Thursday, February 25, 2010

GSK Dumps Santaris, Chooses Regulus

Patents still matter. An impressive demonstration late last year by Santaris and collaborators from a primate center in Texas was not enough for GSK to exercise its option for SPC3649, Santaris’ lead microRNA therapeutics candidate directed at miR-122 for the inhibition of hepatitis C virus (HCV) infection (discussed on this blog here, and in a review by Mark Kay and myself here). Instead, Alnylam/ISIS-backed Regulus announced today that GSK has decided to side with them in the development of such an miR-122 antagonist for HCV. This is an amendment to an earlier inflammatory disease collaboration between Regulus and GSK with undisclosed upfront and ~$150M in potential milestones plus the usual tiered royalties on drug sales.

GSK’s decision not to exercise the Santaris option led to speculations, also on this blog, whether it is maybe the concern that miR-122 inhibition might be tumorigenic or could have other adverse effects on the liver. While one certainly ought to pay attention to such potential, the pre-clinical safety data and the expectation that any such therapeutic would not be administered chronically would have made this an unusually conservative decision.

It now appears, however, that GSK just could not ignore the fact any more that it is Regulus that got the exclusive license to the critically important miR-122/HCV Sarnow patents. The move can also be interpreted such that while LNAs are certainly an attractive approach to inhibiting microRNAs, there are alternative chemistries of similar potency. A new type of locked nucleic acid chemistry developed by ISIS that was inspired by and looks a bit like Santaris' LNAs but for a few adornments, that supposedly also make it less toxic, is one such chemistry that I could imagine Regulus-GSK to select for their IND-enabling studies. It is worth remarking that it is probably less the miR122-related Esau patent that was the swaying factor here, since as issued in the US, the Esau patent called for a fully 2’-MOE antisense molecule against miR-122…probably not a gate-keeping chemistry for microRNA antagonists.

It will be exciting to further monitor the race between Santaris (already in the clinic) and Regulus in developing a microRNA-based HCV therapeutic, including how the IP will be sorted out. In this context, I can only appeal to the players not to engage in big patent fights at this time and let the better arguments win, but outside the patent appeals courts and civil cases. To paraphrase related comments by the new CEO of Silence Therapeutics, Phil Haworth, yesterday, this is an utter waste of money and management attention. Time to wake up!

PS: Another milestone in the deepening relationship between GSK and Alnylam, and GSK’s move into innovative, high-value medicines.

Wednesday, February 17, 2010

The End of the Age of Incrementalism

RNAi Therapeutics aims at addressing high unmet medical need at its genetic root. It is not about dressing existing drugs in new clothes/formulations to extend product life-cycles or worrying about once-a-day versus twice-a-day pills. This belief is encapsulated in the mantra ‘The End of the Age of Incrementalism’ that can be heard in the halls of RNAi Therapeutics companies and fits well into an increasingly risk-averse regulatory environment that further emphasizes more and more comparative effectiveness during the drug approval process.

That this belief is also gaining acceptance in Big Pharma was recently highlighted by GSK’s decision to exit pain and depression and instead focus on developing life-altering drugs for orphan diseases where the RNAi Therapeutics platform should have a competitive edge. GSK’s decision was somewhat surprising given that they had a drug under active review with the FDA for restless legs syndrome (RLS) and that with the right marketing could have come close to blockbuster sales. RLS is a disease that has long struggled to gain acceptance within the medical community and has often been ridiculed as a phantom disease. Nevertheless, there has been some commercial success, not least through GSK’s own efforts. With the life-cycle of its original RLS drug coming to an end, it struck a partnership with Xenoport for a gabapentin pro-drug, an oral small molecule by the name of Horizant (XN13512), that has met all the pre-specified primary and secondary end-points in all 4 phase III trials. This made the author of this blog believe that it should be a smooth approval process if there ever was one.

Gabapentin has long been used for epilepsy and some forms of pain. Rather than developing gabapentin itself for RLS, GSK and Xenoport instead chose the pro-drug version which was designed to be taken up through a different mechanism in the gut, arguing that the changed pharmacology would make for a superior drug. I suspect, however, that the fact that the pro-drug, unlike gabapentin itself, had a long period of patent protection ahead played no minor role in this choice.

Well, this strategy just came back to bite them (and investors) and GSK must have seen it coming [update 2/18/10: Per Xenoport's conference call, the complete response letter was the first time the rat carcinogenicity were brought to the companies' attention by the FDA]: The FDA rejected the drug because rat carcinogenicity studies showed a drug-related increase in a form of pancreatic cancer. The FDA further stated that although it had known for a long time that gabapentin was associated with such a risk, the risk was justified for a condition like epilepsy, but not RLS. Clearly, the FDA was not impressed by a drug that one might argue had largely been developed for its patent profile and for a condition that is not really considered life-threatening. Looking at the RNAi Therapeutics pipeline, chances are low that the FDA will pull similar rabbits out of its hat (see footnote *) for drugs aiming to treat solid cancer, severe hypercholesterolemia, and graft-associated morbidities and mortalities, and should only make the next Big Pharma-RNAi Therapeutics deal come one step closer.

