RNAi Therapeutics Highlighted in Roche Media Day Presentation

Roche can be considered the bellwether of how Big Pharma/Biotech is looking at RNAi Therapeutics for rejuvenating their pipeline, and their moves will be closely watched by the investor community. I therefore found it extremely interesting what a Media Day presentation in 2009 revealed about their RNAi Therapeutics intentions. This presentation was given by Lee Babiss (President & Global Head of Roche Pharma Research) who has been a champion of RNAi Therapeutics at Roche. It is worth noting though that according to musings on the cafepharma discussion board, he has unfortunately left the company.

Roche sees RNAi Therapeutics as an integral part of their personalized medicine efforts conducted in concert with their diagnostics unit and which offers fast development timelines. Notably, doing the math, there were about 5 RNAi Therapeutics research programs in 2008 (0 in 2007) at what appear to be Lead Identification and Optimization stages. Relevant to RNAi Therapeutics, this portfolio of about 100 programs is jammed full with solid cancer programs which is THE poster child of personalized medicine (almost 20% of programs). As has been made public in a previous RNAiNews interview with, Roche intends to work with Tekmira to bring two RNAi Therapeutics programs into the clinic near-term, including one IND in 2010. According to slide 26 this is likely to be a program (‘MVD’) in metabolic disease as this has been the most advanced program (‘phase 0’) at the time of the presentation. ‘MVD’ is categorized under ‘metabolic disease’ and therefore could mean ‘macrovascular disease’. Type II diabetes, respiratory, and solid cancer RNAi therapeutics programs follow not far behind. It would not surprise me that the second program would be one for solid cancer, paralleling efforts at Tekmira (phase I SNALP-ApoB ongoing; solid cancer 2010 IND).

In delivery, it is, however, not all about liposomal delivery. Roche’s dynamic polyconjugates were highlighted in rhesus monkey studies which showed quite significant (>90%) and very prolonged (almost maximal at 3 weeks although this could be repeat administration data) knockdown, albeit at the relatively high doses of 20mg/kg. siRNAs coupled to antibodies were also highlighted in cancer applications which together with SNALP and DPCs may form Roche’s 3-legged stool of siRNA delivery.

Alright, this is it for me in 2009. I would like to thank everybody for reading some of the highly speculative stuff and who have made writing the blog particularly interesting to me either through your comments or by personal contact. I look forward to what promises to be a very exciting year in RNAi Therapeutics.

Rosetta Genomics Business Strategy Back on Track

Following the recent hiring of Kenneth Berlin as CEO, Rosetta Genomics is re-focusing their microRNA diagnostics efforts to leverage their extensive bioinformatic know-how and associated IP in the field. Having emerged as the leader in microRNA diagnostics churning out 3 microRNA tests in short order, initiatives such as betting the future on an early-stage blood-based colon cancer screening test and an one in plant biology ('Rosetta Green') first left investors perplexed and eventually disappointed. According to today’s press release and conference call, however, the company has decided to revisit its proven strategy of going back to their treasure trove of bioinformatic information and select new development candidates.

What is also remarkable is that, for the first time, the company is providing guidance about market size and clarity on pricing. This is a welcome change from previous years that had investors in the dark about expected revenues. In hindsight, it would appear that this was actually was due to lack of strategic planning. It is therefore expected that with the strong commercialization background of Mr. Berlin as former worldwide General Manager for the J&J molecular cancer diagnostics outfit Veridex, this essential aspect of Rosetta Genomics' business can only get better. Predicted revenues of only 2010 $2-4M based on 1,200-2,400 samples processed from their first 3 tests, however, represent business development challenges considering current cash levels of about $10M and a predicted $900k monthly burn-rate in 2010.

Mr. Berlin will therefore largely be measured for his ability to

a) penetrate the following predicted markets (annual numbers of potential tests US/ex-US):

1) miRview Squamous: >60k/250k; 2) miRview Meso: >60k/200k; 3) miRview Mets: >60k/300k;

b) develop 5 tests for which they already have identified the microRNAs, among which were disclosed the following tests and time-line:

1) an extended miRview Mets test (H2 2010 launch); 2) a test predicting superficial bladder cancer to become invasive (late 2011 launch and 350k annual market); 3) a test further differentiating lung cancers from fine-needle aspirates (2011 1M annual market)

c) achieve all this all the while minimizing shareholder diluation

As to the last point, I am curious whether, considering the first 3 tests have obviously been partnered already, Rosetta can further monetize their IP and know-how in the microRNA therapeutics area, and find a Roche-like partner for developing companion diagnostics. Because of trends making it more and more difficult to get reimbursement for home-brew tests, it is surprising that unlike Rosetta’s competitor Asuragen (companion Dx deals with Biogen and Merck), we have heard nothing about their plans in the high-value area of molecular companion diagnostics. Ongoing post-marketing studies for their present tests, however, should partly address the reimbursement challenge in addition to convincing patients/physicians to adopt the tests.

