Merck Paper Reveals Interest in GalNAc-targeted RNAi Therapeutics

Merck’s efforts have to be considered to be the strongest in RNAi Therapeutics among Big Pharma.  Its RNAi Therapeutics strategy so far, however, has consisted largely of trying to replicate the most promising technologies in-house. 

The latest publication by Merck RNAi scientists on the expression pattern of the asialoglycoprotein receptor (ASGPR1; Shi et al. 2013) confirms this as ASGPR is the target receptor of the two most advanced SNALP alternatives for gene silencing in the liver: the trail-blazing DPCs by Arrowhead Research (first use of GalNAc-targeted RNAi Therapeutics in Rozema et al.2007) and more recently the GalNAc-targeted siRNA conjugates by Alnylam (what these really seem to be will be covered in an upcoming blog...so stay tuned!).

Surveying ASGPR expression levels

When developing ligand-targeted therapeutics, it is important that the corresponding receptor is present on the target cell population.  Especially when targeting cancer, it can be difficult to find receptors that are not only present in large quantities, but also throughout the cell population.  

In the case of liver cancer (hepatocellular carcinoma/HCC), this question has occupied the RNAi field ever since the finding that SNALPs work really well for knockdown in normal liver due to uptake mediated by the LDL-receptor.  But is this mechanism also present on cells in liver cancer, a cancer of high unmet need where RNAi Therapeutics could have the biggest impact in oncology near- to midterm?

 

The Merck scientists set out to answer essentially the same question, but instead of interrogating LDL-receptor expression, they wanted to know about ASGPR expression on HCC cells.  Using tissue microarrays, they were able to test an impressively large set of 100s of tissues, including healthy human livers, liver cancer biopsies, and biopsies for other hepatic diseases such as viral hepatitis, chronic active hepatitis and cirrhosis.

Despite some apparent limitations with the tissue microarrays (e.g. normal liver samples were often marked as false negatives despite the known very high ASGPR expression level), the results seem to confirm that liver cancers in general have a tendency towards lower ASGPR expression and that inter-sample heterogeneity is comparatively large (some liver cancer samples had much more ASGPR expression than normal liver).  

This suggests that just as in the case in breast cancer where treatment decisions are often based on receptor expression levels, ASGPR-targeted liver cancer RNAi Therapeutics should also be combined with a companion diagnostics for ASGPR. 

For those curious about the status of ASGPR expression in chronic HBV since Arrowhead’s exciting ARC520 program for this indication involves a GalNAc-targeted melittin-like peptide, relax: livers infected with HBV express ASGPR just as well as normal livers.

Home-brew versus licensing

Based on the literature and conference presentations, it is reasonable to assume that Merck is well behind Arrowhead, and even Alnylam in developing GalNAc-targeted RNAi Therapeutics.  This begs the question, as it has in the case of SNALP, why does Merck not take a license or even acquire the original?

Of course, it always takes two for a deal, but given the financial capabilities of a Merck, if it wanted access, it could get it.

The likely explanation is that Merck's strategy in replicating technologies in-house is to first identify target technologies by validating them and then to develop viable IP workaround solutions.  In fact, in the apparent absence of a broad gate-keeping patent estate around GalNAc-targeted therapeutics, ASGPR has been a recognized drug target receptors well back into the 90's, there should be relatively little restrictions on the use of GalNAc per se.

Instead, a competitive advantage is largely gained through specific know-how like how to best link the GalNAc ligand to the oligonucleotide payload and synthesize the molecules cost effectively. The latter issue came to my attention recently when access to 'proprietary process for manufacturing GalNAc conjugates' from Alnylam was mentioned as the top corporate highlight in the quarterly update provided by Regulus Therapeutics.

For the aficionados, the triantennary GalNAcs as practiced by Alnylam are much more costly to synthesize than single GalNAcs as in the case of the liver-targeted DPC versions (where high affinity through multivalency is achieved by having multiple single GalNAcs along the DPC).