* The reason for characterizing the FDA’s decision this way is two-fold. Firstly, the FDA has known, and even stated as much in its complete response letter, that gabapentin had been known to be associated with pancreatic cancer in male rats. The question arises why the FDA has not told Xenoport from the outset, in their initial meetings, that it won’t accept a gabapentin-based drug for RLS for this reason. Of course, the companies share some of the responsibility for this lack of foresight costing them and investors years and hundreds of millions $$$. Secondly, the pre-clinical carcinogenicity studies are of questionable value now that the experience of gabapentin in humans has, to my knowledge, not been associated with cancer and increasingly looks to be a sex and species-specific phenomenon.

Thursday, February 11, 2010

For Mipomersen, 10% a Big Difference Does Make

I must admit that I have been very wrong about how clinical results from the ApoB-lowering drug candidates by Tekmira (ApoB-SNALP RNAi, phase I) and ISIS Pharmaceuticals (mipomersen antisense, phase III heterozygous FH) would turn around the sentiment for RNA Therapeutics. In both cases, investors sold off the stocks on results that I believe bring both approaches closer to commercialization. It is almost as if the market is one in which home-run clinical results get rewarded with a doubling in share price, while those in-line with expectations are sold off by about 20%.

Compounding the problem for mipomersen is that investors were secretly hoping for slightly better LDL-cholesterol reductions. This was particularly the case because the modest 25% reduction observed in the previous homozygous FH trial had always been down-played by management as being attributable to a challenging patient population, and that we should expect much more from the subsequent phase III trials. Well, technically, the reduction was slightly improved, but with 28% not too much to get anybody excited.

While analysts were largely focused on potential safety problems, particularly questions about the seriousness of observed liver enzyme elevations, I believe that this attention was very much caused by the disappointment about the degree of knockdown. This is because in order for mipomersen to become a commercially successful drug, it needs to tap the market outside the ultra-orphan homozygous FH population, especially the severe hypercholesterolemic patient population that does not respond sufficiently to current maximal drug therapy and are either on LDL apheresis or eligible for it. Tapping this market or not can make all the difference, the difference between $10M and $500M-$1B in annual sales.

The reason why the LDL apheresis population is so attractive is because mipomersen would aim to replace another procedure that specifically aims at lowering LDL-cholesterol, and demonstrating a comparable knockdown should be sufficient to get approval without time-consuming and costly outcome studies. It could also lead to adoption of mipomersen by some of the other high-risk populations, but this would only be gravy.

LDL apheresis is somewhat unpleasant and, depending on the particular apheresis technique applied, removes good stuff like coagulation factors and HDL cholesterol, too. It does, however, do a fairly good job in removing LDL-cholesterol, by about 30-55%, and in order for mipomersen to be considered an alternative, it needs to get into that range. At the moment, mipomersen is on the lower end of this spectrum where safety could tip the balance, and with lots of questions regarding the safety being left unanswered, this can explain the sell-off we have witnessed.

On a positive note, the 28% LDL-cholesterol reduction may be a very conservative number. This is because it does not take into account the +5% increase in the control group and the fact that the number was on an intent-to-treat basis, i.e. they included also those patients in the drug-treated group that dropped out early, so that the adjusted number could well be in the -36% range. Moreover, the natural person-to-person variation could mean that about half of those that stayed on the drug achieved very meaningful LDL reductions which could prove crucial during the regulatory application process. The roll-over rate into the open-label, uncontrolled phase of the study was also said to be ‘good’ and would be quite meaningful as this provides a good measure about the adoption of mipomersen in real life. However, given a slight tendency by ISIS to over-promise, we really have to await the precise numbers that are to be presented at an upcoming scientific conference.

In summary, the heterozygous FH results were very likely not as bad as the market reaction would suggest and eyes are now on the full data presentation and, probably even more importantly, results from the phase III study in the critical severe hypercholesterolemic patient population. The reaction highlights, however, that slight differences in knockdown potencies can make a huge difference for RNA Therapeutics. The upcoming dose-fractionation study by ISIS and Genzyme, while labeled as being for the ‘convenience’ of patients, may actually be driven by the hope it might provide for improved knockdown due to different pharmacology. And finally, for RNAi Therapeutics, and Tekmira in particular, the good news is that the pioneering work by ISIS provides them with a very good idea of what their ApoB candidates will have to achieve in order to be commercially successful. A modest 35-40% knockdown should be well within the grasp of current SNALP-siRNA delivery technology.