With the proposed strategic review, Rosetta Genomics should become a company worth considering again given their strong scientific foundation. It will take some time, however, to re-build confidence in management. After all, the misguided colon cancer ambitions did not seem to have been based on scientific progress from their exploratory published data, and it would not seem fair to blame the former CEO alone for all these blunders. Rosetta Green also needs more explanation. However, I do want to credit former management for having established the scientific engine behind Rosetta and having brought to market 3 out of the 4 currently marketed microRNA Dx tests. Privately-held Asuragen should also receive credit in this regard. This could also mean that people like Victor Ambros, Gary Ruvkun, and maybe 'even' (because plant biologist) my D.Phil. examiner David Baulcombe are even stronger contenders for a Nobel Prize for their discovery of micro- and other silencing small RNAs which in turn could give companies like Rosetta Genomics a significant boost in the near- to midterm.

Silence Therapeutics and Intradigm Merge to Offer Complete RNAi Therapeutics Package

Finally, the long wait has come to an end. The just announced imminent merger of publicly traded Silence Therapeutics (UK/Germany) with privately held Intradigm (USA) illustrates that consolidation has also arrived in RNA(i) Therapeutics. The stated objective is for the financially strapped firms to achieve a size sufficient to be considered a one-stop partner for a Big Pharma/Biotech (BP/P from now on) interested in RNAi Therapeutics, and also partly in a bid to enlarge their investor base. This approach is in part inspired by the successes of Alnylam and Sirna Therapeutics which had and have been very successful in monetizing their broad approach towards RNAi Therapeutics. While there are factors speaking for such an approach, times have somewhat changed in RNAi Therapeutics such that the established players such as the Roches and Novartis' are more likely to take a pick-and-choose approach in their partnering strategies. As such, companies that are focused on fewer technologies, especially in delivery, but then can get it right, may be preferred partners for these BP/Bs.

On the other hand, for a BP/B that views a platform partnership as a way to start testing the RNAi Therapeutics waters somewhat more extensively, I am thinking here of the likes of AstraZeneca, Boehringer Ingelheim and Aventis, an enlarged Silence Therapeutics may be worth a second look at least as a cheaper alternative to the gold-standard Alnylam Pharmaceuticals. The risk, however, that I see is that ‘broad’ could easily equal ‘lack of depth’.

As much as the desire to grow in size, the merger was driven by the desperate need of both companies. It is therefore highly likely that the merger will be approved as described in the Admission Document, also because of the holidays which makes it unlikely for a third party to make a too-good-to-refuse buy-out offer. Net of debt and after parallel fund raisings from existing Intradigm shareholders (about £5M) and a placing led by Nomura (about £9M), the combined company should soon have ~$20M in liquid assets, which will give the company about a year’s time to find a platform partner that is willing to part with some decent upfront cash. Especially with part of their operations now based in the US, it may also make sense to pursue a listing on the US markets for their future cash needs.

While the claim is to be a broadly positioned RNAi Therapeutics company, the combined company clearly has a strong bias towards cancer. Therefore, an alternative headline of this blog post would have been ‘Silence Therapeutics and Intradigm to Form Cancer RNAi Therapeutics Powerhouse’. It is here that I suspect most of the scientific synergies and value drivers of this merger will come from. I do not have to describe here the high unmet medical needs in cancer, and it is the unique promise of RNAi Therapeutics to go after the many thus far undruggable, yet well-validated cancer-related targets that makes it an area of such high value proposition to the industry. Since nanoparticles can be targeted to cancers, delivery should be possible: e.g. via the tumor vasculature, the Achilles heel of solid cancers, and antibodies to haematological malignancies. In this regard, Silence Therapeutics and Intradigm bring to the table cationic lipoplexes as a means to knock down genes in the tumor vasculature directly (Silence Therapeutics), also as supported by the literature, and a less validated cationic peptide polymer-based delivery technology by Intradigm ('PolyTran').

But will the proposed merger address the one area next to viable delivery that has been nagging both companies in the past...intellectual property? Clearly, combining two comparably weak patent estates do not a strong one make. A quick glance over what is issued and pending, however, suggests that the patent estates could synergize to a certain degree.

Silence (as most others) has been mostly troubled by the Tuschl and Kreutzer-Limmer patents that make it very difficult for them to find dsRNA lengths to exploit their issued Atu-siRNA design pattern without having to to get a license from Alnylam: blunt-ended dsRNAs with alternate 2’-O-methyl modified RNAs. Atu-027 for example, the combined company’s lead candidate for solid cancers that has entered phase I studies this summer, is a 23bp dsRNA and Tuschl I should be a formidable challenge here. Intradigm, like RXi/Invitrogen’s non-Dicer dsRNAs before (rxRNA solo/Stealth), chose to minimize Tuschl-KL problems by opting for a 25bp blunt-end design, and Silence’s alternating 2’-O-methylation pattern IP might give those a proprietary touch. As I cannot find any evidence that Intradigm currently has any access to 25bp structure-specific patents aside from Fire-Mello, I wonder how extensive the siRNA design IP access to the Zamore (University of Massachusetts) patent estate really is. Zamore’s seminal discoveries centered around the thermodynamic properties of effective siRNA, and if added to the 25bp dsRNAs could further endow those with a proprietary look. One issued patent relates to the stability of the guide RNA 3’ end with the target mRNA and which has implications for RNAi catalytic turnover. This one, however, would serve more the purpose of a design-around fig-leaf, and not necessarily represent a means to exclude others. The more important discovery by Zamore, however, is related to differential end stabilities of siRNA duplexes themselves, a basic siRNA rule that essentially everybody in the field follows. If Intradigm had indeed access to such IP, it could give them some leverage over other companies [maybe somebody reading this can provide me/the blog with more information on the extent of the Intradigm-Zamore relationship].