This situation is not unlike other areas in the drug development industry.  Take for example antibodies where, despite the various patent battles, there have been a number of commercially viable platforms based on specific libraries or optimization methods.  Nevertheless, despite this apparent freedom-to-operate, the way by which Big Pharma ended up gaining access to monoclonal antibodies was not by way of successfully developing them in-house, but by acquiring them.

I don't expect this to be any different in RNAi Therapeutics.

Mirna Therapeutics Brings First MicroRNA Replacement Therapy into Clinic

It has been a long wait, but 5 years following the initiation of anti-miR122 development for the treatment of HCV infection by Santaris, a second microRNA Therapeutics has now entered clinical development.  MRX34 by Mirna Therapeutics is a miR-34a replacement therapy for the treatment of liver cancer or cancers with liver involvement.  

MicroRNA replacement therapy is technically very similar to RNAi Therapeutics.  It involves adding to cells naturally occurring microRNAs to orchestrate typically a range of processes deemed to be therapeutic.  It is added to cells in the form of double-stranded RNA triggers very much alike RNAi triggers and can therefore largely utilize the same types of delivery approaches.  The delivery technology for MRX34 is the NOV340 SMARTICLE technology owned by Marina Biotech.  This liposomal delivery technology is distinct from SNALP, most notably by employing ionizable ‘amphoteric’ lipids, lipids that can take on both positive and negative charge depending on pH, and not ionizable cationic lipids, lipids that merely become positively charged at acidic pH, as in the case of SNALPs. 

An extensive literature supports miR-34a as an exciting microRNA for replacement therapy in cancer rivaling the most famous let-7.  Accordingly, miR-34 emerged as Mirna Therapeutics’ top priority based on extensive screening conducted at Ambion (now Life Technologies) from which Mirna Therapeutics was spun out.  Under transcriptional control by the guardian of the genome, p53, its pleiotropic effects range from cell cycle inhibition, to counter-acting anti-apoptotic mechanisms and to sensitizing towards chemotherapy.  The choice of liver-related cancers was thus not based on cancer biology (many cancers cancers would apply), but largely a function of where the company thinks NOV340 can deliver to.

This to me raises again the question of whether liver cancer is closer to normal liver or whether it is closer to solid tumors in general in terms of delivery.  MRX34 thus follows Alnylam’s reasonings as manifested by ALN-VSP02, but goes against what is known about blood supply differences between normal liver and liver cancer and what is practiced by the likes of Tekmira and Dicerna.

The phase I study will be a typical dose-escalation study seeking to determine the maximally tolerated dose.  Given the importance of delivery, close attention should be paid to the pharmacokinetic and biodistribution data from this trial.  These should start to become available in the first half of 2014.

The development is positive for at least two other companies.  As the provider of the delivery technology, Marina Biotech obviously stands to gain financially and otherwise from such clinical milestones.  Whether it will be sufficient to pay off its debts that have come due and consequently avert bankruptcy is an open question.  Due to the financial distress, Marina Biotech struggled to enter into deals giving it fair compensation for its technology.

Although the most direct competitor to Mirna Therapeutics, InteRNA as the other major microRNA replacement company in oncology should also benefit from the initiation of the phase I study as it helps to validate its approach.  The question will be whether among its stable of microRNA replacement candidates, there are some with as robust activities as miR-34a.   

BMS Partners with Santaris

In other recent news related to microRNA Therapeutics, Big Pharma Bristol-Myers Squibb partnered with LNA antisense company Santaris under which the Danish company collected $10M upfront.  Although the press release did not specify much the aims of the the alliance, referring to RNA Therapeutics broadly, a few factors speak in favor of microRNAs being involved.  

