Alnylam Wins Important Battle in Kreutzer-Limmer Patent Fight

The Kreutzer-Limmer (KL) patent estate, exclusively owned by Alnylam and very broadly claiming double-stranded RNAs (dsRNAs) for gene silencing in human cells (up to 15-49 base pair dsRNAs), won an important battle at an oral hearing last week at the European Patent Office. This comes after Kreutzer-Limmer has suffered a number of setbacks at the EPO about one and a half years ago. The resuscitation of KL means that Alnylam may leverage this patent as potentially gate-keeping as it looks to enter additional non-exclusive licensing partners for its dominant RNAi Therapeutics patent portfolio, also as a potential backstop in the unlikely event that Alnylam loses the fight for the key Tuschl inventions.

The decision reversed an earlier rejection based on technical objections, mainly by Sirna/Merck and Silence Therapeutics, that some of the claims in the daughter application at issue had been incorrectly drawn from the parental application. In general, the main claim describing the structure of gene silencing short dsRNAs are somewhat clumsily constructed and not as straight-forward as one might imagine. This could be the result of having to take into account the embodiments underlying this claim.

If it were not be so important, reading the deeply philosophical arguments of what it means to be 'double-stranded' can be quite amusing, but unfortunately also very time-consuming and distracting, as are these drawn-out patent back-and-forths. Especially for smaller companies like Silence Therapeutics it may actually be worth focusing on developing the enabling delivery technologies and their therapeutic pipelines instead of having their lead scientists waste their creative juices taking part in these battles.

It is likely that the patent will go back again, but this time to be challenged on other issues such as novelty. Regardless, Alnylam should benefit from last week's decision to uphold Kreutzer-Limmer in that it adds to the appearance that, no matter how the individual patent chips fall, Alnylam has so many patent options that making some kind of licensing arrangement with the company would be required if one wanted to securely develop RNAi Therapeutics. Recent progress in systemic delivery should add to the urgency and may even make waiting for the outcome of the Tuschl Tussle a difficult decision.

ALN-VSP02 Interim Safety and Early Signs of Activity Supporting Continuation of Trial

[This entry has been updated on May 21, 2010]

At precisely 6pm tonight, ASCO released the much-awaited abstracts for presentations at the upcoming cancer meeting. This included one on the interim analysis of Alnylam’s ALN-VSP02for the treatment of liver cancers (my review with Tobias Wolfram on VSP02) targeting both VEGF (antiangiogenic) and KSP (anti-proliferative) and delivered by first-generation SNALP technology*. Importantly, the drug showed an encouraging safety profile with no cases of flu-like hypersensitivity reactions up to the highest dose tested so far (0.7mg/kg, dose-escalation ongoing) and signs of hepatotoxicity up to 0.4mg/kg, the two most important expected potential toxicities for SNALP delivery-based drugs.

There was one death possibly related to the study drug in a patient with a pancreatic neuroendocrine tumor (most others had colorectal cancers) at the 0.7mg/kg dose level. This patient died of hepatic failure after receiving the 2^nd of the 4 bi-weekly 15-minute infusions. While liver patients do often die from hepatic failure and patients in the trial probably have a relatively short 1-2 year life expectancy which means that deaths are to be expected in such a trial just by chance, because of the temporal association of the treatment with the death, a contribution of the drug cannot be excluded. A general hepatotoxicity of the drug or delivery technology, however, is unlikely because, unlike flu-like reactions to liposomal siRNAs, hepatotoxicities should be more uniform, at least in 'average' persons, but were otherwise absent in the 0.4mg/kg cohort as well as in the two other patients dosed at 0.7mg/kg. Similarly, except for a minor grade 2 infusion reaction that responded to slowing of the infusion, there were otherwise no significant adverse events, quite remarkable for such a cancer trial, which supports that the death was an isolated case of which the exact cause remains to be fully determined.

Interestingly, in the deceased patient there was extensive tumor necrosis following drug treatment and this was correlated with decreased blood flow as measured by DCE-MRI, consistent with successful VEGF inhibition. Although it cannot be excluded at this point, it is probably too early to say whether the death may have even had to do with too rapid tumor necrosis due to VEGF and/or KSP inhibition by affecting already impaired liver function in liver cancer patients. Altogether, blood flow was measured in 8 of the 12 patients treated as of December 2009 with 80% of the tumors showing a remarkable >40% decline [in blood flow].

Eyes are now on the more detailed data presentation at the ASCO meeting in June that will include further safety, tolerability and also pharmacodynamic data, possibly at even higher dose levels**. More information on the exact distribution of the declines in blood flow and the neuroendocrine patient history will be of particular interest.

* Second generation SNALP formulation have potencies that are about 10-50 increased compared to the one used in the present trial that had a predicted 50% knockdown potency at around 1mg/kg. This means that at therapeutically relevant doses for future SNALP-based drugs of around 0.05-0.2mg/kg, no significant toxicities have been observed so far in both Tekmira's SNALP-ApoB and Alnylam's ALN-VSP02 trials.