An area where there should be synergies is a broader gene target base in oncology. I had been wary of Silence’s focus on the PI3K pathway which, despite the well-known importance of this pathway in cancer biology, appears to be too closely connected to the history of the R&D staff for comfort and may have caused them to oversee more promising targets. On the other hand, Intradigm has been pursuing a large list of 50 largely cancer-related gene targets, and has obtained a sequence-specific patent on one of those which should give the enlarged work-force plenty to work on. Despite all these genes, after burning through almost $40M and noise around their initial VEGF-directed lead candidate subsided, Intradigm alone would appear to be some a long time from the clinic. With Silence Therapeutics’ Atu-027 and lipoplexes, the new company can boast at least boast one candidate undergoing clinical evaluation and possibly more to come.

In summary, given the bleak alternatives, the merger makes sense. For the field of RNAi Therapeutics, it may also be good to have companies with sufficient size to attract the attention of investors. Long gone are the days that the Alnylam-Sirna rivalry kept attention levels high. There is, however, a lot to be sorted out for the new company before it can gain the hoped-for traction.

RNAi Therapeutics Trends from the International Liposome Society Meeting 2009

Christmas to me means going to London to learn about what liposomes have in store for RNAi Therapeutics. As you know, liposomes have emerged as the leading platform for the systemic delivery of RNAi Therapeutics and offers considerable promise for diseases of the liver, solid cancers, and after that potentially enhanced vaccines, infectious disease and immune cell-related disorders. The fact that liposomal drugs are on the market already should further speed up its development, not least from a regulatory point of view.

The liposome field appears to be split between between cancer and vaccines about 2:1. Especially the success story of doxil (liposomal doxorubicin) has shaped the field and should partly explain why it is attracted so much towards solid cancers for RNAi Therapeutics. The EPR effect, targeted delivery, tumor vascular biology have been recurring topics and research using various advanced imaging modalities nicely informs the application of liposomal siRNAs in oncology. For example, liposomes (not necessarily unique in that regard) are not uniformly distributed within the tumor, but form a concentration gradient around the blood vessels. Strategies to achieve more uniform drug delivery may involve metronomic dosing in which the drug is administered more gradually at lower dosages such that the simultaneous onset of cell killing in proximity to the vessels will relieve intratumoral pressure so that subsequently arriving liposomes are freer to move within the tissue. Hyperthermia or low-dose TNF-alpha were two suggested strategies for increasing the leakiness of tumor vasculature, also to improve liposomal tumor distribution.

Since the highest concentration is achieved around the tumor vasculature and because there are differences between normal and tumor vasculature, targeting genes within the endothelial and supporting cells has become an attractive anti-tumor strategy. This for example is also the strategy that Silence Therapeutics is pursuing and the lipoplex formulation (siRNAs associated with liposome on the outside, not inside as in a SNALP) for some reason might have its application here.

But once within the tumor, mostly the result of the overwhelming EPR effect, the question becomes how to get the liposome into cells and then release the drug cargo into the cytoplasm in the case of siRNAs. Ligand-targeted liposomes are one answer to increase the cellular uptake of the liposomes. Antibodies are an increasingly popular means (immunoliposomes), although they present certain manufacturing challenges. It is still debated how PEG affects cellular attachment. Some claim that you cannot take up a pegylated liposome, some say you can. The answer may be that it depends on the size of the PEG. This is important for example in the context of a targeted liposome and whether to add the ligand to the PEG or directly incorporate it into the lipid bilayer as a lipid-conjugate, and when and how the PEG should leave the liposome. For efficacy and safety, triggerability of membrane lysis is of course important. Too much charge for example can be cytolytic, so you would like to expose membrane lytic activities in a pH-dependent (endosomal acidification), redox or protease-dependent manner. For the liposome field, this typically means pH-sensitive lipids.

Combined chemo-siRNA therapeutics is another trend that will become important for liposomal cancer RNAi Therapeutics. Combining ever more chemotherapeutics appears to be a game of diminishing returns with triple chemo Rx often not any better than double chemo Rx. This is because of limited mechanism of actions of chemotherapeutics and associated resistance mechanisms. RNAi Therapeutics therefore can either target these resistance pathways directly or bring to the table an entirely new anti-tumor mechanism of action.