Most notably, BMS had terminated an phosphorothioate RNaseH antisense collaboration with ISIS Pharmaceuticals.  As Santaris in turn had terminated their PCSK9 phosphorothioate RNaseH antisense program as well, most likely due to kidney toxicity (--> phosphorothioate chemistry as also in Prosensa; van Poelgeest et al. 2013), the first suspicion that BMS sought out Santaris as a more potent PCSK9 alternative becomes less compelling.  Moreover, ISIS Pharmaceuticals is suing Santaris over US patent infringement of RNaseH technology which should hinder the ability of Santaris to enter into relationships with US companies for RNaseH antisense purposes.  Of course, the deal could also indicate that a settlement is in the making...

Cancer RNAi Therapeutics Moving On

As RNAi Therapeutics targeting genes expressed in the liver, especially for orphan indications, have captured much of the recent interest in the technology, it is time to once again pay closer attention to RNAi Therapeutics in the oncology space.

Atu027 and TKM-PLK1 Moving into Phase II

Silence Therapeutics have just announced that they got the green light from the German regulators to test Atu027 in combination with small molecule gemcitabine in pancreatic cancer.  Following a short phase Ib to establish the safety of the combination (note: the original phase I was a monotherapy trial), the plan is to rapidly move into phase II which will aim at establishing proof of concept for efficacy.  Depending on the funding situation, it is possible that the company will expand Atu027 into other indications in phase II, possibly in collaboration with private investigators in the UK.

Clearly, the strength of Atu027 is its safety profile so far, one that has surpassed my own expectation for this positively charged lipoplex formulation.  The clinical attraction meanwhile is that Atu027 aims at a quite novel anti-cancer mechanistic approach by inhibiting metastatic spread by fortifying the vascular endothelia. 

The challenge with Atu027, however, is that the gene target, PKN3, remains a relative black box.  This not only demands a certain leap of faith in mechanistic terms, but also complicates the clinical development as biomarkers and other tools are not readily available.

This is quite different from TKM-PLK1, the cancer RNAi Therapeutics by Tekmira.  Polo-like kinase 1 is a hot target, but specificity/safety issues have so far dogged drug development efforts with small molecules, most notably represented by Boehringer-Ingelheim.  By contrast, the SNALP RNAi delivery approach promises to be more cancer tissue targeted and consequently should avoid some of the dose-limiting toxicity observed so far, especially the hematologic ones.

TKM-PLK1 has flown a bit under the radar and the phase I update late last year did not capture much attention.  This is probably because the response rate did not seem too much to get excited about.  However, as Ian MacLachlan, the CSO of Tekmira, pointed out at the AsiaTides meeting last week, it was a small dose-escalating phase I trial (20-30 patients) in advanced solid cancer patients and only four patients actually received TKM-PLK1 for two or more cycles (6 infusions over 2 months) at 0.6 mg/kg or above, dose and durations you would think would be required for advanced cancer patients to get a chance to respond in the first place. Notably, two out of those four showed signs of efficacy: one partial response and one with stable disease after more than 6 months on study drug.

In light of that, the phase I profile of TKM-PLK1 supports my view that it could be the foundation for an exciting PLK1-related cancer franchise for Tekmira and I look forward to seeing the full results presented at the upcoming AACR meeting.  My attention will be particularly focused at any direct measures of target knockdown.  Beyond that, with the company’s healthy financial situation, I look forward to a rich phase II strategy involving a number of solid cancers and innovative study designs such as patient selection based on genetic criteria.  As good as Tekmira's science is, they now also need to be as proactive and innovative on the clinical front.

ALN-VSP02 and CALAA01 Falling Behind

As TKM-PLK1 and Atu027 are moving into phase II, fellow early movers in oncology RNAi ALN-VSP02 and CALAA01 are falling behind. 

