnylam sees the time has come to return the favor and go after Silence’s issued patents.

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.

Tuesday, November 27, 2007

Alnylam Granted Expanded Kreutzer-Limmer Patent Series in Germany, Signals Its Intention to Enforce Dominant IP Position

Yesterday, Alnylam announced issuance of the new Kreutzer-Limmer patent series in Germany, covering double-stranded RNAs of 15 to 49 base-pairs for gene silencing in mammals. This is quite significant and Alnylam’s accompanying press release made it clear that this should be understood as a watershed event, sending a stern signal to companies like Silence Therapeutics, RXi, Nastech, Dicerna and others that thought to have identified Kreutzer-Limmer as a potential loop-hole in Alnylam’s IP strategy by employing double-stranded RNAs (dsRNAs) longer than Tuschl’s 19-23 base-pair siRNAs and/or making them blunt-ended to emphasize an apparent difference to the classical Tuschl siRNA that features 3’ overhangs. These patent workaround efforts seemed to bear first fruits last year when the original Kreutzer-Limmer I patent series was restricted by the European Patent Office to covering siRNAs between 15 and 21 base-pairs in length (opposing parties: Sirna [now Merck], AstraZeneca PLC, Atugen [now Silence Therapeutics], Janssen Pharmaceutica N.V., and Sanofi-Aventis).

From a partnering perspective, this seemingly small development could have important implications for striking the next major deal, since which company would feel comfortable paying hundreds of millions of dollars for a technology license that appears to be circumventable.

Kreutzer-Limmer was Alnylam’s first line of defense against such blunt-end siRNAs and siRNA precursors longer than 23 base-pairs (aka Dicer substrates) given that, depending on the explicitly granted range of double-stranded RNA lengths, Kreutzer-Limmer would directly cover such structures. Its short-coming, however, is that in 1999, Kreutzer and Limmer did not understand well how these dsRNAs exactly caused gene silencing, which is what Tuschl II is famous for. While I consider Tuschl II, in addition to the ubiquitous Fire-Mello patent, as the fundamental patent series for therapeutic RNAi, due to its excruciatingly detailed explanation of what it takes to effect efficient RNAi in mammalian cells, it is the early priority date of Kreutzer-Limmer’s invention that makes this patent so potentially valuable and dangerous, and explains why Alnylam saw it necessary to remove any uncertainty and obtain exclusive access to it by acquiring Ribopharma AG in 2003.

I found it curious that a number of companies have chosen to take licenses to Kreutzer-Limmer, but not Tuschl II. While that may be interpreted as reflecting the fundamental importance of Kreutzer-Limmer, it was as if by pursuing this strategy, it is almost made implicit that as soon as the scientifically less detailed Kreutzer-Limmer series were curtailed in scope due to heavy opposition, the field for newly patentable RNAi inducers would be wide open. In this case, Alnylam would probably have argued in a second line of defense that, although not spelt out letter by letter, Tuschl II would also cover Dicer-substrate and other RNAi inducers that obviously function either as siRNA precursors (= pro-drugs) or are derived from it, for example 3-stranded siRNAs (meroduplexes). This argument becomes particularly relevant in the case of a weakened Kreutzer-Limmer as this ironically would directly strengthen Tuschl II. In this way, Alnylam holds all the cards and may play them as they wish.

Silence Therapeutics, in particular, will not be very happy with the outcome in Germany, not only because it and others, myself included (to be explained in my next posting), sees itself as a major force in RNAi in Europe, but also since their blunt-end, modified dsRNA is not only the size of the classical Tuschl siRNA, but with Kreutzer-Limmer any gene silencing dsRNA, modified or unmodified, is covered. Silence Therapeutics’ approach could be likened to first taking an invention (here: Tuschl’s siRNAs), then impair its function (here: by flushing the ends blunt), and finally rescue some of the original function by adding further changes (here: by introducing a pattern of RNA modifications). Certainly original, in its own complicated way.

I should disclose here that I largely agree with Alnylam’s view of their IP position and have invested in this company, but at this time I particularly felt like speaking out on all these confusing claims about proprietary RNAi compositions that threatened to hurt investments in RNAi Therapeutics. The acquisition of Sirna Therapeutics by Merck was certainly triggered in part by Sirna’s IP claims which now appear to be weaker than originally hoped for by the buyer and has escalated into a costly and time-consuming mess for a number of companies. In the same vein, I should also emphasize that I am likewise invested in companies that I have strongly criticized in this and other contexts and that I am therefore not wed to any company’s view of the space. It is in this spirit that I hope that Alnylam does not use their IP position to block the evaluation of RNAi inducers that differ from the classical siRNA design in more than just a modification here or an overhang there. Financial incentives should therefore be created for investments in such start-ups without requiring a $1 billion upfront license fee.

PS: In my next posting, barring further developments, I would like to provide the promised company-by-company overview.

Two additional recent developments that I would like to briefly comment on:

1) The FDA removed the clinical hold on Targeted Genetics’ rheumatoid arthritis AAV gene therapy that had been suspected to have played a role in the unfortunate death of a clinical trial participant. I am relieved by this judgment since there was just no good scientific evidence that the gene therapy caused or was associated with the fatality. AAV vectors are currently probably the most potent method to deliver RNAi in vivo and there are a number of indications where AAV-RNAi may be years ahead of synthetic siRNA strategies, and where the benefits outweigh the real risks of gene therapies. One such indication would be AAV-RNAi for treating Huntington’s Disease, where published and orally presented data so far suggests superiority of the AAV approach compared to siRNAs and that Targeted Genetics should now be in a better position to pursue in collaboration with Sirna Therapeutics/Merck and Bev Davidson’s group in Iowa.