I have always found it attractive to apply siRNAs for developing more effective vaccines. While I am not familiar with the literature around virosomes, viral ghost shells, a presentation by Swiss company Pevion Biotech caught my attention in this context. Basically, they are using flu viruses, disassemble them to get rid of the viral RNA and associate protein, and then reassemble them. During the reassembly process, they can then variably add their lipid-anchored antigen of choice and use the re-formulated virus as a vaccine. The fact that our bodies have seen flu virus before apparently helps to obtain a more favorable immunological response. By this, they believe to have broken the potency-toxicity linear correlation that seems to plague adjuvant science. While promising and based on a credible scientific presentation, it still does not appear to be among the most potent adjuvants, so I wonder whether an siRNA targeting the right immunomodulatory gene loaded into the virosome, essentially replacing the viral RNA there, could improve this equation even further. A challenge for nanoparticle-formulated siRNAs for enhanced vaccines is that although they are easily taken up by phagocyotic antigen-presenting cells, the phagocytic pathway usually destroys the siRNA and little escapes into the cytoplasm. The virosome could offer a solution since the flu virus is THE paradigm of how membranes fuse with each other and also explains how an extracellular antigen, the vaccine, can produce a favorable cellular immune reponses in this system. What I noted at the conference is the surprising lack of cross-talk between people using liposomes for vaccines versus those using them for oligonucleotide delivery which is unfortunate in cases like virosomes.

Pevion also seems to be producing good-quality liposomes. For liposomal RNAi Therapeutics, it is probably making the jump from rodents to primates and scalable manufacturing that separates the boys from the men. There are many interesting early-stage strategies, but if the goal is translational medicine, it is of utmost importance to take manufacturing issues into account from the very beginning. Of course, if you have such know-how, this challenge also presents an opportunity/entry barrier that can be capitalized on and should also present a significant barrier for generic competition. A scientist from Gilead told the story of the attempt by a fairly large Argentinian generics manufacturer to copy Gilead’s AmbiSome formulation by just mixing the individual components together and that this went quite wrong.

In order to address that gap between basic and applied science, the opening talk advocated the creation of companies whose sole business model is to take promising academic research and transform it into industrially partnerable technologies. Certainly, this concept appears to be gaining traction and it will be important in how the compete with or complement small biotech companies. The Centre for Drug Research and Development in Vancouver is one such entity that can be put into this category, also with a liposome focus (Pieter Culles e.g.). Vancouver in general can be considered the world's capital for liposomal research, which brings us to the Tekmira, UBC/Alcana, Alnylam triangle.

Pieter Cullis, co-founder of a number of liposomal companies including Tekmira, but now apparently more closely associated with Alcana and Alnylam, gave a nice talk about the rational design of lipids for use within SNALPs. Only small changes in the lipids can have large biological and physico-chemical effects. For example, by just modulating the chemical strength of the linkage of the fatty acids with the lipid head-group, SNALPs of very different potencies are obtained. Yet another objective is the investigation of the pKa-potency relationship which again can greatly impact liposomal performance. Through this, Alcana-UBC, largely driven by two ex-Tekmira scientists have come up with around 100 new lipids which further help to refine the design rules in an interative process. Actually, the rational lipid design approach is the approach Tekmira itself has been taking- probably not surprising given this background and may explain why Tekmira is getting access to these lipids for free without having to fund their development. It also differentiates it from the ‘lipidoid’ approach by MIT-Alnylam. Maybe given the varied performances due to even only subtle changes, huge libraries of lipids are not that desperately needed as was thought just 3-5 years ago. It is also the animal model that would become the bottle neck, as the tissue culture system is of only limited utility in predictively characterizing SNALP performance and mice become the system for the screen. Still, ‘lipidoids’ may offer fundamentally new structures which can be further refined using similar rational design approaches. For the liver, at least in terms of potency, this may not be needed according to Cullis’ back-of-the-envelope calculation that predicts single digit microgram(siRNA)/kg potencies that the technology is approaching for this tissue, an area that is approaching certain theoretical limits.

In summary, the conference, has confirmed my view that liposomal delivery is a good place to be in RNAi Therapeutics at the moment. It may even be time to have a conference entirely devoted to liposomal strategies for RNAi Therapeutics. There is a need, however, to focus on those companies that operate on sound scientific principles and ideally have a track record in being able to produce high-quality/consistent liposomes in a manner that is scalable.

Anti-miR122 Antagomir Successful in Fighting HCV in Chimpanzees

I just wanted to post this video by KENS 5 on the anti-miR122 studies in HCV-infected chimpanzees by Danish LNA company Santaris and researchers from the San Antonio Southwest Foundation for Biomedical Research. The study, just published online in Science, shows surprisingly potent reduction of HCV levels (2 1/2 logs) with an miR-122 antisense inhibitor and, at least equally important, without the emergence of viral escape mutants. While this study only involved four chimpanzees and human studies are still in the early phases (phase I, healthy volunteers), these results justify hopes that such a first-in-class agent could be a valuable component of future combination therapies with increased cure-rates, shortened treatment times, and maybe without the need for current standard-of-care component ribavirin.