The next step for ALN-VSP02 will be a phase I in hepatocellular carcinoma (‘real’ liver cancer) in China under a license by Alnylam to Ascletis.  As noted by Patrick Lu of Sirnaomics at AsiaTides though, it would be a mistake to believe that the Chinese regulatory environment is any less demanding than in the West.  Au contraire, it seems that much more paperwork is required to file an RNAi-realted IND as the authorities there are keen on building their expertise in this new technology.  Expect a delay of at least 6 months from IND application to the green light to go ahead, after all the paperwork has been submitted.  For Tekmira watchers, I expect the $5M milestone thus in 2014, not in 2013.

While ALN-VSP02 still has a pulse, despite of the fact that it lacks the long circulation thought to be necessary for cancer uptake (note: delivery to liver cancer is a very different animal from delivery to normal liver; TKM-PLK1 is a long circulating SNALP), RIP CALAA-01 by Arrowhead Research.  Having said that, the value of CALAA-01 to Arrowhead Research has not been lost entirely as the company has filed for a patent covering a low/high dose strategy of minimizing immune stimulation.  

As the recent high-profile pulling from the market of a pegylated peptide (Omontys by Affymax) due to hypersensitivity, even anaphylactic reactions demonstrate (and also the infusion reactions reported by Alnylam in their SNALP RNAi Therapeutics trials), administration-related hypersensitivity reactions are an issue for the entire drug development arena.  In order to rescue the value of a number of blockbuster franchises, I expect that changing administration schedules could be one area attracting increased attention.  A patent here could have unexpected value.   

A New Crop of Cancer RNAi Therapeutics

Behind TKM-PLK1 and Atu027, a new crop of cancer RNAi Therapeutics is emerging.   Dicerna has signaled that it will finally move into clinical development (Q1 2014) with a liposomal Dicer-substrate formulation with a focus on liver cancer.  Of note, the liposomal formulation process is quite distinct from that of SNALP involving sequential core and envelope formation steps, potentially amenable to ligand-directed targeted delivery.

Another program worth following is another one for liver cancer, the subject of a collaboration of Korean company Bioneer andSanofi-Aventis.  The attraction of that program is that the nanoparticle here, SAMiRNA, comprises a single molecule, even to the extent that the RNAi trigger and delivery components can be manufactured on a single column.  Although I’m opposed to the fundamental notion that the ease of route of administration and the manufacturing process should drive drug development, don’t underestimate the attraction of meeting idealistic product profile when it comes to partnering with Big Pharma.    

The efforts above are a reminder that cancer could be an even bigger commercial opportunity for RNAi Therapeutics than liver-related indications.  The genome-wide targeting opportunity with RNAi Therapeutics has not been lost.  The pharmaceutical industry is watching.

Register for the GTC RNAi Research and Therapeutics meeting in San Francisco today (June 20-21).  Get a free RNAi Therapeutics blog T-shirt and 20% discount on registration by entering discount code 'RNABLG13'.

ALN-VSP02 Was a Good Start, but Directs Focus Towards Improved Formulations

Alnylam recently published detailed phase I results from its RNAi Therapeutic candidate to treat liver cancer, ALN-VSP02.   Although results from this study had already been reported, the new paper by Tabernero and colleagues provides an insightful warts-and-all view of Tekmira's SNALP generation 1.0 (short-circulating DLinDMA particles) in a solid cancer.  The results show that while ALN-VSP02 did achieve a number of important goals, including evidence of tumor response and RNAi activity, it will be necessary to turn to the newer SNALP formulations for more robust efficacy and improved safety.

New Insights on ALN-VSP02 Activity

Previously, Alnylam provided clear evidence of RNAi activity using the 5' RACE assay.  It is now clear, however, that this was only based on 3 positive biopsies out of 15 investigated in this regard, and only for the VEGF, but not the KSP target gene.  Consistent with this less than robust RNAi activity, there was no convincing evidence of target gene knockdown and no spindle abnormalities as would have been expected from KSP knockdown.