Putting the ALN-VSP02 Adverse Event into Context (update May 21, 2010)

It has come to my attention that there is considerable angst, especially among investors, about the one death in the trial. While every such case is unfortunate and has to be studied in detail whether it is linked to treatment with the investigational drug, let me emphasize that the reason why liver metastases are specifically treated for most cancers, including in this trial, is because they turn out to be rate-limiting for many of these late-stage patients. As such, more deaths are to be expected in this trial, also and especially due to liver failure. One caveat, however, is that the patient died after the 2nd infusion, that is between 2-4 weeks after dosing had been initiated and one of the enrollment criteria for the trial was a life expectancy of >12 weeks, so a contribution of the drug is likely.

A good indication about the seriousness of these adverse events as it relates to drug safety and risk:benefit is how regulators react to such reports that by the way have to be made in a timely manner, meaning that if there had been more such reports they must not have been deemed sufficient to stop the trial. Related to this, it will be important to learn whether the regulators allowed further dose escalation or whether the patients that have been recruited since the December 2009 abstract deadline were treated largely at the 0.4 and 0.7mg/kg dosages to further study the drug safety around these dose levels, although the company did say in their press release that the maximum tolerated dose has not been 'reached' yet [update: the company has since confirmed that dose escalation has continued since December 2009, meaning that at the very least there should now be data for the 1.0mg/kg cohort if not higher which reflects favorably on the safety profile thus far].

It is surprising that Alnylam did not comment generally much about the detailed data in the press release about the abstract. It is possible that they consider doing so at this point as being of little value since much more comprehensive and informative data will be presented soon at the ASCO.

In the meantime, unlike drugs developed maybe for restless legs syndrome or ED, deaths are a frequent occurrence of cancer drug development and investors have to live with that without assuming the worst.

Targeting MicroRNA to Increase Good Cholesterol

In back-to-back publications in the journal Science, researchers from Boston and New York demonstrate that microRNA-33 (miR-33) is part of the complex control system regulating cholesterol metabolism (Najafi-Shoushtari et al. and Rayner et al.). The research further suggests that miR-33 is an attractive target for increasing ‘good’ HDL-cholesterol and continues to add to the intriguing possibilities how microRNAs may be exploited for the treatment of a range of major diseases.

Most pharmacologic strategies in cardiovascular risk management today aim at lowering the ‘bad’ LDL-cholesterol, including oligonucleotide therapeutics targeting ApoB and PCSK9 currently in development. It is, however, actually the ratio of the bad to good cholesterol and associated proteins in the blood that provides a better measure of the cardiovascular risk compared to the absolute levels of bad cholesterol alone.

The reason HDL-cholesterol is good for you is because it promotes reverse cholesterol transport from atheromatous plaques in blood vessels back to the liver and excretion via bile. If you come across a colleague in your workplace with an intensely red head, chances are that they have just taken niacin, the most widely used drug to increase HDL-cholesterol. Partly because of the side-effects of niacin and also to even more increase HDL levels, the medical community is extremely interested in additional drugs that can complement the LDL-lowering drugs such as statins with those that elevate HDL-cholesterol. Despite the failed high-profile billion $ gamble by Pfizer on small molecule Torcetrapib, appetite for such agents continues to be high also in the pharmaceutical industry.

Ever since the work by Brown and Goldstein, lipid metabolism has become the prime example for complex, yet robust regulatory networks in biology with lots of built-in feedbacks and redundancies. It is therefore probably not surprising that encoded within an intron (the part of an RNA transcript that is cut out during messenger RNA processing) in one of the master regulators of this network, the SREBP transcription factors, was encoded a microRNA, miR-33, that was co-expressed with SREBPs and similarly functioned in cholesterol regulation. While SREBPs contribute to the synthesis and cellular uptake of cholesterol, miR-33 redundantly works to increase cellular cholesterol levels by targeting members of the ATP-binding cassette transporter family involved in the transport of cholesterol out of cells (loading of cholesterol onto HDL apolipoproteins).

In other words, inhibiting miR-33 function should enhance cholesterol efflux from cells. This would be particularly beneficial for macrophages that by overfeeding on LDL-cholesterol play a central role in forming plaques and clogging up arteries. Indeed, the researchers were able to show that the inhibition of miR-33 with antisense would do just that: increase cholesterol efflux from macrophages. Preliminary data showing the predicted increase in HDL-cholesterol following systemic administration of miR-33 antisense molecules were presented as well.

Although encouraging, in order to firmly validate mir-33 as a therapeutic target for increasing HDL-cholesterol, a more thorough understanding of the consequences of miR-33 inhibition is required as are strategies aiming at targeting miR-33 preferentially in the atherogenic macrophages and possibly also hepatocytes. This, however, should be a quite realistic goal given that phosphorothioate antisense molecules alone as well as a number of oligonucleotide nanoparticle delivery systems are preferentially taken up by these cells anyway.

It is quite exciting to see so many microRNAs emerge as potentially high-value therapeutic targets. It is a testimony to the intense interest these small RNAs have attracted in the scientific community as they are involved in very likely almost all biological pathways. This should both help to establish microRNA Therapeutics as a vibrant industry able to stand on its own feet due to the many therapeutic opportunities, and also benefit RNAi Therapeutics by contributing to our understanding of the molecular mechanisms and functions of the natural counterparts of RNAi triggers.