2) At a recent symposium on RNAi and its targeting in Sonoma, California, Ian MacLachlan from Protiva presented more data on the efficacy of SNALP-siRNA delivery in non-human primates. According to the abstract, more than 90% gene silencing of ApoB, with silencing lasting for several weeks, could be achieved by single-dose intravenous administration. These are impressive numbers and the task is now to minimize the toxicities associated with cationic liposomes. I am quite impressed by Protiva’s past work not only on RNAi delivery (in collaboration with Sirna Therapeutics and Alnylam), but also on dissecting the causes for the toxicity, and would expect them to be the first to find a solution for this problem. Unfortunately, the ownership and know-how of SNALP delivery technology is highly contested and I can only urge the involved parties to consider working together on this promising technology. During a recent conference call by Tekmira it was apparent that a lack of suitable scientists caused delays in the development of SNALP technology. I would even venture as far and propose that Alnylam’s delays on their systemic delivery programs have probably cost the company more in terms of reagent, labor, time and market cap than the combined market cap of Tekmira and Protiva.

Friday, November 23, 2007

The Confusing World of AtuRNAi, Stealth siRNAs and mdRNAs (Part II)

(RNAi IP discussion continued from previous entry)

If long dsRNA had worked exactly in humans as it did in the worm and plants, then you would have expected an immediate flood of publications reporting the same. Long dsRNA for gene silencing, however, were impractical for most vertebrate cell applications due to the induction of non-specific cytokine responses that essentially shuts down most gene expression and therefore does not allow for targeted gene silencing. An exception may be embryonal cells which lack an interferon response and for which long dsRNA was reported to induce specific gene silencing first in zebrafish in 1999 (Wargelius et al. Biochem Biophys Res Commun. 263:156) and then in mice (a mammal) in late 2001 by the Filipowicz group (Basel, Switzerland).

These latter findings, however, were overshadowed earlier in 2001 by a publication from the Tuschl group in Germany, representing the culmination of a body of work he first started as a post-doc in the Bartel/Sharp labs during his time at the MIT, and then as an independent investigator at the Max-Planck Institute in Goettingen. While small RNAs were then known to derive from long dsRNAs, their molecular role in guiding the recognition and destruction of target mRNAs was only hypothesized and their structure mostly unknown. A breakthrough towards this understanding came by establishing a biochemical system in Drosophila (fly) lysates that recapitulated RNAi in the test tube (1999, MIT). One year later, they reported that during this reaction both the long dsRNA as well as the target mRNA is cut at 21-23 nucleotide intervals (MIT, 2000), thereby providing a link between dsRNA processing and mRNA targeting. In early 2001, then at the MPI, Elashir and colleagues in Tuschl’s lab further delineated the relationship between dsRNA processing and target mRNA cleavage in the Drosophila system, including the observation that the mRNA is cut around 10 nucleotides from the 5’ end of a 29 base-pair dsRNA (kind of Dicer-substrate). Importantly, by sequencing the 21-23 nucleotide RNAs by borrowing a cloning technique developed for the discovery of microRNAs around the same time, they found that the small RNAs were clustered consistent with long dsRNA processing into 21-23 base-pair DUPLEX RNAs. Moreover, the small RNAs were found to contain 5’ monophosphates and 3’ hydroxyl groups all consistent with the notion that long dsRNA was processed by an RNase III enzyme into 21-23 base-pair duplexes (reported to be the Dicer enzyme by Hannon in Cold Spring Harbor in the same month).

This led them to test whether small duplex RNAs were crucial functional intermediates between long dsRNA and mRNA cleavage, by synthesizing duplex RNAs and adding them to the Drosophila system. I quote: “Perhaps the 21-nt RNAs are present in double-stranded form in the endonuclease complex, but only one of the strands can be used for target RNA recognition and cleavage”. Indeed, this prediction turned out to be correct and synthetic duplex RNAs could silence mRNAs in this system, and duplexes with 2-3 nucleotide 3’ overhangs, the hallmark of the hypothesized RNase III-type processing, worked best. These data then formed the basis for the Tuschl I patent series to which Alnylam, RXi, and Sirna Therapeutics obtained co-exclusive licenses. My guess is that Alnylam actually would not mind if this patent wasn’t issued after all, since for some obscure reason UMass, unlike the Whitehead Institute, MIT, and MPI decided to grant RXi and Sirna co-exclusive licenses. Equally curious is the fact that while in the January 2001 paper the duplex RNAs were shown to work only in fly lysate, in the Tuschl I series, out of the blue, human cell studies are described. I could well imagine that the ultimately issued Tuschl I patent will be solely focused on the fly data, so that the first human siRNA description would be exclusive to the Tuschl II series (I would encourage you to read my 27 May, 2007 Blog “2007RNAi Therapeutics IP: The Importance of Being Tuschl” on this issue).

Clearly, work in human cells was ongoing at the time in Tuschl’s lab, and they conclude the fly paper in Genes and Development with the ominous statement: “The siRNAs may be effective in mammalian systems, where long dsRNAs can not be used because they activate the dsRNA-dependent protein kinase (PKR) response (Clemens 1997). As such, the siRNA duplexes may represent a new alternative to antisense or ribozyme therapeutics.” The compositions, methods, and uses of synthetic siRNAs in human cells are described in excruciating detail in the Tuschl II patent series, much of which has issued in the EU and US and is exclusively licensed to Alnylam.