New Insights on ALN-VSP02 Safety  

The present paper also provides valuable insights into the safety of a SNALP 1.0.  As was reported before, infusion reactions were seen in a minority of patients and this was readily addressed in practical terms by slowing the rate of infusion.  Interestingly, the infusion reactions appear to be related to complement activation, not cytokine activation.

Some cytokines were also upregulated (rarely TNF-alpha, however), especially above 0.4mg/kg, despite the use of modified siRNAs.  The evidence thus increasingly points towards the DLinDMA lipid itself to be the culprit.

Finally, ALN-VSP02 caused considerable spleen toxicity, especially with prolonged dosing.  In the one complete responder in the study, 90% of the spleen was reduced after receiving 50 doses of ALN-VSP02.  This is consistent with the preclinical monkey data.  Somewhat reassuringly for SNALP 1.0, this effect might be due to on-target efficacy, as the same lipid formulation with a luciferase siRNA did not have this effect in monkeys (note: the spleen is not an essential organ in humans, and I’d much rather have my spleen removed than cancer).

SNALP-based Cancer RNAi Therapeutics in the Future

Based on the above limitations, it is important that future SNALP-based cancer RNAi Therapeutics include the following improvements, essentially all of which have been achieved over the last 6-7 years since ALN-VSP02 had been selected as a development candidate: firstly, a more potent and less immunogenic lipid is required (there should be multiple to choose from now); secondly, the SNALP particles should be less than the 80-100nm with ALN-VSP02 and include more stable PEG-lipids for improved, i.e. prolonged circulation times to better take advantage of the EPR effect of solid tumors (TKM-PLK1 includes such stable PEG-lipids); and finally, as spleen safety might also be affected by lipid accumulation along the endosomal-lysosomal pathway, enhanced lipid biodegradability may be desirable.

ALN-VSP02 has been put on hold by Alnylam for development in the Western markets.  Last year, however, Alnylam licensed development and commercialization rights to Ascletis for the Greater China market where liver cancer (hepatocellular carcinoma, HCC) is prevalent.  The next clinical study by Ascletis is expected to involve more HCC patients who, unfortunately, have been almost entirely missing in the phase I study of ALN-VSP02.  Despite the challenges that ALN-VSP02 will face in HCC, innovative RNAi Therapeutics have an important role to play in the future treatment of HCC, a disease that is proving again and again hard to manage by more conventional approaches as Celsion’s disappointing results (radiofrequency ablation with liposomal doxorubicin) today showed. 

ASCO for RNAi Therapeutics in Line with Expectations, but Curious Nevertheless (Part I- ALN-VSP02)

Alnylam and, finally, Silence Therapeutics have by now disclosed the latest results from the phase I studies of their RNAi Therapeutics candidates, ALN-VSP02 and Atu027, for solid cancers at this year's ASCO.  As the studies have thus more or less come to a conclusion, one has to say that, although clearly nothing spectacularly positive came out of them, these two candidates warrant further evaluation and their safety and pharmacokinetic profiles augur well for the other candidates based on the same SNALP and AtuPLEX delivery platforms.  

With most of the results known, I will therefore focus my discussions of the RNAi Therapeutics ASCO 2012 on the new insights and include some comments on recent corporate maneuvers of the companies involved.

  

ALN-VSP02 Provides First Insights into SNALP Repeat Dosing (and yes, SNALP technology belongs to Tekmira and AlCana is an instrument of Alnylam)

The extension study with ALN-VSP02 that was the subject of Alnylam's ASCO poster presentation included 7 patients with cancers involving the liver that had been rolled over from the main study (results presented a year ago here) and provided more insights into the long-term safety/tolerability of SNALP delivery technology.  It was good to see that even very sick patients could be given 1^st generation SNALP formulations at dosages of 0.7mg/kg or more every other week for quite prolonged periods of time (up to 2 years).  As the phase I results with ALN-PCS02 indicate, such concentrations will allow for solid gene knockdowns in normal liver cells with some of the improved SNALP formulations. When I first got excited about SNALP delivery technology 6 years ago, one of the main risks that I saw was around repeat-dosing including antibody formation against the PEG component and other immune stimulations upon repeat dosing.  On that front at least, the ALN-VSP02 study, especially based on the PK data, was a clear pass. 