Understanding the Biological Basis of SNALP Delivery to Broaden Potential of Technology

Almost daily, there is important research being published documenting the progress towards making RNAi Therapeutics become a reality. In addition, I am again and again surprised by the revelations of key regulatory roles of the natural counterparts of the RNAi triggers, the microRNAs, in many important diseases which lays the foundation for a healthy microRNA Therapeutics industry in its own right. In my next two postings, I would therefore like to highlight two such quite nice stories published recently, one about broadening the potential of SNALP RNAi delivery technology by understanding its molecular biological basis (this posting), the other about microRNA-33 as an exciting new target for increasing the ‘good’ HDL-cholesterol (next posting).

Towards Actively Targeted SNALP Delivery

As you know well, SNALP (stable nucleic acid-lipid particles) has captured my imagination as the systemic delivery system that has the potential to become the main value driver of RNAi Therapeutics in the next 5-10 years. There should be 5 SNALP-RNAi candidates for which an IND will have been filed by the end of this year, all this accompanied by a healthy stream of pre-clinical research and, importantly, clinical data especially surrounding applications for the liver, SNALP's low-hanging fruit.

The liver, however, is not where it stops. Solid cancers have emerged as another major opportunity for SNALP for which today’s technology may already be adequate for a number of indications. Due to the flexibility and inherent potential of the platform, however, there is much room to increase the important therapeutic window not only for solid cancers, but also for other indications such as inflammation, infection, and hematologic abnormalities. The Holy Grail towards this is ligand-targeted delivery which also requires increasing SNALP circulation times partly by limiting their uptake in the liver and making them invisible to undesired phagocytic cells ('stealth').

A paper by by Akinc and colleagues at Alnylam points towards how this might be achieved. In an important first step, the apolipoprotein E was (ApoE) was found to be critical for the functional delivery of basic, i.e. not actively targeted ionizable SNALP liposomes (iSNALP). Note here that SNALPs typically come in 2 basic shapes: those that are positively charged at physiologic pH (‘cationic SNALPs’ to which e.g. the ‘lipidoid’-containing SNALPs belong to), and those that are neutrally charged at physiologic pH, but that become positively charged at acidic pH (‘ionizable SNALPs’ which Tekmira focuses on, while Alnylam develops both types). SNALPs of the latter kind have a number of characteristics that make them preferable for most systemic RNAi applications. One is that the positive charge makes them inherently more 'sticky' biologically which is not only detrimental to their pharmacology, but can also cause a number of toxicities, including the damage of cell membranes. iSNALPs, by contrast, do not suffer from these drawbacks.

On the other hand, positive charge is important for efficient encapsulation of the siRNAs into the liposomes and also beneficial for cell transfection by first interacting with the negatively charged cell exterior and subsequently disrupting the endosomal lipid bilayer. Because iSNALPs become positively charged at low pH (hence 'ionizable'), they can still be efficiently formulated by transiently decreasing the pH during packaging, and they can still efficiently escape from the acidifying endosome into the cytoplasm for the same reason. One outstanding question, however, has been how iSNALPs, for which the potencies now approach the single digit microgram/kg range (about 100-fold more potent than 1^st generation SNALPs and about 1000-fold more potent than RNaseH antisense), make critical contact with target cells in the first place without the positive charge.

Employing an impressive array of genetic cell and animal models, Akinc and colleagues show that it is the plasma protein Apolipoprotein E (apoE) that allows, by associating with the lipid layer of the SNALP, the iSNALP particles to recognize cells carrying cognate receptors of the LDL-receptor family. Confirming the distinct uptake pathways involved in cationic and iSNALP delivery, the ApoE-LDLR interaction was not rate-limiting for cationic SNALP delivery. As a side note, because the LDL-R itself seems to be the major receptor in the liver, this needs to be taken into account when using iSNALPs for the homozygous familial hypercholesterolemia population in the upcoming SNALP hypercholesterolemia clinical trials.

Hints that ApoE could be involved in iSNALP delivery actually came from earlier studies on neutral liposomes furthermore illustrating how the wealth from decade-long prior liposomal research is now benefitting the development of the SNALP delivery platform.

In order to demonstrate ApoE-independent targeted delivery of iSNALPs, the researchers then decided to increase the stability and density of the exterior PEG layer, generally used as a stealth strategy, such that ApoE would not be able to easily associate with the lipid layer of the particles, and also to increase the circulation times such that iSNALPs would have sufficient time to distribute throughout the body. To replace ApoE’s function in cellular uptake, another ligand could now be added, in this case to the end of the PEG-lipids, such that they re-enabled uptake, but in an ApoE/LDL-R independent manner.

Despite this fundamental demonstration of re-targeting iSNALPs, the main challenge now is to do so without losing knockdown potency. In the present study, increasing the stability and density of the PEG layer decreased knockdown potency by about a log, from the sub 0.1ug/kg now routinely achieved with ApoE-targeted iSNALPs to close to 1mg/kg, probably due to either of the following 2 factors: 1) despite cell surface receptor recognition of the particles, the dense pegylation inhibited subsequent endosomal uptake; 2) the re-targeted iSNALPs were taken up, but then remained inert inside the endosomes because of inefficient shedding of the PEG layer thus leaving the endosomolytic lipids unexposed.