Wednesday, February 10, 2010

RNAi Therapeutics Portfolio Update: Sell Targeted Genetics, Buy Benitec

As I intend to manage the RNAi Therapeutics portfolio more aggressively for performance rather than being more of a representation of the state of RNAi Therapeutics investments, I have decided to take advantage of yesterday’s strength of Targeted Genetics on news that it completed its asset sale to Genzyme. Despite exciting results from their ocular Leber’s Congenital Amaurosis program and what this may imply for the use of AAV in ocular RNAi Therapeutics, at this point there is too little evidence that RNAi Therapeutics will play an important role in the foreseeable future of Targeted Genetics to justify its place in the portfolio, especially in light of almost non-existent active research and development and talent outflow.

The proceeds will be re-invested in Benitec as there are signs that this company could emerge as the only proper surviving DNA-directed RNAi Therapeutics player. News from last week that Pfizer will continue to develop a HCV ddRNAi Therapeutics in which Benitec has a significant stake and a recent patent grant for an hairpin with a long loop are very encouraging.

Disclaimer: Investments in RNAi Therapeutics are highly risky and not suited for most people.The purpose of the blog and model portfolio is to convey a sense of the dynamics in the field and is NOT an endorsement for making related investments. I also have financial interests in some of the companies included in the portfolio. I do not, however, have short positions in any of those.

Tuesday, February 9, 2010

RNAi Therapeutics Delivery Wave Continues to Build

After listening to the presentations of a number of RNAi Therapeutics companies at the BIO CEO & Investor Conference, I came away thinking that while we are rightfully focused on discussing the challenges of RNAi Therapeutics, underneath of it there is a powerful wave of RNAi delivery solutions building and that is making its way into the clinic.

As a number of the presenters rightfully pointed out, there is not one universal delivery solution, such that this wave consists of various approaches addressing one cell and tissue type after another. Given that a cell/tissue is relevant for many diseases and given that there may be more than one suitable cell/tissue type for most diseases, this means that RNAi Therapeutics is quickly becoming broadly relevant for drug development. It is also true that while 21-23bp siRNAs will be the sweet spot for most applications, different siRNA lengths may work best for different delivery solutions, and which is facilitated by the remarkable robustness of RNAi in our cells that efficiently recognizes many forms of dsRNAs as substrates for RNAi.

To mark the occasion, I have compiled below a list of organs together with what I consider the leading respective delivery technologies. The list does not seek to exclude other technologies that could also be used for a given organ, but which are somewhat behind in development. The list is in order of clinical maturity with green highlighting those that I consider ready for prime-time, orange for those that are just on the verge, and red those that look very promising, but will require more research before I would feel confident about their clinical success.

The orange category is particularly exciting, because RNAi in immune cells looks like a low-hanging fruit waiting to be picked. Particularly when it comes to phagocytic cells such as dendritic cells that are important for vaccines, the opportunity lies in taking advantage of the natural tendency of siRNA nanoparticles to be taken up by such cells while there is increasing evidence that with the right chemistry you can get functional release of siRNAs into the cytoplasm instead of their degradation in phagosomes. I similarly feel that a renaissance of RNAi for the eye is in order given that DNA vectors and siRNA-conjugates are just begging to be applied as the unmet need grows in the exponentially ageing population.

Liver: cationic liposomes

Solid cancer proper: cationic and targeted liposomes

Endothelial cells: lipoplex/Atuplex

Phagocytic cells: liposomes

Other immune cells (incl. cancer of blood): targeted immunoliposomes

Eye: lipophilic-siRNA conjugates and ddRNAi (AAV and lenti)

Respiratory epithelia (incl. lung): modified siRNAs (inhalation; topical)

Brain (instillation): lipophilic-siRNA conjugates and ddRNAi (AAV and lenti; instillation)

Injured skin: lipophilic-siRNA conjugates (topical)

Heart and intestine: lipophilic-siRNA conjugates in reconstituted lipoproteins (intravenous)

Kidney: modified siRNAs (intravenous)

Thursday, February 4, 2010

Pfizer and GSK to Invest more in RNAi Therapeutics

GSK launches rare diseases unit: Today, GSK announced that it will move more aggressively into addressing rare diseases and set up a unit for this. The rationale is that developing medicines for diseases that have largely been neglected by the pharmaceutical industry in the past will have a good pharmaco-economic profile and ultimately be more profitable. More and more, selling pills to the masses does not appear to be the answer to Big Pharma’s patent woe and productivity cliffs. This should be very good news for RNAi Therapeutics, which offers a direct and cost-efficient way to such (often genetic) diseases, and maybe we will see this deal between GSK and Alnylam later this year.