Certainly, there remains controversy around whether the precautionary transient pre-treatment with immunosuppressives (e.g. corticosteroids) that was applied here and may have aided in achieving these results is a realistic product feature of a RNAi Therapeutics.  As I have said before, personally I believe that for severe indications such as many cancers, this is mostly a minor practical inconvenience; it may, however, impede their adoption for less severe conditions in larger patient populations.

One new safety-related item worth mentioning is the reported effect of prolonged ALN-VSP02 administration on spleen size and Alnylam’s interpretation thereof.  As we know, the spleen is a non-essential organ in humans and the vast majority of us would not notice if we were without one.  Thus, the reduction in spleen size observed in this trial with prolonged treatment was not considered a severe adverse event or dose-limiting toxicity.  Spleen toxicity would not really be surprising for nanoparticle-formulated drugs such as ALN-VSP02 (and also phosphorothioate-based oligonucleotide therapeutics) as they are often observed in animal studies.  I therefore considered it possible that it could be a platform-based toxicity.  

Alnylam, however, put forward a more optimistic interpretation by claiming that it is more likely an on-target toxicity, meaning that knockdown of the proliferative gene KSP in the spleen was responsible for it.  Considering the biodistribution of SNALP and the recently highlighted data on SNALP and other LNP-mediated gene knockdown in immune cells that are enriched in lymphoid organs such as the spleen, this is certainly a plausible explanation as well and is further supported by the new monkey data presented at ASCO.  Adding to this confusion is the fact that ‘Alnylam’ has been developing 3^rd generation SNALP technology to ameliorate spleen toxicity.  So if the spleen shrinkage was no general effect, why bother with 3^rd generation?

Aside from these scientific developments, including a very nice complete response by RECIST in an endometrial cancer patient with multiple liver mets which had already started to respond during the main phase of the trial, my interest was equally piqued by the fact that the press release by Alnylam lacked any mention of the fact that ALN-VSP02 involves critical delivery technology by Tekmira and that key IP of a siRNA ingredient may also well belong to Tekmira, pending the outcome of an Interference proceeding in the US.  If you look for even more support for Tekmira’scontention that Alnylam has been aiming to marginalize the company, and as yet another of Alnylam's main rivals, Marina Biotech, looks like it is going out of business (my bet is that they will participate in some kind of consolidation to survive in another form), look no further.  The PR is only rivaled in silliness by the misleading one issued by Alnylam’s Canadian subsidiary, Alnylam Canada (AlCana), two weeks ago on the status of the Tekmira-Alnylam showdown in Canada (the misleading part being in quite obviously trying to suggest that the Canadian courts reversed all earlier decisions that went against AlCana, including handing over the allegedly stolen documents back to their rightful owners, Tekmira). 

But the real clincher of that PR was the following: despite of pointing out the elephant in the room, namely that the Canadian proceedings are part of a wider confrontation between Tekmira and Alnylam, Alnylam Canada hilariously and awkwardly at the same time failed to mention Alnylam at all- in the entire press release!  If they believed that this would make AlCana, a de facto academic lab financially supported and strategically instructed by Alnylam all along, any more independent of Alnylam, try again (note: a key element in the litigation is that Alnylam established an ‘independent’ company- AlCana- to gain insights into key Tekmira trade secrets and know-how thereby minimizing legal liability if caught).  More unintended humor was provided by stating that the Canadian case had been post-poned until resolution of the US case between the ‘corporate’ parties, implying that neither AlCana nor the courts consider AlCana to be a true, independent corporation.  Although I have not had the chance to read the primary court documents myself, the decision strongly suggests that the Canadian court took pity on the AlCana scientists and does not like to see them being thrown under the bus by their de facto employer, Alnylam.