By using techniques such as fluorescence microscopy, it should be fairly straight-forward to figure out which one of the two possibilities it is, if not a combination. Whatever it is, optimizing the stability and density of iSNALP pegylation, the location of the targeting ligands (outside on the PEG and/or on the lipid bilayer), and exploiting alternative mechanisms of shedding the PEG layer (e.g. through the incorporation of proteolytic cleavage sites or pH-dependent shedding), should be just some of the approaches that should solve these challenges, and may indeed differ depending on the disease indication. Moreover, in practice it may not be necessary to completely abrogate ApoE-mediated uptake to sufficiently re-target iSNALPs.

I am sometimes reminded, also by readers of this blog, that there are also other siRNA delivery technologies besides SNALP. That’s certainly true, and some of them have shown encouraging data indeed, including in humans, and are important components of the RNAi technology landscape as RNAi Therapeutics will ultimately involve the use of more than one delivery platform. On the other hand, there is little arguing that the development of SNALP technology in the last 6 years has been breathtaking and of the highest scientific quality and its success should be to the benefit of the entire RNAi Therapeutics space.

Milestones in SNALP siRNA delivery

2005: SNALP delivery in rodents; recognition of innate immune stimulation

2006: SNALP delivery in non-human primates with EC50s of around 1mg/kg

2007: siRNA modification abrogates SNALP-siRNA triggered TLR immune stimulation

2009: SNALP delivery for (non-liver) metastatic solid cancer; demonstration of RNAi mechanism of action

2010: SNALP potency approaching single-digit microgram/kg potencies

2010: biological basis for SNALP delivery being elucidated, laying the foundation for increasingly wider applications of the technology

mdRNA and RXi Coming Out with their RNAi Triggers (Part II)

In the second part of my review of mdRNA’s and RXi Pharmaceutical’s new trigger designs (part I: specificity of mdRNA's usiRNAs), I will try and somewhat de-mystify RXi’s ‘self-delivering rxRNAs’, the industry’s best-kept secret. First insights into the structure and chemistry of sd-rxRNAs were recently provided at the ARVO meeting in Florida and the publication of a related patent application (International Publication number WO 2010/033247 A2).

Shown above is a typical example of an sd-rxRNA. Sd-rxRNAs are based on a so-called ‘asymmetric siRNA’ structure in which the double-stranded region of the RNAi trigger is relatively small (less than 15bp) whereas the length of the guide strand is maintained around 19-23 nucleotides to maintain silencing efficacy. Although on average these RNAi trigger designs are significantly less potent than traditional Tuschl-type siRNAs, extensive screening in some cases allows for quite potent asymmetric siRNAs to be found with picomolar activities.

The double-stranded region is kept short because it is thought that structurally rigid double-stranded nucleic acids don’t wiggle well through the plasma membrane. Single-strand nucleic acids, however, as practiced in the antisense field can enter the cells more readily possibly because of the increased flexibility and an exposed nucleobase that is relatively less charged.Another benefit of keeping the dsRNA region below 15bp is that it avoids conflicting with competing IP estate such as Alnylam's Kreutzer-Limmer series, although in this case the scientific rationale, as just pointed out, should be strong enough to stand on its own feet. Having said that, I can remember conference presentations by a Korean group that reported on similar asymmetric siRNAs, and there was also a publication on 'asymmetric siRNAs' in Nature Biotech2 years ago, although in that case the dsRNA region was more centrally placed, whereas in the case of RXi’s sd-rxRNAs the shorter passenger strand base pairs with the 5’ end of the guide strand.

The guide strand carries a synthetic 5’ phosphate group. This is probably intended to address the less efficient phosphorylation of highly modified, conjugated siRNAs of unusual structure compared to traditional RNAi triggers that undergo rapid phosphorylation following introduction into the cell, a step that is necessary for activating the silencing potential of an siRNA.

In addition to asymmetric structure, there are a number of chemical bells and whistles that render the RNAi trigger more lipophilic (‘fat-loving’) and therefore membrane permeable. One important strategy here is to replace a number of the phophodiester bonds in the RNA backbone with phosphorothioate linkages, especially in the long 3’ overhang of the guide strand. Phosphorothioate backbones are well known in the oligonucleotide therapeutics field and e.g. widely applied to RNAseH-type antisense molecules and have the property of being 'sticky' and contributing to favorable pharmacologies. It is possible that these phosphorothioylated 3' overhangs may not only enhance the membrane permeability of the molecules, but harness specific oligonucleotide uptake receptors thought to play a role in RNaseH antisense delivery.

Based on my understanding of the effect of phosphorothioylation on RNAi performance, and also evident from a number of datasets in the patent application, such modifications should not be used too extensively as this often will compromise knockdown efficacy (best limited to the 3' overhang). Phosphorothioylation, like the extensive 2’-O-methyl and 2’-F modifications of the nucleotides, has the added benefit of stabilizing sd-rxRNAs which in many applications will be directly exposed to body fluids (note: such modifications may also be used to prevent innate immune stimulation and to enhance guide-strand specificity).

In addition to phosphorothioylation, the conjugation of a lipophilic group such as a cholesterol to the 3’ end of the passenger strand is intended to also enhance membrane permeability and cellular uptake. This, of course, is very similar to the cholesterol-conjugated siRNA delivery approach taken by Alnylam and first published 5 years ago (Soutschek et al., 2005). For the same reason, lipophilic groups may also be added to within the RNAi trigger structure itself.