A lot of the claims by other companies such as Silence Therapeutics and Invitrogen’s Stealth siRNAs center around the fact that Tuschl II emphasizes the 3’ overhangs of siRNAs, and that blunt-end siRNAs are therefore not subject to Alnylam’s IP estate, but completely ignore the fact that Tuschl, both in his fly and human work indeed tested blunt-end siRNAs, just that they did not perform as well as the overhang siRNAs. I speculate that the reason why Alnylam has not come out and spelt out this fact is because they may think that their equally exclusively licensed Kreutzer-Limmer patent series (use of short dsRNAs for gene silencing in mammals) provides even better coverage for the use of blunt-end siRNAs. Alnylam’s competitors, including Merck, have therefore focused their efforts of fighting Alnylam’s IP dominance on narrowing the scope of Kreutzer-Limmer, particularly in Europe, and have succeeded in doing so last summer to reduce the covered length to 15-21 nucleotides. However, a so called divisional patent application based on the Kreutzer-Limmer patent was granted in Europe in 2005 and has even broader claims than the original patent (15 to 49 base-pair duplexes). It is further ironical that a weakening of Kreutzer-Limmer would only strengthen Tuschl II’s scope. In addition, Tuschl II further covers modifications and conjugations to siRNAs, a claim which Alnylam has cemented by obtaining an exclusive license to the Crooke modification patent estate from ISIS.

While RXi may have marketed their recent StealthTM siRNA license from Invitrogen for therapeutic purposes, in my mind “StealthTM” siRNAs are nothing more than a marketing gimmick disguising the fact that these are 25 base-pair, blunt-end siRNAs with a supposedly magical pattern of base modifications. I would therefore not be surprised if Stealth failed to fulfill the non-obviousness criteria, in addition to the fact that I have not seen any evidence that Stealth, per se, performs any better, if not worse than the classical Tuschl siRNA design. What RXi probably won’t tell you is that Invitrogen has deemed it necessary to gain access to the Kreutzer-Limmer patents through a licensing agreement with Alnylam for the use of Invitrogen’s siRNAs for research applications only.

With regards to Dicer-substrate, licensed by both Nastech and Dicerna, I see practical value in that Dicer-substrates may be beneficial for RNAi delivery purposes in that they provide increased flexibility in covalently conjugating the Dicer-substrate to the delivery carrier, while siRNAs have to be reversibly conjugated, e.g. via disulfide linkages, to achieve the same. However, this does not guarantee the uniqueness of Dicer-substrate since a lot of the duplex length and the conjugation idea is subject to the pre-dating Kreutzer-Limmer and Tuschl patent series. Moreover, in his fly experiments with 29 base-pair duplexes, Tuschl already demonstrated “RNase III-substrate”. Hannon should also have relevance for Dicer substrate in that he was the first to describe Dicer to be the enzyme that mediates dsRNA processing in flies, and likely humans.

Hannon continued his work on the practical implication of dsRNA processing and was one of the first to explicitly describe the use of Dicer-substrates in humans in the form of DNA-directed hairpin expression cassettes driven by a Pol III promoter. While this 2002 paper in Genes and Development was as much inspired by the newly emerging knowledge on microRNA processing as much as by Tuschl’s 2001 findings, “Tuschl and colleagues first showed that short RNA duplexes, designed to mimic the products of the Dicer enzyme, could trigger RNA interference in vitro in Drosophila embryo extracts”), the European group (Brummelkamp et al.) that published in Science on the same subject the same month were mostly inspired by Tuschl: “We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells.” In any case, both Alnylam and RXi have gained access to the Hannon patents which touch on both DNA-directed RNAi and Dicer-substrate (synthetic or DNA-directed).

In a confusing turn of events, however, Hannon reported in 2005 that hairpins, in this case synthetic versions though, with longer duplex regions often worked better (= more potent and reliably) than the classical 19 base-pair hairpin design. This could be explained by the observation that the efficiency of RNAi should be enhanced by requiring a Dicer processing step since this is coupled to the RiSC-mediated gene silencing step. The fact that the original 19 base-pair hairpins, first thought to be processed by Dicer, were found to be inferior could be explained by their inefficient processing into siRNAs by some RNases not normally related to RNAi. Essentially the same conclusion was reached by a paper in the same issue of Nature Biotechnology from John Rossi’s group at the City of Hope, this time, however, by employing synthetic 25-30 base-pair duplexes (licensed to Nastech and Dicerna) instead of synthetic hairpins. For a discussion of Dicer-substrate science and IP, please refer to my October 31, 2007 Blog: “A new player in RNAi Therapeutics: Dicerna Pharmaceuticals”.

Besides RNA polymerase III-driven small hairpins, DNA-directed RNAi can also be initiated through more microRNA-like constructs. This was enabled by the elucidation of the microRNA silencing pathway and the trick basically is to design DNA vector constructs that will mimic one of the RNA intermediates during microRNA processing. These methods have the advantage that RNA polymerase II promoters can be employed with potential tissue-specific or other regulation. This should allow for potentially safer DNA-directed RNAi, although RNA polymerase III constructs have extreme knockdown potencies. Brian Cullen’s (Duke) and particularly Narry Kim’s (Seoul, Korea) groups have spear-headed these efforts, but I have not heard from companies yet specializing on the use of such RNAi constructs for therapeutic purposes. It is further likely that the original DNA-directed RNAi patents will be quite important for the commercialization of these later methods.

In addition to employing RNAi triggers that funnel into the RNAi pathway upstream of siRNAs, it is also theoretically possible to make use of at least two more intermediates functioning downstream of siRNA generation: single-stranded guide RNA that recognizes target mRNA within RiSC and a 3-stranded intermediate in which the passenger (=non-targeting) strand is interrupted based on findings from a number of groups, again almost simultaneously about 2 years ago, that the passenger strand was cut prior to guide RNA RiSC loading, analogously to how target mRNAs are cleaved.