In part 2, I will cover the latest data on Atu027 and share some thoughts on the confusing corporate strategies of that company.

MicroRNA-26 yet another Small RNA in the Fight against Liver Cancer

When you combine the fact that current systemic small RNA delivery technologies should work best for the liver and solid cancers, that RNAi opens up many of the well validated, but hitherto un-druggable cancer targets, and that endogenous small RNA regulatory pathways (microRNAs in particular) turn out to play central roles in cancer biology, then it should not come as a surprise that various Small RNA Therapeutics approaches are poised to greatly advance the care of liver cancer patients. Surgical resection is the most common treatment, while the pleiotropic small molecule inhibitor Sorafenib that has been shown to increase median survival from 7.9 to 10.7 months is the most advanced drug for this treatment. Clearly, in light of this and the over 600,000 deaths from primary hepatocellular carcinoma (HCC) alone each year, many of them in East Asia (HBV-related), and many more deaths from the metastatic spread of other cancers to the liver, the unmet medical need is significant.

This potential has not been lost on the RNAi Therapeutics industry. There are now a number of RNAi Therapeutics liver cancer programs already in or approaching the clinic, most prominently Alnylam’s VSP-02, but also others such as one by mdRNA. At the same time, there is increased validation for microRNA-based therapeutics for liver cancer both of the antagonist type (e.g. Rosetta-Regulus collaboration) and the agonist type. In the case of the latter, two recent high-profile papers in Cell (Kota et al., 2009: Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model) and the New England Journal of Medicine (Ji et al., 2009: MicroRNA Expression, Survival, and Response to Interferon in Liver Cancer) suggest that microRNA-26 mimicry is a serious contender.

Kota and colleagues got started after making the observation that miR-26a is strongly down-regulated in a mouse model of liver cancer driven by the myc oncogene. Importantly, miR-26a levels were subsequently found to be also reduced by about 50% in liver cancer samples compared to the matched healthy liver tissue. While global down-regulation of microRNAs is a well known general property of cancer, what made miR-26a a particularly attractive candidate for microRNA replacement is that a) it is a broadly expressed microRNA and over-expression in non-target cells therefore should be well tolerated; and b) its over-expression in a liver cancer cell line decreased cell proliferation which was attributed to miR-26a directly targeting the cell cycle regulators Cyclin D2 and E2.

They then chose AAV technology to deliver a microRNA mimic to the livers of the same mouse model. AAV delivery to normal liver can be extraordinarily efficient, with essentially 100% transduction using so-called self-complementary vectors in mice. The track record for HCC has been more mixed, but using a self-complementary vector of the AAV8 serotype the authors achieved more than 90% transduction. To their great satisfaction, tumor growth was greatly retarded following systemic administration of the mimic compared to an expression vector in which the microRNA cassette had been deleted (60% tumor burden to 20% tumor burden). Importantly, whereas there was wide-spread apoptosis in the liver cancer, the surrounding normal tissues and more distant tissues with a high proliferative index (e.g. testis) appeared to be untouched. This was the first demonstration of efficacy for a systemically administered microRNA mimic that was not targeted against the cancer-initiating oncogene itself.

Further support that miR-26a is a good candidate for mimicry in liver cancer comes from a bioinformatic study just published in the New England Journal of Medicine which confirmed in a population of HBV-related HCC in China the down-regulation of miR-26 in liver cancer. Moreover, as predicted by the Kota et al. study, liver cancer patients with the least amounts of miR-26 had a worse prognosis. On the other hand, it is the same miR-26 lo patients that benefited most from interferon-alpha therapy. Such a finding may have its clinical use since for a drug with borderline efficacy like interferon in liver cancer. Being able to exclude those patients that will not benefit from a given drug may give them an opportunity to try other drugs or at least be spared of the side-effects. When used during drug development as a companion diagnostic, of course, such a test would moreover have major benefits for increasing the success rate of developing a drug candidate. Companies in the microRNA Rx/Dx space like Rosetta Genomics, Asuragen, and Regulus may want to take a look at being part of a companion Dx-Rx development program where patients are selected for a miR-26 mimicry liver cancer trial based on their miR-26a status. More generally, I shall look forward to seeing a wider adoption of microRNAs as companion Dx and, looking out from my soapbox, even a tighter integration of microRNA Dx/Rx capabilities.