The real question, of course, is how all this translates to silencing in vivo. Unfortunately, the presentations contained only very little in vivo efficacy data. The abundance of tissue culture experiments, however, indicate that compared to lipid-mediated transfection, much higher amounts of sd-rxRNAs are required to achieve similar silencing (50-1000 fold higher). There is, however, one dataset that shows that ~50mg/kg sd-rxRNAs can knock down a gene expressed in a mouse liver (compare this to the 1000-fold lower dosages required to the latest SNALP liposomes), but further improvements in potencies are required before sd-rxRNAs, without further formulation become therapeutically relevant.

Compared to the 2005 Nature study by Alnylam, I agree that sd-rxRNAs may be a little bit more potent on average. Consequently, the patent application is littered with references about the superiority of sd-rxRNA over Alnylam’s siRNA-cholesterol conjugate technology. This, however, is a little bit unfair in my opinion given that it appears to be Alnylam that was really the innovator in this field and that its conjugate technology should have progressed since the initial publication.

Maybe a little bit more surprising, or then again maybe not, were also regular references about the technical superiority compared to Dharmacon’s Accell self-delivering siRNAs. To be clear, references of technical superiority per se are nothing unusual in the patent literature. It becomes noticeable, however, when such references are quite frequent and apparently politically motivated. As discussed on this blog before, I have speculated that Accell is likely to use lipophilic chemistries, too, not least because there are patent applications by Dharmacon covering lipophilic siRNA conjugates. (e.g. WO/2008/036825). I don’t want to speculate any further here, but RXi Pharmaceutical would probably do well to take potential conflicts of interests serious and take the necessary steps to address them before yet another IP drama erupts.

In summary, sd-rxRNAs are an example of how one would aim to render siRNAs more membrane-permeable without sacrificing too much silencing efficacy. The concept and the means employed may not be that revolutionary, but credit has to be given for extensively testing which structures and chemistries could work. For now, sd-rxRNAs are probably most promising in direct RNAi applications such as for the skin (see recently announced TransDerm collaboration), eye, and lung (by inhalation) where the ability to administer large amounts of unformulated siRNAs to the target organ may outweigh the potency disadvantages compared to traditional siRNAs formulated in polyconjugate or nanoparticle formulations.

Drug Development Upside Down: Tekmira Getting Paid as BMS Supports Pre-clinical Development

This morning, Tekmira Pharmaceuticals announced a quite unusual type of drug development deal with Bristol-Myers Squibb (BMS). In this expansion of their existing relationship, BMS will have continued access to Tekmira’s industry-leading RNAi delivery technology, SNALP, for target validation. What makes this such an unusual deal, however, is the fact that BMS will share the resulting data with Tekmira which then can turn around and use it to develop its own proprietary RNAi Therapeutics, and on top of that getting paid for it…!

Over the years, the relationship between Tekmira and BMS has taken on a more and more therapeutically flavored character. The partnership initially started out as a type of services relationship whereby Tekmira, then as Protiva, monetized on their liposomal siRNA delivery technology by helping BMS validate drug targets. However, another not insignificant benefit to Tekmira already then was that such services work was highly synergistic with their focus on drug development because such work allowed it to further develop its expertise in SNALP delivery at the partner's expense.

The stickiness of the alliance alone can be considered as evidence that BMS must have been quite pleased with Tekmira's technology. The relationship was first expanded in 2008 to develop SNALP delivery for tissues outside the liver. The following year, BMS then exercised their option to extend the collaboration for another year. This to me was the first indication that BMS might even be interested in RNAi Therapeutics as a drug modality itself and consider SNALP as a promising means to facilitate it. Why otherwise would BMS help develop the basic capabilities of a delivery technology instead of just waiting until Tekmira or any other company could offer a technology ready for such target validation?

On the surface, today’s news could be overlooked as simply extending an already existing relationship in which Tekmira keeps helping BMS validate drug targets. What is very new, however, is that Tekmira can now use gene target-specific data generated during this collaboration, including those by BMS (!), for developing their own RNAi Therapeutics. I have certainly noticed that Tekmira has become a bit cautious about which targets to exercise their 7 picks under Alnylam IP for. For small biotechs that depend on Big Pharma collaborations to fund late-stage trials, the danger of picking a target not desired by Big Pharma is very real. One of the general advantages that Big Pharma has over many of the smaller biotech companies, including Tekmira, is that they can afford to invest in technologies that allows them to pick better targets. This deal thus provides Tekmira with access to the target picking capabilities of a Big Pharma.

One thing that I am curious about is how the deal negotiations evolved. One way to interpret the deal is that BMS is still primarily concerned about target validation and if Tekmira wanted to compete with BMS on some of the targets, so be it...although at the not so inconsiderable expense of instantly granting Tekmira a significant development lead (no need to go through the lengthy small molecule lead discovery and optimization process) and the loss of exclusive target IP. On the other hand, it is possible that BMS approached Tekmira because it was primarily interested in developing RNAi Therapeutics themselves with Tekmira replying ‘yes, but only after you let us pick the best targets as we could use your target picking expertise but also wouldn’t want to help other companies in competing with us for targets that they validate with our own technology.’ Whether this was the case or not, one should not underestimate Tekmira’s ability to say ‘no’ if a technology license would invite too much competition (btw, this is the reason why I would not expect a simple Novartis-Tekmira licensing deal, because Novartis sits higher than Tekmira on the target picking pecking order).