The single-strand siRNA method is mostly investigated by ISIS for commercial purposes, probably because it feels that their IP position on single-stranded antisense RNAs would make them the dominant player in single-stranded RNAi. It should be kept in mind, however, that it was again the Tuschl group, known to be close to Alnylam, that first reported on single-stranded RNAi inducers (Martinez et al., 2002) and patents have been filed. Moreover, evidence so far suggests that single-stranded RNAs are only very inefficiently recognized by the endogenous RNAi machinery and it is doubtful that any potential advantages of single-stranded RNAs versus duplex RNAs would ever make up for the inferior potency. Patents covering 3-stranded siRNAs have been filed for by Nastech, although they have not been associated with any of the initial reports on siRNA passenger strand cleavage. It will therefore be interesting to determine the priority dates of the various discoveries, and probably more importantly, data as to the efficiency of these “meroduplex RNAs” (same initials as Nastech’s planned RNAi spin-out mdRNA) compared to other RNAi inducers. Such 3-stranded siRNAs may offer certain advantages with regard to conjugation chemistries and the fact that short RNA strands are cheaper to synthesize than larger ones, but until this is proven it will look just like another thinly disguised patent work-around attempt.

In summary, it is clear that it was Fire and Mello’s discovery on long dsRNAs as RNAi inducers in worms and Tuschl’s extensive body of work leading to the delineation of the classical duplex siRNA that opened up RNAi for therapeutic use. Many of the other developments are directly derived from both of these fundamental discoveries and require appropriate IP licenses. The combination of Tuschl II, Kreutzer-Limmer, and all the other patents it has either exclusive or non-exclusive access to, makes Alnylam the gate-keeper of RNAi Therapeutics. The exact terms of companies wishing to commercialize RNAi Therapeutics will vary depending on their co- or non-exclusive access to some of these fundamental patents, how far removed their exact siRNA derivatives are from the classical siRNA design as well as the ability to prove their utility. While this dominant IP position by Alnylam may make them unpopular and almost look like a bully, one should not forget that concentration and clarity of IP encourages investments particularly in the risky business of drug development and therefore will increase the likelihood of maximizing the therapeutic potential of the technology. It should also be said that Alnylam has been pretty good in de-risking RNAi technology, thereby benefitting the whole field of RNAi Therapeutics, in addition to granting access to RNAi technology through their licensing policy, although the terms will increase the longer you wait. Outside this core RNAi IP, other IP, particularly relating to delivery, but also access to validated targets will prove valuable, albeit much more fragmented.

In my last blog on this RNAi IP series, I would like to briefly discuss individual companies according to technology strength and IP position. Alnylam’s view on this issue will be presented in a special IP-focussed investor presentation on November 28 at the 19th Annual Piper Jaffray Health Care Conference and can be followed live or recorded by webcast on the company’s website.

Erratum: Please note that in my discussion of Dicer-substrate in the October 31, 2007 Blog: “A new player in RNAi Therapeutics: Dicerna Pharmaceuticals”, I mistakenly stated that Hannon’s long hairpin RNAs were DNA-directed, when they actually studied synthetic versions of these hairpins.

Monday, November 19, 2007

The Confusing World of AtuRNAi, Stealth siRNAs and mdRNAs (Part I)

“Next-generation” RNAi is all the rage in the world of the ambitious RNAi start-up. The definition (note that everything is allowed under the guise of the satire): “An RNAi-inducing molecule derived from the classical Tuschl siRNA design, however with a magical pattern of modifications and variations in the exact length of the RNA duplex, sometimes an NA duplex, with overhang or not. As important as the chemistry that may sound impressively inventive to the lay (investing) public is that a proper name is chosen to further accentuate its apparent uniqueness. This is intended to suggest freedom-to-operate with the ultimate aim of attracting investments from people hoping the company will eventually catch up to the market cap of Alnylam (why would you invest in any of their direct rivals otherwise?).”

Considering that it has become commonplace to hear CEOs talk about their RNAi being so unique and advanced that they are now operating in parallel universes, the staid Tuschl siRNA must have really lost its relevance for the development of RNAi Therapeutics. While I think that some select siRNA derivatives given names such as StealthTM RNAi or Dicer-substrate definitely warrant further investigation, as it is yet unclear how well they will perform relative to the simple, but fundamental siRNA design, what I would like to do is to cut through the marketing fog and provide a brief overview of the types of RNAi inducers currently being used at the bench or in the clinic and how I think they relate to each other in terms of IP. In this post I will lay the foundation by giving a summary account of the history of RNAi as a tool, including some of the fundamental patents (and applications), before dissecting some of the Next-generation siRNA designs in a follow-up posting.

Studies on RNAi-related gene silencing really started in the early 90’s in plants with the observation of co-suppression whereby genes that share sequence similarity inhibited each others’ expression. Usually, this was triggered by the inappropriate processing of one of the gene products, typically from an introduced designer gene that is recognized as aberrant and therefore as a threat by the plant RNAi surveillance system. While the mechanism by which this occurs is a scientifically very interesting question, it cannot be used for gene silencing in humans and therefore has little or no relevance to RNAi Therapeutics IP. Parallel work on gene silencing in worms by Fire and Mello, of course, discovered that it was long double-stranded RNA (dsRNA) that was central to inducing RNAi and patents were filed covering dsRNAs longer than 25 base-pairs for gene silencing. This patent can be licensed non-exclusively by almost anybody that wants it, and despite it being based on work in worms and the long dsRNA nature in the stated claims, it is nevertheless considered to be a license that you should add to your IP portfolio anyway, I guess just because it has proven so fundamental to the understanding of RNAi in general and nobody would want to argue that. I also think this highlights the fact that real fundamental scientific insight will be credited by the patent courts even if the exact length of the duplex or modification pattern was not spelt out in the claims letter by letter.

Shortly after Fire and Mello published their research, Kreutzer and Limmer from the University of Bayreuth in Germany reasoned that short dsRNAs may have similar gene silencing effects in mammalian cells. This prediction, as we know, turned out to be true and now forms the basis of the Kreutzer-Limmer patents claiming short dsRNA of around 15-49 base-pairs for the induction of gene silencing in mammalian cells, although the exact length is the subject of patent challenges, including Merck’s opposition in Europe. This early work was considered important enough by Alnylam for them to acquire Ribopharma AG, the company founded on the Kreutzer-Limmer patents. Although I consider Tuschl’s subsequent work to be quite a bit more fundamental to the use of RNAi in mammals, Alnylam understood that it was important to remove any uncertainty as to the dominance of their RNAi IP position given the relative timing and overlapping content of Kreutzer-Limmer and Tuschl.