In summary, miR-26a mimicry is yet another promising small RNA approach to treating a disease with a high unmet medical need, not just sometime in a distant future, but very tangible. Again, the lesson here is that once a given organ can be addressed by small RNA delivery, a flood of therapeutic targets immediately become available to keep Small RNA Therapeutics busy for some time on this one organ alone.

A Framework for Progressing RNAi Therapeutics into the Clinic

This week was quite busy for the RNAi Therapeutics space with important corporate developments for CytRx/RXi (succesful spin-out/IPO), Nastech/mdRNA (announcements of patents filings and scientific advisory board), Silence Therapeutics (extension of AstraZeneca collaboration to Atuplex delivery), and Arrowhead Research/Calando (filing of first cancer IND). I hope to provide a wrap-up soon, but as I am contemplating the further developments in delivering RNAi to the liver, I would like to use the clinical development of liposomal RNAi as a case example of how one might think about development timelines for RNAi Therapeutics in general. While this discussion focuses on SNALP/liposomal-siRNAs just because it is an area that I am following more closely than others, it can easily be translated to for example to PEIs and delivery to the lung, or for that matter AAV/lentivirus and delivery to the brain.

RNAi Therapeutics are thought to speed up drug development, because once a target is identified, finding an appropriate siRNA should be a straight-forward process. However, it is clear that right now developing ways to get the RNAi triggers into the cells is the rate-limiting factor, meaning that the benefits of rapid drug development won’t necessarily all be realized for a good fraction of the first crop of RNAi Therapeutics. In order to assure that the next batch may more readily move through development, it will therefore be important to establish a number of predictable delivery technologies for the different target tissues. Once such a delivery system has been identified for a given tissue, virtually any gene of interest could be targeted in a manner that reduces development risk mainly to target risk (should be quite low given the virtually unlimited target space of RNAi) and safety profile of the particular RNAi trigger (particularly due to the less predictable off-targeting effect).

The question for Alnylam now is whether to choose liver cancer or hypercholesterolemia as the indication for their first SNALP/lipidoid-RNAi IND. Assuming that they feel quite confident with the gene targets and that they intend to use the same delivery formulation since the target tissue is fairly similar (normal versus tumour liver tissue), why choose one over the other? As we know, safety and finding a SNALP/lipidoid formulation with a good therapeutic index is as much of a concern as is efficacy. One could argue that starting with liver cancer may be a less risky business decision since the length of dosing and nature of the disease should be more forgiving in terms of the tolerated safety profile. By contrast, to derive a cardiovascular benefit through cholesterol lowering requires long-term chronic treatment which is why tolerability is so important for this indication. On the other hand, the PCSK9 program would provide a host of information as to the efficacy of this novel delivery system. Circulating PCSK9 levels could be readily measured and this information be used to adjust dosing strategies not only for the late-stage development of this particular program, but also all the other programs, including liver cancer, making use of the same delivery system.

I can imagine that the decision won’t be an easy one to make, but with improvements in the therapeutic index of SNALP-RNAi, the PCSK9 program becomes relatively more attractive. If successful, it should be possible to realize the anticipated advantages of RNAi Therapeutics in terms of increased development speed. Although true for drug development in general, from a technology and business development point-of-view it is therefore particularly important for the field of RNAi Therapeutics that its early clinical candidates, using unproven delivery systems, target genes where proof-of-concept can be established at an early stage.