In summary, I believe that this strategic deal was done by a company focused on building a biotech company that can stand on its own feet, giving away almost nothing in return for enhancing their ability to develop their own therapeutics pipeline...and also adding some cash to its coffers, always welcome these days. Having said that, Tekmira’s 7 other SNALP technologies licensees and others in Big Pharma still on the sidelines will have to realize that the longer they wait, the higher the price. To paraphrase the CEO of GSK, quality in small biotech still has its price, despite the economic turmoil.

[Disclosure: DH owns shares in Tekmira Pharmaceuticals]

mdRNA and RXi Coming Out with their RNAi Triggers (Part I)

In conference presentations this week, both mdRNA and RXi Pharmaceuticals finally lifted the veil of mystery shrouding the nature and performance of their claimed proprietary RNAi trigger designs. Independent of what they may mean in terms of IP, they are certainly worthy contributions to the science of RNAi triggers: the use of unlocked nucleic acids to increase the specificity of RNAi gene silencing (mdRNA), and the convergence of delivery and RNAi triggers through nucleic acid chemistry (RXi).

At the ‘RNAi and miRNA World Congress’, mdRNA presented data illustrating how selectively spiking siRNAs of the traditional Tuschl structure with a number of unlocked nucleic acid residues (usiRNAs) can reduce the level of off-targeting commonly provoked by ‘naked’ siRNAs. While similar strategies have been reported before and are broadly attempted in the industry, what differentiates mdRNA’s approach is that this particular nucleic acid analogue does not involve the addition of modifying chemical groups. Instead, in an unlocked nucleic acid the ribose ring is simply broken without making the residue any bulkier, but this is enough to significantly affect the interactions of the siRNA with its target mRNA and the Argonaute protein, the effector protein of the RNAi silencing pathway.

The simplest, and therefore widely practiced first step in reducing off-targeting is to modify the 5’ end of the passenger strand with a variety of chemistries. Because the 5’ end is important for the Argonaute protein to recognize and bind an RNA as the guide strand, half the off-targeting can be eliminated based on the sensitivity of this interaction to chemical alterations. It so happens that a UNA at the 5’ end of the passenger strand is similarly not tolerated…as has in effect recently also been shown in a publication by Sirna Therapeutics/Merck (Kenski et al., 2010). In the example provided by mdRNA at the conference, this meant a reduction in the number of genes that were changed in their expression by a factor of more than 2-fold (generally 2-3 fold) from ~390 to ~ 180, while on-target gene silencing activity was maintained at around 95% (data from RNA microarray experiments).

I should add, however, that additional usiRNA residues in the 3’ overhangs of both passenger and guide strands somewhat complicate determining the exact contribution of the passenger strand 5’ modification in reducing off-targeting. This is because it is known from structural studies that the position of the 3’ end of the guide strand relative to the Argonaute protein changes upon the transition from the microRNA-off-target (in the so called ‘PAZ pocket’) to the on-target cleavage (dislodged from the ‘PAZ pocket’) confirmations. 3’ ends that do not fit into the PAZ pocket may therefore change the ease with which the enzyme adopts either conformation and therefore affect the propensity of microRNA-like off-targeting. Structural studies with UNA residues at the 3’ end may be instructive here.

More interesting is mdRNA’s observation that when, on top of the above modifications, one normal base is replaced with its corresponding UNA analogue in the seed (= the first 2-8 nucleotides), the off-target activity is further reduced to about 35 genes that are changed by at least 2-fold, while again maintaining on-target efficacy. This was theorized to be due to the changes in guide RNA-target mRNA hybridization energies within the seed: the usiRNA lowered this energy sufficiently such that a microRNA-like interaction that is reliant on the seed becomes prohibitively weakened. However, the extended length of the guide RNA-target mRNA base-pairing compensates for the lost energy thus still allowing for Argonaute conformational change and on-target cleavage to ensue.

Previously described modifications have similarly been thought to function in reducing microRNA-like off-targeting by modulating seed energies. usiRNAs, however, could be a particularly attractive approach, because its small size should make it less likely to be detrimental to on-target activity. This, of course, would have to be shown with more examples, preferably in a peer-reviewed publication, and the Sirna Therapeutics paper has shown at least some limitations in where the UNA can be placed in the seed. Similarly, it remains to be demonstrated how much of the reduction in the off-target activity was due to eliminating microRNA-like interactions instead of a reduction in the innate immunostimulatory potential of these siRNAs as UNAs were also shown by mdRNA to mitigate such responses.

I believe this story shows how RNAi trigger design has matured to a point that RNAi Therapeutics truly lives up to its original promise of specificity which had temporarily (2003-6 period) been tarnished by the discovery of wide-spread microRNA-like off-targeting and the innate immunostimulatory potential of exogenously delivered synthetic siRNAs. It also demonstrates that at least on the level of RNAi trigger design mdRNA could certainly be a valuable partner to most in Big Pharma.

In my next entry (link here), I will provide my thoughts about RXi Pharmaceutical’s ‘self-delivering rxRNAs’ for which, until now to the best of my knowledge, the company has created so much expectations without actually disclosing the chemical nature thereof.