Around the same time, Hamilton and Baulcombe discovered that small RNAs were generated during plant RNAi. While they were prescient in predicting that these may mediate RNAi, they did not formally prove it and the structure of the siRNA that was detected in those experiments remained unknown. A world away, in Australia, DNA-directed hairpin vectors for reliably inducing RNAi were being described by Waterhouse and colleagues from the CSIRO. Based on the utility and impact of these vectors on plant research, the patents derived from these studies should give the CSIRO a strong position in the agricultural uses of RNAi. In many ways, the commercial development of plant RNAi is more progressed than therapeutic RNAi as traits can now be altered relatively quickly without having to resort to lengthy breeding and selection. I guess the most important question will be how uniform these knockdown phenotypes will be across a field of crops. The CSIRO patents also form partly the basis for Benitec’s claims to the therapeutic uses of DNA-directed hairpin RNAs. The Graham patents form the other pillar of Benitec’s contested patent estate describing the use of DNA cassettes driving the expression of various forms of dsRNAs, although I find these patents to be quite theoretical in nature and wonder whether most of the described non-Pol III expression cassettes would actually work for gene silencing in most mammalian cell types (to be continued…).

Two noteworthy developments last week that I would briefly like to comment on:

1) ISIS released further phase II data for their ApoB-targeting antisense compound mipomersen. The 200mg/week dose reduced by about half the level of bad cholesterol in patients already on stable statin therapy. This looks quite impressive and if no safety issues come up in the larger phase III trials, then this has the potential to become a blockbuster. I’ve been quite critical about mipomersen in the past, particularly due concerns about fatty liver which many scientists in the field would have expected to observe following ApoB knockdown. Safety data for the 200mg dose, based on liver enzyme measurements, however do not indicate this to be a problem. Ultimately, the proof is in the pudding and I would be happy to ultimately have to admit to have been wrong on this issue. ISIS explains the absence of fatty liver due to transcriptional compensatory changes in fat metabolism. Overall, these data augur well for the development of all RNA-targeting platform technologies, including RNAi Therapeutics, as it suggests that minor off-targeting should be well tolerated in many cases.

2) Pfizer announced the acquisition of Coley Pharmaceuticals for almost triple of Coley’s market cap before the offer. Coley Pharmaceuticals is an oligonucleotide therapeutics company that exploits the immunostimulatory properties of oligonucleotides for applications such as boosting vaccines or in the fight against cancer. Actually, I’ve been quite impressed by their OTS presentation in Berlin, particularly their vaccine program. This comes only days after a blog this month where I asked the question when Pfizer will make its big move in RNAi Therapeutics (11 Nov 07 Blog: “When Will Pfizer Finally Make its Big Move in RNAi Therapeutics?”). It is notable that Coley is a Massachussetts company and I would like to think that the proximity to Alnylam will not be an impediment to Pfizer’s new biotech initiative that also appears to more and more focus on oligonucleotide therapeutics. With the new oligo expertise in-house (note that Alnylam does not have another subsidiary to throw into the next deal) and plans to add more staff to a research facility in Cambridge (the headquarters of Alnylam) in addition to a possible biotech incubator near Boston, the plot thickens.

Wednesday, November 14, 2007

Will “Target Protector” Technology Come In Play?

Last month, the Schier lab from Harvard extended their impressive work on the function of microRNAs in the zebrafish model of development (Choi et al.: “Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430”. Science 318: 271).

Antisense oligos to microRNAs are one method to experimentally infer microRNA function through loss-of-function analysis. However, this method becomes problematic when the intention is to study the biology of a particular microRNA-target interaction given the multitude of predicted targets for a given microRNA, typically estimated to be around a hundred per microRNA.

In order to overcome this problem, the investigators designed morpholino antisense oligos (morpholinos are quite popular with the fish community), which they termed “Target Protectors”. These are supposed to hybridise to a defined microRNA target site thereby blocking the function of the microRNA acting on that particular target site. This actually turned out to work quite well in the zebrafish and future publications should demonstrate the versatility of this new methodology.

The reason why I suddenly blog about it is because I had the pleasure today to listen to a presentation by Dr. Alex Schier in which it became apparent to me that there may be investor interest in commercializing the technology. This would complement the microRNA antagonist (“antagomirs”) approach currently pursued by the likes of Regulus, Rosetta Genomics, and Santaris, that aim at therapeutically blocking the microRNA. Given the many targets of a given microRNA, blocking them for therapy is therefore based on the belief that evolutionary pressure should have ensured that their targets are tied to a common biologic phenotype. Target Protectors, on the other hand, may be more specific in upregulating only one transcript that when de-repressed should be therapeutically beneficial. As such, one could imagine targeting the miR-122 target site of HCV, thereby avoiding potential side-effects due to inhibiting the most abundant microRNA in the liver.

It will be interesting to see who will take an interest in this emerging IP estate. It is also notable in this context that Rosetta Genomics, the microRNA target company, has previously mentioned their foresight to patent not only their discovered microRNAs, but also predicted microRNA target sites.

Update (23 November 2007): It has come to my attention that Target Protector has come into play indeed:

Tuesday, November 13, 2007

Nastech’s RNAi Ambitions Hit by P&G Break-Up

Since Nastech announced that Proctor & Gamble would drop their collaboration on a intranasal spray of parathyroid hormone (IN-PTH) for the treatment of osteoporosis last Wednesday, the stock has been falling ever since and lost about 2/3 of its value in less than a week. This is not surprising as it shatters investor confidence in the nasal peptide delivery technology that the company was founded on one and a half year after Merck cancelled a similar agreement with Nastech for the intranasal delivery of an anti-obesity peptide.