Curiously in light of these promising results, it appears as if GSK let an option to the Santaris compound expire. While Big Pharma may have multiple reasons why it does not want to pursue a certain compound aside from the science (e.g. the fact that HDL-cholesterol was lowered as a result of miR-122 inhibition could warrant caution), for example product portfolio considerations, one possibility is that IP concerns and GSK's relationship with Regulus were important factors: Regulus has an exclusive license to the IP surrounding the fundamental work by Jopling and Sarnow on the role of miR-122 in HCV replication. While this may be good news for Regulus, I was made aware that the broad Esau and Tuschl patent applications on microRNAs as therapeutic targets and held by Regulus are being narrowed down considerably by the patent offices.

Note: For a more detailed discussion of this paper, please read the review by Mark Kay and myself in Molecular Therapy (click here).

RNAi Therapeutics 2010: The Year of the SNALP

While the broad markets have enjoyed a considerable rally since the lows earlier this year, with the S&P500 up more than 65% since March, shares in RNAi Therapeutics companies have only initially participated, but then reversed course. This is unfortunate since this does not make establishing a broad drug development platform any easier with no products on or close to the market. I want to be clear though that one cannot blame it all on a market that 'does not get it' or just bad luck: strategic mistakes have been made, false expectations raised to a point that the market, and this might include Big Pharma, is saying ‘show me credible non-human primate, or even better, human data before I believe you’.

On the other hand, chaos brings with it opportunities, especially for companies that can emerge from this confusion with pre-clinically well-validated technologies and unambiguous proof-of-concept data for therapeutically relevant gene knockdown in humans. A lot will therefore depend on whether a single delivery technology, SNALP, can achieve such results. Results from both SNALP-ApoB (Tekmira), expected at the end of Q1 2010, and ALN-TTR (Alnylam) later in the year, provide opportunities for demonstrating efficacy in relatively small patient populations. Safety, of course, will be equally important to watch.

There are other RNAi Therapeutics candidates in the clinic among which maybe Benitec’s HIV program may provide molecular indications of antiviral activity with the rHIV-shI-TAR-CCR5RZ triple RNA(i)Rx combo. Cancer-related clinical results will mostly focus on safety, although ALN-VSP02 results could go into more mechanistic depths. Quark Pharmaceuticals’ candidates, of course, are far ahead of the field- sometimes I ask whether possibly too far ahead in light of what we have learned about the uptake of naked siRNAs and innate immune stimulation. Beyond RNAi Therapeutics, progress with mipomersen, DMD exon-skipping, and miR-122 inhibition for the treatment of HCV could help return optimism to RNA therapeutics drug development in general.

Taken together, I believe that 2010 could indeed be remembered as the RNAi Therapeutics Year of the SNALP, although it is always possible that a MEGA-deal, possibly inspired by the ApoB-TTR results could divert some of the immediate attention. Some of you may remember that I called out 2008, also for reasons related to SNALP, as the RNAi Therapeutics Year of the Liver. I still believe that this would have been possible if the attention had been focused properly on the exciting development path of this technology for liver applications some of which are now entering the clinic, instead of the somewhat broader messages the market received and is now struggling to cope with. After SNALP, cancer is a strong runner-up, and may in fact drive some of the major business developments of 2010. 2011 or 2012 may be the RNAi Therapeutics Year of Cancer outright.

Given my obvious fondness for liposomal delivery and to stay on top of the exciting scientific developments in this area, e.g. targeted delivery, I am already looking very much forward to be attending the annual International Liposome Society meeting in London next week. All the while next door Cancer RNAi Therapeutics company Silence Therapeutics, which also works on somewhat related lipid-mediated siRNA delivery, should be discussing their merger at the General Meeting and is just one more reason to go.

RNAi Therapeutics Portfolio Update

As we approach the New Year, I decided to take a look at the RNAi Therapeutics portfolio and finally take out gene therapy company Oxford Biomedica. This is not because I have lost faith in gene therapeutics, recent clinical data strongly suggest otherwise and the ocular/neuro applications approach that Oxford Biomedica takes, also in partnership with Aventis, should make this one of the companies in the field to watch. Oxford Biomedica, however, has done too little in RNAi Therapeutics drug development to justify its place in the portfolio. I still wonder how ocular DNA-directed RNAi Therapeutics for example could be institutionalized- maybe as part of a more general gene therapy company such as Oxford Biomedica, or an eye-focussed RNAi Therapeutics company employing both synthetic and ddRNAi techniques. Maybe even packaged into a re-formulated Targeted Genetics, yet another company that provided clear gene therapy clinical efficacy data for a rare eye disease. Until more strategic clarity is provided, however, including their continued interest in RNAi Therapeutics or not, I decided to sell some of TGEN as well.

The proceeds from these sales were put into ISIS Pharmaceuticals whose shares I believe have been oversold in the wake of the somewhat lukewarm phase III homozygous FH mipomersen results. Considering previous clinical results obtained with mipo as well as the overall favorable lipid profile changes as a result of ApoB knockdown, chances are that the upcoming phase III results in the other severe hypercholesterolemic populations will look better and signal the start of the manic phase of the manic-depressive mipo story.

