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.

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.

Conclusion

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.

Calando’s RONDEL RNAi Therapeutics Delivery Promising, but CALAA-01 Prematurely Entered into Clinic

[Part 1 of a 3-part collaborative series with Tobias Wolfram on the first notable attempts at RNAi Therapeutics for solid cancers that have entered the clinic]

When during the company's R&D day the CEO of Alnylam, John Maraganore, highlighted Calando’s cyclodextrin-based siRNA delivery technology (RONDEL) as one of the noteworthy non-SNALP systemic siRNA delivery technologies out there, it certainly piqued my interest. This is not least because any cash infusion and longer-term commitment by a partner like Alnylam would do wonders for the parent company of Calando, Arrowhead Research which is a conglomerate of early-stage, IP-focused business units and has just barely scraped by bankruptcy through a diet of cutbacks. While I always remembered the maturity of RONDEL, developed in the Mark Davis lab at Caltech, to be years behind SNALP, Tobias and I decided to to take a closer look at the development path of CALAA-01, the first clinical RONDEL delivery candidate and also investment focus of reorganized Arrowhead Research.

When Tobias first heard of the technology, it struck him as a very elegant, because simple, and modular method to formulate targeted nanoparticles. In fact, there are not many targeted nanoparticle siRNA delivery approaches where the components supposedly can be assembled by the pharmacist just before patient administration. RONDEL siRNA delivery consists of mixing together siRNA, a short cyclodextrin-containing polycation, and adamantane-coupled PEG stabilizers some of which carry a transferrin ligand, so as to create 60-80nm particles. These particles were rationally conceived to satisfy a range of pharmacologic and formulation considerations. Their suitability for solid cancer relies on the enhanced permeability and retention (EPR) effect of nanoparticles with reasonably long circulation times (here supposedly achieved by PEG stabilization), the ability of the particles to be taken up into cancer cells by transferrin receptor-mediated endocytosis and their subsequent release into the cytoplasm in a pH-dependent manner.

Unfortunately, what we soon came to realize was that while the concept is a very nice one indeed, the particles, particularly CALAA-01, remain to be better characterized both physically (shape, uniformity, storage and biological stability etc) and for their RNAi knockdown ability in vivo. For example, knockdown of RRM2, the target of CALAA-01, and subsequent tumor inhibition have not been demonstrated in a convincing in vivo system. Instead, knockdown efficiencies have largely been limited to in vitro studies. Moreover, these involved siRNAs that were selected with what today would be considered outdated methods and probably as a result were not very potent. The in vivo studies were essentially limited to pharmacological investigations, with the combination of in vitro efficacy and in vivo pharmacology forming the rationale for moving CALAA-01 into the clinic. Moreover, even when considering only the pharmacology, measures such as biodistributions and circulation times did not fit the model which may be explained by nanoparticle instability in vivo, something that really needs to be investigated further. Also, since the siRNAs were unmodified it strikes me as rather strange that no innate immune induction and only moderate adaptive immunity were reported.

What I found to be a valuable take-home message from those studies, although according to Tobias’ liking resting too much on indirect evidence, but not necessarily data obtained with the CALAA-01 clinical candidate, was that the utility of the targeting ligand appeared to be in increasing the cellular uptake of particles with little positive surface charge, less so in skewing the biodistribution towards the solid cancers per se. This could also be because the particles were cleared relatively rapidly from circulation, mostly into the kidney and bladder, which raises further questions about the purity and integrity of the particles. In this light, the mention of nanoparticle assembly by a pharmacist may also be interpreted a necessity due to storage problems of fully formulated particles. Nanoparticle assembly is notoriously sensitive to even slight changes in parameters such as temperature, speed of mixing etc so that it would be preferable for the physician to just administer the drug without the need for prior handling. It is therefore unfortunate that results from long-term storage and robustness of the formulation method were not presented. Nevertheless, an enhanced cellular uptake through the addition of a ligand could critically increase the therapeutic index of a cancer RNAi Therapeutic, and the modular nature of the RONDEL system should easily facilitate such additions.

Our assessment that CALAA-01 was probably entered into the clinic too early naturally rests on the publicly available data only. The publication dates of the relevant data, however, strongly suggest that they indeed represent the relevant data points and considering the financial situation of Calando, it would not appear that much were to be gained by holding back on positive data. This is by no means to belittle what otherwise is very rich science. Unfortunately, it is here that the tension between corporate demands for advancing a pipeline and the need to sufficiently advance the science is most evident and in the end risks harming both objectives. Optimistically, completion and evaluation of the CALAA-01 phase I trial will allow for valuable insights into the performance of RONDEL delivery in man for the benefit of any follow-up programs. Without any such strong data, it is questionable however whether the promise of RONDEL as a differentiated and flexible platform for RNAi Therapeutics delivery alone will be enough to make Arrowhead Research a good RNAi Therapeutics investment.

PS: To expand on the latter point, Tobias and I also discussed that, in general, it is easy to caution against entering RNAi Therapeutics candidates into the clinic early and dismiss such as a move of desperation. On the other hand, the case can be made that, when it comes to RNAi Therapeutics as a broadly applicable platform, clinically evaluating candidates which for example would not be expected to effect large knockdowns can be justified in that the data coming out of these studies may provide timely data invaluable for follow-up programs using very similar delivery approaches. Although investors will rightly fear the costs of a failed trial particularly for small biotech companies, such data may be valued more highly by a potential Big Pharma partner. This argument receives added weight in an environment like now where it is very difficult to raise capital from the public markets, and partnering is the primary means for small biotech to obtain capital at acceptable terms.

Combining Forces to Understand RNAi Therapeutics Inside Out

This entry is first a PR on a business development of the blog itself, but also reflects what should be the next major value-driver in RNAi Therapeutics after the liver: solid cancers.

Solid cancers are a very attractive target for RNAi Therapeutics because of its unmet needs and because of the so called Enhanced Permeability and Retention (EPR) effect of solid tumors which means that it should be possible to target them with siRNA-containing nanoparticles. Getting there, of course, is only half the story. The particles need to navigate their way through the extracellular matrix of tumors, latch onto the cancer cells, be taken up, and then finally be released into the cytoplasm. This requires a good understanding of nanoparticle-related chemistry and physiology, the biology of the extracellular matrix of cancer tissues, and cancer cell membrane biology.

Enter Tobias Wolfram. I have known Tobias since my days studying biology in Heidelberg (10 years ago now!) and have since been impressed by his enzyclopaedic knowledge of not only biology, but also history, psychology, economics and what not. He spent much of his time as a teenager with science projects and in molecular biology labs and won national prizes, at a time I did not even know that PCR existed. Since then he has become a truly multi-discliplinary scientist spanning the subjects of biology, chemistry, and material physics. Right now, he is at the Max-Planck-Institute for Metals Research in Stuttgart, the home of German engineering, where Tobias employs precisely engineered nanometer-patterned substrates for studying the interaction of cells with the extracellular matrix and their use for cell-based diagnostics. I visited him there two weeks ago to talk about working more closely together on the topic of RNAi trigger delivery.

In an experiment, we have decided to combine his expertise in getting molecules to cells with my understanding of the molecular biology of RNAi inside cells, to hopefully provide more insightful blog entries on the topic of RNAi Therapeutics delivery, with an initial emphasis on solid cancers. With Calando, Alnylam, and Silence Therapeutics having active programs in solid cancers, we will, over the next couple of weeks, start by taking a look at each of the applied technologies.