Nastech’s experience highlights the risk of investing in biotech companies that are centered on a single, as yet unproven technology. It is therefore worth keeping in mind that RNAi Therapeutics is only one clinical trial or adverse event away from being shaken by similar woes.

With the benefit of hindsight, Nastech always wanted to be everything to everybody and doomed to fail. It does not take a degree in Economics to see that too many clinical programs, including some based on not very well validated peptides, and Blue-Sky Science Projects (like projecting that it would take another 10-15 years to develop an RNAi Therapeutics) were a recipe for financial disaster. While it is good to take pride in your science, the odds are stacked against you in trying to develop technologies all on your own, even in RNAi, an area where so much of the innovation will come out of academic laboratories and you may be better off licensing those while focusing your resources on drug development.

No matter how impressive you think it may sound that your RNAi (Dicer substrate) is so potent that it works at homeopathic doses, that you have found the Holy Grail to off-targeting (Ribo-T), and “solved” the delivery problem (peptide-conjugation), to those in the Art it sounds too good to be true, particularly when data in investor presentations lack critical controls and in the absence of appropriate peer-reviewed publications to support these claims. The burden is now, as they aim to spin out their RNAi unit (mdRNA) for money and visibility, on Nastech to prove to the investing public that there really is some value hidden in their RNAi. Otherwise, it will sound more like yet another pipe dream rather than reality of a company that would like to think that it alone can achieve what the rest of the scientific world is struggling with.

Nastech employees may represent a further RNAi-related value not reflected in the current ~$120M market cap (with ~$58M in cash). After Sirna Therapeutics was bought by Merck, there was an exodus of experienced oligonucleotide scientists that ended up working for Nastech in Bothell, WA. Although I doubt that Nastech’s RNAi IP will be valued very highly at this juncture, their know-how acquired in the process may be viewed as an asset by a larger company looking to jump-start their own RNAi Therapeutics work, similar to the acquisition of Alnylam’s Kulmbach, Germany, operations by Roche in July. Of course, employees may prove to be a fickle asset at a time when money is tight and Alnylam is relocating their European operations back to Cambridge, Mass. The coming days and weeks will be critical for the future of Nastech and their RNAi ambitions.

Sunday, November 11, 2007

When Will Pfizer Finally Make its Big Move in RNAi Therapeutics?

This week, Pfizer crossed my RNAi Therapeutics radar twice again. One has to do with their much-anticipated biotechnology initiative, including an innovation center in the San Francisco Bay Area which, according to news reports, should have RNAi Therapeutics as one of its focus areas. As we all know, Big Pharma has come under pressure with upcoming patent expirations, declining drug approvals due to a conservative FDA and outdated pipelines, but actually has the cash to invest in much needed innovation. The other event was a recurring rumor about their interest in Silence Therapeutics which led to a rapid 25% increase in the shares of the UK/German RNAi Therapeutics outfit in a matter of only two trading days.

Just watching Pfizer’s actions in RNAi Therapeutics from a distance is quite curious. Initially, they appeared to be toying around with DNA-directed shRNAs, but then engaged in a triangular relationship for developing an RNAi Therapeutics for AMD with Quark and Silence based on Silence’ AtuRNAi-type of siRNAs and Quark’s identified target gene. Pfizer also appears to be scouting out RNAi delivery solutions with a deal earlier this year with Mirus Bio that was followed by a neat publication on hepatocyte-specific RNAi delivery in PNAS (see 24 July 2007 Blog: ”Mirus Scientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes”). Around the same time, I counted at least seven delegates from Pfizer at thie year’s leading RNAi Keystone conference who, I suspect, weren’t there just to satisfy their scientific curiosity.

It seems prudent for Pfizer to learn more about the potential for RNAi Therapeutics first-hand through smaller collaborations with groups that have demonstrated know-how in RNAi Therapeutics before committing more significant resources. On the other hand, if they really saw potential in the technology and given their balance sheet, it would appear that the longer they wait the costlier the licenses that Novartis, Roche, and others regard as a must for freedom-to-operate. Particularly with the recent Alnylam-Roche deal, the pressure among Big Pharma is only going to increase. With Merck’s chair now empty at Alnylam’s table, Silence up for sale, and the their biotech initiative taking shape, I wonder whether Pfizer is going to get serious and make their move soon.

Sunday, November 4, 2007

The Risk of Rushing RNAi Therapeutics into the Clinic

RNAi has always caught on very fast. It took only nine years for the Nobel Committee to recognize the importance of RNAi in medical biology and award The Prize for its discovery, and only 6 years following the discovery that siRNAs induce RNAi in mammalian cells by Tuschl to become an indispensable tool in the basic and applied studies of human gene function. Equally astounding is the fact that there are now close to 10 RNAi-based therapeutics that have entered the clinic since.

I have extensively described here before why I think RNAi has the potential to be the next great drug development engine, including the prospect of faster development timelines due to straight-forward mechanism of action and platform reproducibility. However, in the wake of Alnylam’s Q3 conference call announcing an insignificant delay in their RSV program, but a more open-ended delay in their liver programs, what I would like to do today is to point out the dangers of rushing RNAi Therapeutics into the clinic mainly borne out of the tension that exists between applying the best and safest science and satisfying investor demand for gushing clinical pipelines.

From the clinical perspective the ultimate danger is obvious: putting trial participants at risk, and disappointing patients’ expectations for a cure of their disease. From the perspective of running an early-stage biotech business that needs to raise money fairly regularly, the issues easily become more complicated. Although I admire the honesty and scientific intent that underlie statements like that by Nastech that one should not expect RNAi Therapeutics from your company until another 15 years, it certainly won’t capture the imagination of Wall Street. The easy way would be therefore to set your bar a little bit lower and signal to your potential investors that you deserve more money since you’ve been able to put so many drugs into the clinic in such a short period of time. A sophisticated biotech investor would know that these companies can be a good investment, although you do not necessarily want to stick it out until the Day of Reckoning comes.