ALN-VSP02 with Potential to Become Valuable Targeted Component in Liver Cancer Therapy

In the final part of our 3-part series looking at the 3 most advanced RNAi Therapeutics candidates for cancer (part 1: CALAA-01; part 2: Atu-027) Tobias Wolfram and I have been analyzing ALN-VSP02, Alnylam’s candidate for the treatment of cancers with liver involvement that has entered the clinical stage of development in the first half of last year. ALN-VSP02 is a 2-pronged strategy to push back liver cancer comprising of 2 siRNAs packaged in Tekmira’s SNALP delivery formulation, one directed against the well validated vascular endothelial growth factor (VEGF) to choke off the nutrient and oxygen supply to the liver and the other against kinesin spindle protein (KSP) to disrupt cell division and induce apoptosis. Overall, based on the pre-clinical data and scientific rationale of the approach, we consider VSP02 to be a solid clinical candidate with potential to become an important component in the fight against a disease for which new options, especially molecularly-targeted ones are desperately lacking.

Liver cancer, both primary and secondary, is one of the most underserved cancers for which new therapeutic approaches are urgently needed. According to the 2009 issue of ‘Cancer Statistics’ by Jemal and colleagues, almost as many people die of primary liver cancer as are diagnosed. Primary liver cancer (aka hepatocellular carcinoma/HCC) has only recently risen to prominence in Western societies with ~22,000 newly diagnosed cases in the US alone in 2009, partly the result of an increased Asian immigrant population and the hepatitis C wave starting to take its toll. It is an even much larger problem worldwide with about 500,000 annual new cases particularly in the rapidly growing countries in Asia where hepatitis B infection is so prevalent. In addition to primary liver cancer, it is metastatic liver cancer that is the cause of much mortality arising from cancers of non-liver origins. Often more aggressive than primary HCC, such cases account for about 50,000 of newly diagnosed cases in the US with colorectal metastatic to the liver accounting for about 80% of those.

If you are lucky, your liver cancer is a candidate for surgical resection which will extend your life expectancy significantly and in a few cases is even curative. Unfortunately, due to the disseminated nature of liver cancer and the poor health of the liver at the time of diagnosis, surgical resection is not possible for the majority of cases (only 10-20%). One objective for the development of new drugs has therefore been to shrink the cancer sufficiently that patients become eligible again for resection. Despite some success with (systemic) chemotherapy, these often suffer from dose-limiting toxicities and are not curative. Liver transplants and relatively crude methods involving burning up the cancer tissue with heat or radioactivity are frequently used alternatives, but as you can guess, are either rather desperate attempts at fighting liver cancer or not practical for large patient populations.

There are, however, also reasons to be hopeful. Systemically administered sorafenib (aka Nexavar; first approved for kidney cancer) for example is the first ‘targeted’ therapy approved for primary liver cancer, a small molecule 'targeting multiple’ kinases with varied functions including angiogenesis. Illustrative of the high unmet need in HCC, a pivotal trial with this drug was halted pre-maturely to make the drug rapidly available to patients after it has shown an increase in median overall survival from 34.4 weeks on placebo to 46.3 weeks and median time to progression from 2.8 to 5.5 months. Unfortunately, most other small molecules and chemotherapeutics have not met with similar success largely because of drug resistance and systemic toxicities.

The avoidance of systemic toxicity is also the reason why a recent development in liver cancer has been quite exciting and that is poised , together with surgical resection, to set a newstandard-of-care for many types of liver cancers: a regional therapy for chemotherapeutics (and possibly other agents) that is repeatable and works by isolating the hepatic circulation and thus allows for bathing exclusively the liver in cytotoxic agents. The developer of this drug-device combination, Delcath Systems (ticker: DCTH), has just shown very promising top-line data in a phase III study for melanoma metastatic to the liver (another one of those cases where the liver metastasis is the major cause of mortality) extending hepatic progression free survival from 70 days with ‘best alternative care’ to 217 days with the company’s percutaneous hepatic perfusion system, aka PHP(detailed data to be presented at ASCO in early June; disclosure: DH owns DCTH shares). Other studies using this system for primary liver cancer and secondary colorectal and neuroendocrine are currently in phases II and III of clinical development. Possibly a lesson for Alnylam for future studies is that Delcath plans to conduct much of its late-stage clinical development for primary liver cancer in Asia, both for patient access and eventual market, while the trials for metastatic liver cancer largely take place in the US. Nevertheless, even if PHP will become part of a new standard-of-care for liver cancers, the problem is far from solved with hepatic disease in many cases eventually recurring and non-hepatic sites becoming rate-limiting. For cancer, monotherapy is seldom the answer, and for liver cancer this could mean a combination of surgical resection, regional high-dose chemo, plus one or two targeted therapeutics such as ALN-VSP02.

In this context, an RNAi Therapeutic should aim to either reduce/remove residual tumors after surgery and/or chemo, (re-)sensitize tumors to chemo, or contribute entirely new mechanisms of actions to the treatment of liver cancer. In addition, because a SNALP particle such as in ALN-VSP02 has selective delivery (main sites: liver, and other sites of solid cancers including lymph nodes) and the siRNAs target genes specific for cancer, it may be a systemic therapy with potential to also address sites of extra-hepatic disease.