Otherwise the portfolio should be well exposed to the potentially major value creating force in RNAi Therapeutics 2010 with Tekmira now making up the largest position in the portfolio, largely the result of the relative weakness in ALNY (-40%). Silence Therapeutics should warrant a re-evaluation after they make public their merger partner. AVI Biopharma remains on the radar for their involvement splice modulation (DMD foremost) and other areas that could provide them with near-term revenue, especially if they should move closer with mdRNA which could make for an attractive combination. Just fresh from the press is also the announcement that ISIS Pharmaceuticals will play a more active role in splice modulation.

Has mdRNA Found a Way around Alnylam’s RNAi Trigger IP Wall?

mdRNA has to be The RNAi Therapeutics corporate success story of 2009. At the brink of bankruptcy at this time of last year after problems with their former nasal delivery business sent the company into a breathtaking tailspin, the new management, critically made up of former Sirna Therapeutics executives, was able to save the company by quickly cashing in on some non-core assets, attracting upfront payments of about $12M in two non-exclusive RNAi Therapeutics deals with RNAi heavy-weights Novartis and Roche, and an opportunistic financing shortly after final approval of a partnered legacy generic (calcitonin-salmon nasal spray for osteoporosis) caused an irrational spike in mdRNA’s share price. Although I perceive mdRNA’s lack of RNAi Therapeutics publications as a clear weakness, also in light of its history of later unsubstantiated scientific claims, these developments are signs that not only is the management apparently well connected, but that the company has been able to establish a quite decent RNAi Therapeutics drug development platform and with IP that cannot be dismissed out of hand. Since the Novartis deal on delivery involved not only IP, but also the transfer of liposomal formulation know-how, but the Roche deal on RNAi triggers appears to be an IP-only deal, I have looked more into mdRNA’s claim of having freedom-to operate with its ‘proprietary’ RNAi trigger designs. Out of fairness, I should add here that a lot of this change had been set in motion with the old management.

It has become a popular game in the RNAi Therapeutics industry to design ways around Alnylam’s dominant RNAi trigger patent portfolio. While corporate strategy is the driving force behind these supposedly proprietary designs, given that intellectual property is not always guided by the spirit of science and since new trigger designs might have unexpected beneficial properties for certain applications, it is prudent to pay attention when Roche is willing to pay mdRNA non-refundable $5.0M for a non-exclusive license to mdRNA’s RNAi trigger IP claims after having just paid more than $300M for much less of Alnylam’s RNAi trigger IP. Of course, one might also view this deal, which was also largely free of downstream obligations, as a move by Roche to cover all their bases- just in case.

At the time of that deal, mdRNA had three RNAi trigger designs in its stable: certain rights to City of Hope’s Dicer substrates, the three-stranded meroduplex siRNA design after which mdRNA takes its name, and unlocked nucleic-acid-modified siRNAs (usiRNAs). Subsequent to that deal it emerged from regulatory filings that, perhaps because Dicerna appears to own most of the Dicer-substrate IP, that mdRNA has dropped Dicer substrates from their portfolio. Furthermore, since Dicerna would probably have been quite happy to make a deal with Roche, it would appear that Dicer-substrates, although part of the deal, was not the main motivation for Roche. This leaves us with meroduplexes and usiRNAs.

If you assembled five PhDs in molecular biology for a weekend, presented them with the main claims of Tuschl I and II, and asked them to come up with RNAi trigger design-arounds, in principle the result may have looked similar to what mdRNA arrived at. That the various designs should also likely to be functional biologically, at least to a certain degree, is a testament to the robustness of the RNAi pathway when it comes to relatively short double-stranded RNAs, a realization that has only sunk in subsequent to the Tuschl publications, but that was already heralded by the findings of Kreutzer and Limmer.

Both Tuschl I and II essentially claim double-stranded RNAs of 21-23 (T-I) or 18-24 (T-II) (contiguous?) NUCLEOTIDES in length. Besides size and overhangs which had been the main theater of the RNAi trigger IP war thus far, one might argue that an RNAi trigger that does contains a number of NON-nucleotides or in which the dsRNA is made up of three, and not two strands, does not literally violate the Tuschl claims.

I have long been puzzled why mdRNA would think that using unlocked nucleic acids (UNA) per se would be the solution, as I considered UNA just another nucleotide-modification alternative in the armamentarium of the siRNA synthetic chemist: UNAs are simply nucleic acids in which the C2-C3 bond has been disrupted (that is apparently also the process by which they are generated). But when the company’s CEO Michael French at the BMO Capital Markets Focus on Healthcare Conference made a point that it is the fact that UNAs supposedly are not nucleotides is indeed what underlies mdRNA’s interest in UNAs. A quick, non-representative survey among scientists that I know, however, was consistent with my initial assumption that UNAs clearly have to be considered ‘nucleotides’ as they look and behave like such. Moreover, there are numerous quotes by the inventor of UNA, Jesper Wengel of Denmark, and by the Danish company RiboTask from which mdRNA has exclusively licensed the UNAs for therapeutic applications that UNAs are modified nucleotides. The question therefore appears to boil down to whether potential partners first and then the patent offices can be convinced of that a ‘nucleotide’ that does not contain an intact ribose or deoxyribose strictly is a nucleotide no more. Although I am skeptical that any one nucleotide modification is sufficient to get around the Tuschl patents, I could imagine that the possibility alone of their literal interpretation could motivate others to come up with similar non-nucleotide siRNAs.