Pfizer exercises option for Tacere’s HCV candidate: DNA-directed RNAi company Tacere, a Benitec spin-off, announced today that Pfizer has exercised its development-commercialisation option for Tacere’s HCV drug candidates. Pfizer obtained the option in the deal 2 years ago with Tacere (see blog entry here). This follows pre-clinical studies in rodents and monkeys that, according to the press release, have shown the AAV vector to penetrate the liver well and to be generally well tolerated. Pfizer will now collaborate with Tacere and fund the IND-enabling studies. This triggers undisclosed milestone payments, some of which will be due to technology licensor Benitec which, in addition, has an equity stake in Tacere. There is hope for ddRNAi Therapeutics!

RNAi Therapeutics Portfolio Update: Silence Therapeutics (Buy)

Considering the recent merger between Silence Therapeutics and Intradigm (see here) and following the review with Tobias on Atu-027 and the technologies behind it, I have decided to use the proceeds from the recent sales of mdRNA and RXi Pharmaceuticals to purchase shares in Silence Therapeutics for the fantasy RNAi Therapeutics portfolio. At 13.5 pence, giving the company a market cap or slightly less than 40M UK pounds, there should be good upside if Silence’s management can succeed in the certainly demanding task of integrating the two companies while at the same time establish RNAi drug discovery partnerships- all within the one year cash runway that the latest financing gave them.

I have long been wary about the value of Silence Therapeutics’ technologies. Readers of this blog might still be familiar with the angry outburst in the comments section of this blog from what appeared to be a Silence employee (just speculating) about 1 ½ years ago. Having appreciated some of the good science behind Atu-027, I can understand the frustration. But good science does not always mean that IP claims are valid, and in the case of Silence, their credibility had suffered with their bold claims of having broad freedom-to-operate in the RNAi Therapeutics trigger arena.

In the end, Silence had to settle with significantly less than they hoped for and probably led investors to expect, namely patent protection for blunt siRNAs containing alternate 2’-O-methylations opposite of unmodified nucleotides and that are 15-23bp long (length could be subject to the Tuschl outcome; in a twist of irony, it would be an Alnylam-Max Planck victory that would be beneficial to Silence). Although this is a far cry from getting protection for a much wider range of modification patterns and types of modifications as it looked possible at one point, they should be able to live with the outcome because I was even surprised that they were granted these patents at all, apparently on the basis that these patterns unexpectedly allowed for siRNA stabilization without killing efficacy. Some reward from being an early mover characterizing the properties of modified siRNAs, with a lot of their reported findings actually standing up to the tests of time.

My gut feeling tells me that while Atu-siRNAs have their use for RNAi Therapeutics at present, the evolution in RNAi Therapeutics trigger design is likely to render them obsolete in 10 years’ time, with Atu-siRNAs DNA/claim language not being able to adapt to the changes. Also, an extensive head-to-head comparison between the selection process for Atu-siRNAs versus Tuschl-type siRNAs, similar to what Alnylam has been doing for Dicer-substrate versus Tuschl-type siRNAs, would be helpful in getting a better feel for Atu-siRNA’s real value. The literature would suggest that the efficiency should be considerably impaired because the modification options are so limited.

Intradigm’s contribution to RNAi trigger assets are certain 25bp siRNAs, slightly longer than both Tuschls and reminiscent of RXi’s/Invitrogen’s ‘Stealth’ siRNAs. It is, however, still a mystery to me what these look like exactly and why these should be patentable structures. Similarly, the the scope of their licensed Zamore rules for siRNA design are somewhat uncertain apart from the one that has issued, and there is generally the questions of how it should be possible to enforce design rule patents when they depend on thermodynamic features rather than distinct siRNA structural motifs.

More than RNAi triggers, and a slight departure from earlier days, the company now emphasizes their relatively diversified approach to siRNA delivery. This is understandable as their is no question that financial value should follow scientific need. Silence's delivery technologies include the lipoplex-siRNA system is used for their lead candidate Atu-027 and that was shown to render endothelial cells of blood vessels accessible to RNAi. Here as well, I have long been quite skeptical about the strength of the data, because relative to other systems their characterization, including formulation method, had been quite sketchy, and I don’t believe that all of this is because they want to keep these details as trade secrets. Nevertheless, the apparent tolerability and efficacy of lipoplex-siRNAs from rodents to monkeys is very encouraging and should get them onto the radar of potential Big Pharma partners. Beyond lipoplex-siRNAs, Silence now lists peptide-based, targeted delivery (HKP) and delivery to the lung. While HKPs look nice in theory, the literature was not able to convince me of their immediate clinical utility and seems to be a little bit behind the lipoplexes. And for the lung-related delivery, I have yet to see the data. I plan to provide should I be able to get meaningful answers to some of these questions.