ALN-VSP02 has the potential both to sensitize liver cancers to chemotherapy and to hold the cancer in check by itself by starving it and inhibit cell proliferation. Much of the discussion that I had with Tobias was centered not so much on delivery, but on whether the VEGF siRNA component was the best choice. There is little doubt from the broad success with Roche’s VEGF blocking monoclonal antibody Avastin in a variety of solid cancers that the angiogenic (blood vessel growth) factor VEGF should be a good target also for the typically highly vascularized liver cancers. Our question, however, was that since there is already Avastin, maybe a gene more uniquely suited for RNAi Therapeutics may have been preferable. On the other hand, as the first VEGF targeting RNAi Therapeutic, ALN-VSP02 is not simply a me-too drug because of a different mechanism of action: preventing VEGF from being made locally instead of blocking it once made and then also systemically (which causes additional safety issues). This could lead to unanticipated treatment benefits, but by the same token of course also potentially unanticipated failures. Questions that remain to be answered are for example how a tumor will respond to a maybe 50-70% overall knockdown with intratumor variations in silencing efficiencies as can be expected for SNALP delivery to solid tumors. And even if it ‘only’ had comparable efficacy to Avastin in terms of inhibiting angiogenesis, the fact that VSP02 addresses two targets at once means that it has the potential to substitute for Avastin as there are only that many drugs that a given patient can take.

The siRNA targeting kinesin spindle protein (KSP) is the less controversial component of VSP02, although it also falls in the category of a ‘druggable’ target under traditional definitions. Accordingly, almost all pharmaceutical companies to speak of have created their own, often me-too KSP-targeting small molecules, a number of which are in phase I and II clinical development for a variety of cancers. KSP is an attractive target because its tubulin-organizing function is thought to be specific to mitosis (the last stage of cell division) and its inhibition is not expected to cause side-effects typically associated with commonly used anti-cancer drugs that bind tubulin directly (e.g. neurological and hematological side-effects). Compared to these small molecules, however, ALN-VSP02 should have the added benefit of enhanced specificity and potency also because of its more selective delivery to solid cancers compared to small molecules. Moreover, many of the small molecules have IC50s in the high nM and low microM range, significantly higher than ALN-VSP02.

ALN-VSP02 has been very well validated in pre-clinical studies demonstrating expected pharmacological effects in mouse models of primary and metastatic liver cancer: compromised spindle bodies (‘monoasters’) from KSP knockdown and a decrease in microvessel density and vascular leakage from VEGF inhibition. In addition, Tobias was immediately struck by the maturity of the SNALP delivery system underlying VSP02. Rodent studies, of course, are necessary to evaluate the pre-clinical activity of an anti-cancer drug, but SNALP distinguishes itself in that its safety and efficacy has been routinely confirmed in non-human primates, dating back to 2006.

Liver cancer is a peculiar application for SNALP delivery technology for sure. On the one hand ‘liver’ suggests that first-generation, short circulating SNALPs may be appropriate. On the other hand, liver cancer tissue is different from the normal liver parenchyma in that it is quite a bit more heterogeneous, poorer in cell content and higher in extracellular matrix, and is thought to be primarily supplied with nutrients and oxygen by the hepatic artery rather than the portal vein as is the case for normal liver. It is possible for this reason that the PEG-lipid anchor in VSP02 which largely determines circulation times has a long carbon chain (18C). Such a long-chain lipid anchor has the added benefit in that it may also be able to address co-existing cancer outside the liver. In fact, research published last year by Tekmira showed that in the case of liver cancer short and long chains have comparable efficacy, whereas for cancer outside the liver long chains are preferable. The ability of VSP02 to also address non-liver sites of cancers was then also demonstrated by Alnylam late last year [erratum: while we originally believed VSP02 to comprise a C18 long-chain PEG-lipid anchor based on the schematics in Alnylam's poster presentations, the pharmacologic data presented at ASCO 2010 and the actual formula- PEG2000-cDMA- strongly suggest that it is in fact a short-circulating C14 myristyl anchor].

Tobias noted, however, somewhat critically that ALN-VSP02 is not an actively targeted therapeutic to which my response was that at least it is passively/pharmacokinetically targeted, and that Tekmira and Alnylam are working hard on next-generation formulations with targeting ligands that promise increased targeting selectivity and lower doses. This, however, is at the expense of more complex formulation methods and will take time to develop. We then quickly got into a discussion about the importance of developing such first-generation drug candidates in general, and we soon agreed that their value also lies in providing the foundation for more potent and specific follow-ons (not only targeted SNALPs, but also more potent lipids with a larger therapeutic index), at the end of which there may be cancer treatment strategies without the need for chemotherapeutics altogether.

The ongoing phase I trial is an open-label, non-randomized study aiming to enroll about 55 patients with primary and secondary liver cancers. First results on the pharmacology and biomarkers are to be presented at the upcoming ASCO meeting. This presentation should provide important insights into the functional knockdown of VEGF and KSP, and the future development path of this drug candidate. In addition to differentiating between primary and secondary and geographic differentiation of future trials (-->Takeda for Asia?), additional biomarkers (e.g. based on mutation status of a range of cancer-related genes or more dynamic biomarkers such as microRNAs) may help to further dissect the patient populations into those that are expected to respond best to VSP02 and select the most promising companion drugs for phase II studies. In the end, while the rationale and pre-clinical results are sound, only clinical experience will tell to what extent an innovative drug candidate such as VSP02 will be able set in motion the complex chain of events leading to the reduction or even destruction of liver cancer.