Aside from the IP considerations of usiRNAs, it is premature to judge the scientific value of this modification over others. The fact that ‘unlocking’ a nucleotide is a quite distinct modification should allow it to endow an siRNA with unique properties with respect to recognition by the RNAi machinery, target specificity, the potential to be recognized by innate immune receptors etc. What can be said based on the limited publication record (Bramsen et al., 2009: Large-scale screen....; Kenski et al., 2009: Analysis of acyclic nucleoside modifications...), UNAs like many other modifications are best tolerated in the passenger strand and can reduce off-targeting by the passenger-strand, whereas the use in the guide strand is much more position-dependent. Whether its judicious incorporation can indeed largely abrogate innate immune responses and/or allow for a better differentiation of on-target cleavage over microRNA-like off-targeting as claimed, remains to be seen and especially published (also because mdRNA/Nastech has a track record of making similar claims which tend to silently disappear over time). On the other hand, since Sirna Therapeutics (Merck) is evaluating UNA-modified siRNAs (Kenski et al., 2009), despite its access to Tuschl I, may be interpreted as a sign that there may indeed some unique scientific merit to usiRNAs, and mdRNA may derive value from controlling its use for RNA therapeutic applications.

Moving on to 3-stranded siRNAs, the other pillar of mdRNA’s RNAi trigger strategy, from an IP point one has to probably say that this stands a better chance of surviving Tuschl scrutiny with Tuschl II explicitly claiming double-stranded RNAs consisting of two strands. The double-strandedness of Tuschl I, however, could be more broadly interpreted in that a nick should not make a difference in determining the length of a double-stranded region. In this case, it will be interesting to find out how 3-stranded siRNAs with a gap instead of a nick would perform.

When I first heard of the 3-stranded siRNAs in 2006, I thought that this was based on the observations by a number of labs at the time that the passenger strand of the siRNA is cleaved as part of RiSC activation and that the objective of 3-stranded siRNAs thus was to hi-jack the RNAi machinery downstream of the intact siRNA. However, looking at the history of the relevant patents, it appears that this was not the case, but was merely a coincidence. There is, however, an interesting twist to the story. RiboTask, which happens to also be mdRNA’s partner in usiRNas, filed for almost identical patent protection, but with a priority date that appears to be a couple of months ahead of mdRNA’s. In the case of RiboTask, which calls the 3-stranded siRNAs not meroduplexes but small internally segmented interfering RNAs/LNAs (sisiRNA/sisiLNA), I do not want to be as dismissive about their scientific motivation, as they address a major concern of 3-stranded siRNAs related to their potential instability. Since short segments of double-stranded RNAs should be relatively unstable, they demonstrated (Bramsen et al., 2009: Improved silencing properties using small internally segmented interfering RNAs) that it is possible to restore ‘duplex’ stability by modifying it with locked nucleic acids, the mirror-image of UNAs and curiously invented by the same people. Moreover, they show that while heavy modification of both strands renders most siRNAs inactive (with the exception of 2’F and 2’-O-methyl), converting such a modified siRNA into a 3-stranded siRNA often restores at least some of the knockdown activity (also quite interesting from an RNAi mechanism point of view). The ability to heavily modify an siRNA without losing activity may be particularly useful for applications where the siRNA is exposed to nucleolytic degradation such as for conjugate-siRNA approaches. From a scientific perspective, I should also mention that the 3-stranded siRNA design is a way to abolish passenger strand microRNA-type off-targeting, although this is no more a considerable challenge for RNAi Therapeutics. It should also be of interest to evaluate how nicked, and particularly gapped siRNAs are recognized by the various innate immune receptors. The above demonstrations may also help RiboTask and mdRNA in their way through the patent offices, particularly in the US and in light of the mentioned academic research that might have started before RiboTask’s work, and I am curious whether the usiRNA relationship between RiboTask and mdRNA will be extended also to sisiRNAs/meroduplex technology.