In summary, having invested tens of millions over the last 6-10 years into RNAi trigger and delivery research and with the bench depth provided by the merger, Silence Therapeutics should be an interesting partnership play, with Atu-027 as an innovative approach to cancer treatment that can be showed off to investors and partners, and maybe some positive surprises from their interests in RNAi compounds being tested in the clinic by Quark Pharmaceuticals and Pfizer.

Disclaimer: Investments in RNAi Therapeutics are highly risky and not suited for most people.The purpose of the blog and model portfolio is to convey a sense of the dynamics in the field and is NOT an endorsement for making related investments. I also have financial interests in some of the companies included in the portfolio. I do not, however, have short positions in any of those.

Please read the important disclaimer at the bottom of the page.

Tuesday, February 2, 2010

For Silence Therapeutics’ Atu-027 Cancer RNAi Therapeutic, the Proof is in the Pudding, not the Theory

This is the second part of the three-part series in which Tobias Wolfram and I are looking at the scientific merits of the first three notable RNAi Therapeutics candidates that have entered the clinic for solid cancer indications. Solid cancer is arguably the one therapeutic area where RNAi Therapeutics currently have highest value because of a) the ability to target many of the important, but otherwise undruggable cancer-related genes; b) systemic delivery technologies that ought to be able to reach solid cancer tissues; and c) a patient population that is essentially indifferent to the exact mode of administration (e.g. oral vs intravenous infusion).

In Part I of our series we looked at Calando’s CALAA-01. We came out slightly disappointed with that program because it seemed stuck in the quite early, non-biological/physico-chemical characterization stages and did not provide too much bona fide evidence of anti-cancer activity in pre-clinical animal models. Neither was Calando’s RONDEL delivery technology convincingly shown to facilitate in vivo gene silencing in the first place. We are therefore relieved, and I personally also somewhat pleasantly surprised at the same time, to be able to report a considerably better validated cancer RNAi Therapeutics candidate in Silence Therapeutics’ Atu-027. While the precise theory behind Atu-027 remains somewhat uncertain, we are encouraged by the scientific rigor with which the target had been validated as a component of the well known PI3-kinase pathway and the careful demonstration that lipoplex-siRNA delivery can be used for knocking down genes in the vascular endothelia from mice to monkeys.

The Target: PKN3, a Downstream Component of the Cancer-related PI3K pathway

The precursor of Silence Therapeutics, Atugen AG, was a child of the millennium genomics era involved in gene discovery and target validation. The technological workhorses underlying this work were a combination of gene knockdown approaches such as ribozymes, antisense (‘GeneBlocs’), expressed RNAi that were combined with RNA expression analysis and proteomics.

For some reason that may be related to the prior training of Atugen’s scientific staff, Atugen specialized in studying the genetic components of the PI3-kinase pathway. This pathway has attracted wide attention because it plays important roles in various biological processes and has consequently been implicated in numerous diseases, particularly in cancer where a number of genetic abnormalities that affect a range of phenotypes ranging from cell growth to metastasis have been linked to it. While clearly an attractive target for cancer therapy, and there has been some success in drug development in that regard, its many roles in molecular biology means that targeting the pathway upstream, for example at the PI3 kinase level, is likely to be associated with a poor therapeutic index. Atugen therefore reasoned that discovering targets downstream of PI3K could result in equally potent, but also more specific therapeutics for cancer.

Applying their genomic tool kit that placed a high priority on specificity controls, they came up with a handful of candidate target genes that they then nicely validated in subsequent in vitro and in vivo tumor-related assays. From this, PKN3 eventually emerged as a gene whose expression and phosphorylation was not only PI3 kinase-dependent, but when knocked down also abolished the ability of PI3 kinase-activated cancer cells to grow under culture conditions thought to reflect the natural tumor environment. When such cancer cells were modified with a DNA-directed RNAi construct against PKN3 and then introduced into mice in an orthotopic prostate tumor model, the primary tumor formation was, somewhat unexpectedly, not much affected whereas metastasis formation was significantly inhibited. Thus, the authors speculated that it is PI3 kinase-dependent functions like cell motility and/or cell-cell/cell-matrix contacts that are disturbed following PKN3 knockdown, and that the substrate-dependent growth defect observed in vitro may reflect deficiencies in tissue invasion that are shared with the facilitate metastasis in vivo. Consistent with a role for PKN3 in real-life cancers, tissue immunohistochemistry from prostate cancer patients revealed PKN3 to be upregulated in the cancer, but not surrounding normal tissue.