The danger to the field of RNAi Therapeutics is therefore that as some of these rushed candidates come to a stage where they have to prove their safety and efficacy in large-scale clinical trials, a good number of them will fail, essentially because some of the Best Practices were not followed, including addressing cytokine induction issues, off-targeting profiles, RNAi delivery, and pre-clinical safety and efficacy studies that ideally include non-human primates.

Acuity Pharmaceuticals (now part of Opko Health) dazzled everybody when they came out of nowhere and can now claim to have been the first to put an RNAi candidate (for wet AMD) into the clinic. Unless they have changed the composition of their drug since study initiation, Cand5 appears to be an unmodified siRNA injected straight into the eye. This alone makes me wonder whether an optimized compound has been put into the clinic, and I have more confidence in a program run by Allergan and Sirna Therapeutics (Merck) targeting the same pathway for wet AMD, but with a modified siRNA formulation intended for slow release.

SiRNAs that induce cytokine responses may also have a number of additional biological properties, some of them even potentially beneficial for the disease at hand. Gunther Hartmann from Bonn, a scientist with a cytokine angle on oligonucleotide therapeutics, has even proposed at the recent OTS meeting to purposefully combine the immunostimulatory potential of RNAs (isRNA) with siRNA design. Cancer and infectious disease may be good areas to test this concept as isRNAs are thought to help the immune system in fighting related these diseases.

There has been similar discussion whether there would indeed be any harm if an RNAi therapeutic targeting the Hepatitis C Virus (HCV) had some concomitant interferon response. Isn’t interferon (and RNAi) nature’s first answer to viral infections and the mainstay of current HCV treatment regimens anyway? Similar arguments may also apply to RSV.

The fact that Alnylam is now focusing RSV-01 on adult populations makes me therefore wonder whether this was driven at least in part due to concern that the tender infant respiratory system may be more prone to overreact to a potentially immunogenic siRNA molecule than a lung hardened by years of air pollution. This siRNA is probably unmodified as it was this June that the first Alnylam compounds using ISIS modification patents moved into IND-enabling studies. Being unmodified from a pharmacokinetic perspective may not be that bad or even desirable in RSV, as RSV is an acute infection and long drug exposure may therefore have the potential to do more harm than good.

I should emphasise, however, that the early rodent RSV studies that form the basis of Alnylam’s RSV-01 and which have supposedly been replicated by the company, demonstrated sequence-specific antiviral activities. Furthermore, from Alnylam’s presentations one can assume that RSV-01 was carefully screened for cytokine induction in a number of human cell lines and animal models. I should add as well that the slight delay of the RSV experimental infection model studies is not the result of any of these considerations, but more simply reflects the fact that finding volunteers to be infected with a virus that gives you flu-like symptoms and requires you to be locked away from the outside world for a couple of weeks, is not that easy. However, 74 of the 88 subjects, I suppose mostly students, have already been recruited and we should hear top-line data early next year.

Alnylam’s conservative approach to drug development is further demonstrated by their delay of filing INDs for their liver programs, for hypercholesterolemia and liver cancer. While there is no doubt that with current systemic delivery capabilities it is possible to achieve potent gene knockdown in the liver, the safety and dose-response data so far would explain Alnylam’s caution into committing to a particular formulation by year-end as originally guided. Instead, I agree with their assessment that with new chemistries coming online, such as MIT’s lipidoids which formed the basis of the recent microRNA saturation data in Nature, it is wise to keep testing all of to find the formulations that offer the best therapeutic index. It would not be the first time that a drug for treating heart disease would fail in a large-scale trial because of unacceptable side-effects seen in a handful of participants. For what it’s worth and mindful of the business considerations about demonstrating human proof-of-concept of an RNAi Therapeutics with the hypercholesterolemia program, I wonder whether Alnylam should not go first with liver cancer anyway.

Needless to say, this cautious, data-driven approach not only benefits Alnylam the Science, but also Alnylam the Business. The importance of their scientific credibility through publications and conference presentations cannot be underestimated when it comes to their ability to execute on their business development goals, mainly in the form of lucrative license deals and access to enabling technologies. With a cash position of $468M, Alnylam is in a stronger position than ever to focus on the long-term success of the company and its shareholders.

Rosetta Genomics on Track to Bring the First Clinical RNAi-related Product to Market

Almost unnoticed in the microRNA diagnostics space, Rosetta Genomics reported this week that it had completed the pre-validation phase for its first microRNA diagnostic product scheduled to come into the clinic in the first half of next year. It would be exciting to see the first RNAi-related product have a direct clinical impact and, if successful, will fund Rosetta’s microRNA diagnostics and therapeutics programs with minimal shareholder dilution. The microRNA diagnostic is designed to differentiate between squamous and non-squamous lung cancer which is not always possible to tell under the microscope and an area of particular importance now that Genentech’s VEGF-targeting MAb Avastin showed life-threatening side-effects particularly in subjects with squamous cell cancer.

While RNAi Therapeutics has attracted most of the RNAi attention, microRNA-based diagnostics are set to become the first commercial success of RNAi-related products in the clinic. Their differential expression, scalability, and, equally important, potential relative stability advantages compared to protein and larger mRNA biomarkers means that microRNAs have the potential to become the biomarker platform of choice. The (near) future should tell.

Wednesday, October 31, 2007

A new player in RNAi Therapeutics: Dicerna Pharmaceuticals

IN VIVO Blog ( reported today that a new RNAi Therapeutics company, Dicerna Pharmaceuticals, is about to debut. According to the same source, a $13M Series A financing round is expected to be announced in November. This company is aptly named after an enzyme in the RNAi pathway, as it is founded on the slightly unorthodox way to induce RNAi by providing synthetic Dicer-substrate siRNAs (D-siRNAs) of 26-30bp in length to effect gene silencing.