The theory of why PKN3 RNAi might have utility as a cancer treatment had to be modified when it was found that the delivery system did not target the cancer cells in which the initial target validation experiments had been carried out, but instead endothelial cells of the blood vasculature. The late change in the theoretical framework for Atu-027 that occurred in the otherwise very impressive 2009 landmark Atu-027 mouse-to-monkey validation study (Aleku et al., 2009), is probably the major weakness in Atu-027’s rationale. The question arises why take such a target risk at all when there are many other well-validated, but undruggable cancer targets already out there to choose from? Is PKN3 a suitable cancer target after all, and, even if it were, are they knocking it down in the right cell types? It is here that the relative lack of knowledge on the biology of PKN3 (all the functional studies on this gene were conducted by Silence Therapeutic) turns out to be a disadvantage and may complicate things like the development of biomarker tests to monitor early clinical efficacy.

On the other hand, the fact that PKN3 emerged after having considered a range of targets, suggests that based on practical experience Silence had highest confidence in the effectiveness of PKN3 as a cancer target. This argument is supported by what Tobias and I felt to be very well controlled target validation experiments, including the use of multiple positive and negative control oligonucleotides to confirm that the observed phenotypes were on-target. In that regard, I would score the relevant dataset generated by Silence in the top 5% of the RNAi literature. Maybe not surprising because of Silence's roots in gene discovery.

Being the only one working on a gene, however, has a few benefits, too. As such, Silence has made inroads in obtaining broad patent protection for the use of PKN3 as a target in PI3 kinase-related diseases. Given the large interest in the PI3 kinase pathway, this could be a valuable asset to the company and somewhat further justifies what otherwise would look like a disproportionate investment for a platform company into a single pathway!

The RNAi Trigger: A 23 base-pair Atu-siRNA

Although Atugen’s background was more in ribozymes and antisense, it decided to make drugs based on synthetic siRNAs after its transformation into a drug development company. This was around the time that RNAi had just been discovered in humans by Tuschl. In the case of Atu-027 this meant that while antisense, expressed RNAi, and small molecules acting on the PI3 kinase pathway were used for target validation, the active ingredient would be a 23 base-pair blunt-end siRNA in which alternating 2’-O-methyl nucleotides would face unmodified residues, a pattern that also coincides with the eventual scope of the issued Atu-siRNA patents in the US and Europe.

Since siRNAs may have anti-tumor activity independent of RNAi, the result of non-specific innate immunostimulation by some siRNAs, it is notable that such extensive 2’-O-methylation was shown only later in the field to result in siRNAs with no or very little innate immunostimulatory potential. This not only lends credibility to their pre-clinical efficacy data, the choice of 2’-O-methylation at such an early stage can therefore be more generally regarded as a very lucky one considering that the scope of the Atu-siRNA patents would eventually only encompass 2’-O-methylations. Speaking of RNAi trigger IP, a strong Tuschl II would actually benefit the blunt-end Atu-027, while a strong 21-23 base-pair/nucleotide covering Tuschl I could cause problems.

Lipoplex-siRNA Delivery to Endothelial Cells of Blood Vasculature

The delivery system for Atu-027 was developed in parallel to identifying the target siRNA. It is a pegylated lipoplex-siRNA system where the siRNA sits on the outside of positively charged, peylated liposomes ('Atuplex'), instead of the more commonly used SNALP-like liposomes where the siRNA is encapsulated inside. Because of this fundamental difference, the two systems vary considerably in their biological properties. Whereas SNALPs deliver their cargo mainly to the liver, but can be further stabilized to reach tissues beyond that, lipoplex-siRNAs have a preference for the endothelia lining the inside of blood vessels pretty much throughout the body.

In careful labeling studies, Silence Therapeutics scientists were able to show convincingly that they can achieve bona fide RNAi knockdown in the 50-70% range when administering around 1mg/kg- from mice to monkeys. Moreover, the invariable pharmacokinetics following repeat administration support that there is little or no cytokine activation. It is curious that most of their in vivo studies involve repeat administration. This is in contrast to what for example Tekmira and Alnylam are practicing which carefully study both single and repeat administration both of which together should inform much better on eventual dosing schedules, causes for toxicities etc. Similarly, only one cationic lipid, AtuFECT01, is used throughout the years, and a relatively simple opportunity to improve the therapeutic index has probably been missed. There is also a lack of a detailed description of the precise formulation process and early-stage physico-chemical characterization, although part of this may be for competitive reasons.

Apparently limiting themselves to delivery systems developed in-house, the performance of the lipoplex-siRNA technology means that in order for Silence to pursue their long-standing interest in cancer, they have to address the disease where it interfaces with blood vessel endothelia. Importantly, the concept of starving cancer cells by inhibiting neoangiogenesis is well accepted and one only has to look at the commercial success of Roche/Genentech’s anti-VEGF monoclonal antibody Avastin. The more pressing task therefore is to find the right target genes.

Pre-clinical safety and efficacy

The lipoplex-siRNA formulations were generally well tolerated at dosages predicted to be therapeutic. No significant differences in body weights were observed with various siRNAs. Immune responses were also not detected. This is particularly important in studies like these as it further suggests that the observed anticancer activity was not due to an off-target immune-related mechanism. Further supporting such a lack of immune responses was, as noted above, the fact that the pharmacokinetics did not change with repeat administration. Immune reactions would have likely facilitated antibody formation against the PEG component and caused rapid clearance of the lipoplex-siRNA upon repeat administration. I originally had been skeptical of Silence’s claims that there were no immune issues since they never seemed to have paid much attention to this issue, but they may have just gotten lucky in their early choice of extensive 2’-O-methylation as part of the Atu-siRNA architecture.

There were, however, some toxicities noted that mostly involved the liver and included 2-3 fold minor elevations in liver enzymes with an PTEN siRNA (this could be an siRNA-specific response different from Atu-027) and an apparent increase in DNA replication in livers of mice treated with control lipids alone which may be indicative of liver cell damage (Santel et al.,2006). Liver safety and immune reactions may therefore be the two expected dose-limiting adverse events for Atu-027.

In terms of efficacy, Atu-027 had ED50s in mice and monkeys of 1 and 0.3mg/kg using liver and lung endothelia as surrogate tissues. Since the samples include non-endothelial contaminants that could express some PKN3, this may actually somewhat underestimate the true extent of the knockdown in the target endothelial cells. This knockdown efficacy translated into a 60% reduction of the primary tumor in an orthotopic mouse prostate cancer model, and an even greater 80% reduction in the lymph node metastases. It should be noted that while the effect on the primary tumor is to be welcome, the results somewhat differ from those in the initial characterization of PKN3 as a target 5 years earlier where an effect was only observed for the metastases (Leenders et al., 2004). The anti-tumor response was confirmed in a range of other tumor models. Taken together, the safety and efficacy profile of Atu-027 are in support of initiating phase I studies.

Target and Delivery: A match made in heaven, or marriage of necessity?

As alluded to in the target validation segment, we are somewhat concerned about target risk since, as a result of the emerging properties of the lipoplex-siRNA system and in vivo PKN3 knockdown phenotypes, the rationale for PKN3 as an anticancer target has undergone multiple transformations, first validated as playing an important role in the cancer cells itself, but then thought to act directly on blood vessel and eventually lymph vessel formation.

Cynics could say that once they got stuck with the lipoplex-siRNA system, they would have done anything to force the model to fit their data. Realizing this dilemma, they did, however, undertake bridging studies and were for example able to show that PKN3 knockdown in isolated (non-cancer) vascular and lymphatic endothelial cells disturb well known endothelial phenotypes such as ability to form tubes in tissue culture.

Despite these twists and turns, from a 30,000 foot level, PKN3, due to its well-validated role in the PI3 kinase pathway, does look like a credible anticancer target in endothelial cells. Are there potentially targets that are better suited for the lipoplex siRNA system? Most likely yes, as it ought to be a general rule in RNAi Therapeutics that ‘target follows delivery’. Thus armed with knowledge about the ‘Atuplex’ delivery system, the next target-delivery combination by Silence has a better chance of looking like a match made in heaven.


In the end, we came to the conclusion that, theoretical concerns notwithstanding, by targeting a skillfully validated PI3K pathway gene with a delivery system that facilitated bona fide gene knockdown from mice to monkeys and the clear anticancer phenotypes observed in a number of rodent cancer models, Atu-027 is a promising clinical candidate for which the pre-clinical proof was simply in the pudding. Given the pioneering and innovative nature of this particular approach, it should also be able to draw the attention of the likes of Novartis and Pfizer that should be able and willing to take such risks as they move into RNAi Therapeutics.

Atu-027 may also be viewed as the start of a franchise that focuses on attacking cancer from its endothelial side with all the unique advantages of RNAi, for example the almost unlimited choice of targets. The merger with Intradigm, which also has developed expertise in neoangiogenesis and cancer, should add depth to this by, most importantly, expanding the pool of potential targets and pathways so that potentially less risky target-delivery matches can be found. There should also be ample scope for improving the Atuplex delivery system as it is questionable whether the first cationic lipid that they published and held onto, Atu-FECT01, is really the non plus ultra of lipid chemistry.