Dicer is this cool enzyme that digests ('dicerna' in Malay means: 'digested') long double-stranded RNAs into the shorter 21-23bp siRNAs with 3’ overhangs, the structure discovered by Tuschl and colleagues to efficiently induce gene silencing by RNAi in mammalian cells. Once delivered inside the cells, D-siRNAs are then processed by Dicer into 21-23bp effector siRNAs which then are incorporated into the RiSC complex to mediate gene silencing. Tuschl-like 21-23bp siRNAs are currently by far the most widely used method of inducing RNAi in human cells and fairly well understood.

In an elegant Nature Biotech paper in 2005 (Kim et al.: “Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy”), Drs. Kim and Rossi from the City of Hope, in collaboration with Mark Behlke from the nucleic acids synthesis company IDT, found that D-siRNAs can effect remarkably potent RNAi in human cell culture, often more potent than siRNAs of the same sequence. Importantly, in this and follow-up work they worked out some of the basic rules that would make D-siRNAs more practical inducers of RNAi such as better predicting strand incorporation and blocking one end of the dsRNA with non-RNA residues and modifications to force directional Dicer processing.

This Nature Biotech paper was accompanied by a similar paper from the Hannon group in Cold Spring Harbor which found that DNA-directed small hairpin RNAs (shRNAs) with double-stranded RNA stems longer than minimal 19-21base pairs similarly make them often more potent inducers of RNAi. Like Rossi and colleagues, it was speculated that this is due to biochemical coupling of Dicer processing to the RiSC effector complex. In addition to certain advantages in terms of potency, which I feel need further validation on a larger scale, D-siRNAs may in some instances facilitate RNAi delivery where covalent linkage of parts of the RNAi delivery system with D-siRNA is helpful as the active siRNA would be freed from the carrier by Dicer cleavage, although again it remains to be shown that the covalent attachment of e.g. peptides by itself is not inhibitory to Dicer processing.

[Erratum: The 2005 Hannon paper described the use of synthetic, not DNA-directed hairpins, with extended duplex length.]

In addition to these potential biological advantages, certainly a big part of the motivation that went into founding the company from an investors’ perspective is that Dicerna should be sufficiently distinct from the Tuschl siRNAs, a space clearly dominated both in terms of IP and know-how by Alnylam Pharmaceuticals. One can therefore expect that the new chairman and co-founder Douglas Fambrough from Oxford Bioscience Partners will do his best to make Dicerna his second Sirna Therapeutics, which he and his partners sold to Merck last year for a whopping return on their investment.

However, like with Sirna Therapeutics his claims of having freedom-to-operate will likely be clouded by uncertainty as there are a number of areas where Alnylam’s pre-dating IP will significantly overlap with Dicerna’s claims. This is not helped by comments, also cited in the IN VIVO Blog, of new CEO James Jenson stating that Tuschl’s landmark work had been conducted in flies, when Tuschl II –which by the way has issued and is exclusively licensed to Alnylam- is all about RNAi in mammalian cells, all this after laying the groundwork in work described in Tuschl I (also claimed by Sirna and CytRx, but has not issued) through amazing biochemical work in flies: Tuschl the prolific!

Importantly, Tuschl’s work as described in Tuschl II essentially discovered that RNAi operates in mammals and defined the basic rules of synthetic siRNAs. This, in my mind, should go a long way in the patent courts. In its worst case, D-siRNAs could therefore be regarded as simple pro-drugs of siRNAs. This also includes the 3’end overhangs which are thought to be beneficial for D-siRNAs since they are an important recognition element for Dicer.

Kreutzer-Limmer is another important cornerstone of Alnylam’s IP strategy, indeed important enough for them to buy the company (Ribopharma AG) that owned it very early on. Kreutzer-Limmer pertains to dsRNA-mediated gene silencing in mammalian cells, including predicted Dicer substrates, and although less well known in the scientific community due to lack of scientific publication, it is actually thought to have been the first demonstration of such gene silencing. Scientifically, my heart is with Tuschl’s detailed work, but Alnylam played it safe by just removing the uncertainty.

In addition, I would not be surprised if there wasn’t a note-book entry or publication that made use of long siRNAs either by design or accident. This would not be unlike early in the shRNA arena where scientists have made use of shRNAs with minimal and relatively long dsRNA stems alike.

Practically, the relatively small field of D-siRNAs will have to achieve what thousands of researchers around the world have done for siRNAs, namely coming up with siRNA design rules that consider all of potency, off-targeting potential, and the induction cytokines, as Rossi’s work has shown that these rules may differ from that of siRNAs. For these and other reason, I expect the complexity of developing D-siRNA therapeutics to be probably increased.

Nevertheless, I am curious to see more data come out that carefully characterize and compare the potencies of siRNAs and D-siRNAs. Comparative gene tiling studies would be an obvious experiment. This reminds me of the finding of hyperfunctional siRNAs, i.e. the odd siRNA that will be active in the low to mid picomolar range, and I could imagine a situation where efforts to find such siRNAs prove difficult for certain genes, while a D-siRNA is hyperfunctional, and vice versa.

I certainly look forward to Dicerna as a new member of the RNAi Therapeutics community. The science is certainly sound and innovative, and should be tested for use as a human therapy, which we all know would not happen without patent protection.

PS: This new development makes me wonder where that leaves Nastech Pharmaceuticals which has built so much of their RNAi program on Dicer substrates and is about to spin out mdRNA as their pure play RNAi Therapeutics subsidiary. It is clear that COH granted them 5 exclusive targets, but I am less sure about the other rights to Dicer substrates they had obtained.

Sunday, October 28, 2007

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

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

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

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

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

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

Monday, October 15, 2007

The Race to Knocking Down Cardiovascular Disease

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

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

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

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

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

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

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

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

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

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

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