Meanwhile, Atu-027 has started clinical development half a year ago with a multi-dose phase I study in patients with advanced solid tumors. First results are eagerly awaited.

Monday, February 1, 2010

Clutching at Straws, Whitehead Attempts to Force Unwitting Scientist to Testify

According to recent court filings, the Whitehead is facing stiff opposition in getting a scientist to play a role in their defense in the Tuschl case.

The scientist in question is Brenda Bass from the University of Utah. In April 2000, she speculated in a review article that the small RNAs reported in the scientific publication underlying the Tuschl I patent application had been generated by an RNase III enzyme. Since RNase III enzymes are known to leave 3’ overhangs, the defense wants to use this speculative review article as proof that 3’ overhangs had been common wisdom in the gene silencing field at that time. This is designed to substantiate their claim that small RNAs 3’ overhangs were already implicit in Tuschl-I.

While I take my hat off to her remarkably prescient insights, the relevant remarks were clearly only speculations. Even if those speculations came from a well-respected scientist, most speculations in review articles do not turn out to be true to the same extent and for these reasons such speculations are generally not adopted as gospel in a scientific field, or by ‘those in the art’.

Maybe the Whitehead should start looking in the notebooks of Zamore, Bartel, and Sharp, or any other scientist for that matter, whether, since it was apparently so obvious, they were already in the know about the 3’ overhangs at the time (April 2000). This may be better than trying to annoy another person to become involved in a legal action. It essentially inconveniences her for being an insightful scientist.

If Whitehead is now alarmed that Dr. Bass does not really like to play according to their tune, then they really seem to be clutching at straws and continue to alienate the scientific community in their quest to lay claim on what belongs to the inventors of Tuschl II. And as a technicality, even if (unlikely) the judge could be convinced of this review article to have made 3' overhangs common wisdom, then given the filing dates it is still likely that Tuschl would be able to show that he first conceived of the overhangs before the April 2000 date.

Partly responsible for the alarm could be the fact that Max Planck and Alnylam are seeking, in addition to the permanent injunction, treble damages from the Whitehead and UMass for willfully conspiring to undermine the interests of Max Planck and Alnylam. If the $1.1B price tag for Sirna Therapeutics is any guide, it could get quite expensive. I just cannot get my head around Whitehead's eagerness to side with UMass, who along with their licensees Sirna/Merck and RXi seem to be the only financial beneficiaries should Tuschl I emerge as the fundamental RNAi trigger patent. In fact, Whitehead would even suffer financial harm by doing so, if it is true that Whitehead, MIT, and Max Planck would share equally in the combined Tuschl I-II royalties as per the Therapeutic Agreement. The only reason provided by them and Alnylam/Max Planck seem to be the political damage that siding with Max Planck instead of fellow UMass would entail. Clearly, there must be more to this than meets the eye.

Excerpt from the review:

Does PTGS by dsRNA Involve an RNase III–Like Enzyme?

Although the identity of the RNAi nuclease has not been determined, the characteristics of the short 21–25 nucleotide RNA pieces suggest they were generated by RNase III or a highly related enzyme (see [16] and [1], and references therein). RNase III is the only characterized nuclease known to cleave dsRNA at specific sites to generate dsRNA fragments of discrete sizes. For RNase III to stably bind a dsRNA, it must be at least two helical-turns in length, consistent with the observation that RNAi and transgene-induced silencing yield stable fragments of 22 base pairs. RNase III can produce fragments <22 class="apple-converted-space"> Figure 1, fragments less than 21–23 base pairs would not have been observed in the recent experiments because they would not remain stably bound to the enzyme and thus would be more accessible to degradation by other cellular nucleases.

Given the similarities between the cleavage products of RNase III and the RNAi nuclease, I have incorporated properties of the RNase III enzymes into the model of Figure 1. For example, RNase III makes staggered cuts that leave 3′ overhangs of two base pairs, as shown for the 23-mers of Figure 1. If RNAi involves an RNase III-like enzyme, it might explain why the small RNAs observed by Zamore and Tuschl range from 21–23 nucleotides. The initial cleavage might produce dsRNAs comprised of sense and antisense 23-mers, but the 3′ overhangs would be more accessible to single-strand–specific nucleases present in the extract, and trimmed to 21 and 22 nucleotide pieces. Zamore and Tuschl observe that cleavage of the dsRNA, unlike mRNA cleavage, does not absolutely require ATP. However, dsRNA cleavage is faster in the presence of ATP, and without ATP the pieces are predominantly the longer 23-mers. Certainly this is a clue to the role of ATP in this in vitro reaction, but at present its meaning is unclear.
By Dirk Haussecker. All rights reserved.

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