Since IP is subject to interpretation and therefore fraught with some uncertainty, it remains to be seen whether mdRNA can achieve certain freedom-to-operate in the RNAi trigger space. Although usiRNAs are the current focus of the company, keeping the meroduplex as an alternative design option open seems prudent. More broadly, RNAi Therapeutics in general can only gain from investments in evaluating new modifications and designs as unforeseen beneficial properties could emerge from this. Until then, however, it is unlikely that any Big Pharma would want to risk their RNAi Therapeutics future entirely on mdRNA’s RNAi triggers. This also explains why mdRNA sees more partnership potential in its RNAi delivery efforts, the currently more pressing need in RNAi Therapeutics and for which the financials should continue to grow. mdRNA urgently needs such a partnership since although the company had just averted bankruptcy, with cash running out over the next 3 months, the struggle to get out of the bankruptcy quicksand has not stopped. Based on management, while a number of early-stage technology evaluations are ongoing, no significant upfront payments from partnerships can be expected until the middle of next year by which time it hopes to have more non-human primate data to satisfy a more discriminating Big Pharma audience. Given the positive developments of the company over the last year and management’s track record, investor interest should be sufficient to support a PIPE within the next 30-45 days so that the company gets a shot at achieving its partnership goal. If one indeed wanted to make the gamble on mdRNA’s management, from an investment perspective, waiting until more is learnt about the terms of the financing may be prudent, unless valuations continue to fall much further.

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.

Merging Antisense with RNAi Therapeutics to Create Fitter Companies

Yesterday’s long awaited data presentation on phase III results from ISIS Pharmaceutical’s lead antisense gene knockdown program was met by disappointment in the investor community: ISIS down over 15% on the day. While the exciting news is that the drug, mipomersen, looks like it is very close for approval for the severe, but very rare condition of homozygous familial hypercholesterolemia (hoFH) and importantly also possibly other forms of highly elevated LDL-cholesterol, part of the disappointment may be related to the long-term outlook on antisense for gene/mRNA knockdown: a 27% reduction in ApoB levels following 6 months of 200mg weekly injections with a safety profile (a number of cases of elevated liver enzymes, injection side reactions) that may be adequate for the severe cases of hypercholesterolemia, but not necessarily for less severe diseases. And this is for an organ, the liver, which has one of the best pharmacologies for antisense. Also, while a 27% knockdown may be therapeutic for a few targets, especially in metabolic disease, for most other targets it is insufficient.

Lack of high-quality gene knockdown opportunities would lead to an inefficient use of its capital that includes an enviable $600M+ cash pile and an equally remarkable cash-flow from their ‘satellite businesses’. Maybe partly because of this realization, but also of course because it believes that its IP is transferable to all areas it considers antisense, ISIS is not standing still and continues to innovate in areas such as RNAi Therapeutics and the promising gain-of-function antisense technologies of splice modulation and microRNA inhibition.

On the other side of the fence, RNAi Therapeutics is also facing a challenging investment environment. Capital is particularly difficult to raise for early-stage platform technologies. While some companies such as Alnylam, Tekmira, Sirna Therapeutics (historical example), and mdRNA are making good strides in establishing broad RNAi drug development platforms, this alone does not make up for lack of steady newsflow from mid- to late-stage clinical results that can support rich valuations in biotech. This is also not helped by some obvious mis-steps within the industry itself with the main sins being spending money on lawsuits and prematurely entering programs into the clinic, ironically not least in an attempt to keep investor interest levels high.

By merging with antisense, both the pipeline maturity profile could be enhanced and resources spent more efficiently by avoiding the temptation of entering programs into the clinic prematurely and otherwise weeding out programs that serve to artificially fill pipelines. The latter point, of course, would also apply to the antisense company, and the antisense company would further benefit from gaining access to the most potent and therapeutically promising gene knockdown technology known, RNAi Therapeutics. As the intricate relationship between Alnylam and ISIS Pharmaceuticals or the acquisition of Coley as a launch pad for Pfizer’s RNAi Therapeutics ambitions demonstrate, the scientific barriers for such mergers should be relatively minor. One risk, however, that cannot be ignored, particularly for those companies that have been built for sale, is that by combining various drug development platforms, the new entity may become a less attractive candidate for a Big Pharma acquisition. When it comes to mere survival, however, a combination should be the lesser evil.

I will now briefly discuss three fantasy combinations, at least one of which I would speculate to see within the next year.

1) Alnylam Pharmaceuticals and ISIS Pharmaceuticals (probably not within the next year). Without a doubt the most influential and potent leaders in RNAi Therapeutics and antisense, respectively. Already highly entangled through their IP cross-licensing agreements and microRNA therapeutics spin-off Regulus, a combination would create an almost cash flow-positive dream team with over $1B in cash, blocking IP, unmatched expertise in nucleic acid chemistry, clinical pharmacology, and RNAi Therapeutics. First revenues from the sales of mipomersen and continued IP licensing revenues would support a solid pipeline consisting of mipomersen label extensions, RNAi Therapeutics opportunities for liver and solid cancers, full ownership of the miR-122 program for the treatment of HCV, and transitioning antisense towards splice modulation, microRNA inhibition (with Regulus), and possibly other gain-of-function antisense applications. As part of the re-organization, some programs could easily be sacrificed without punishment by the markets. John Maraganore would be the CEO of the combined company, allowing Stanley Crooke to follow his passion in the science of oligonucleotide therapeutics.

2) mdRNA and AVI Biopharma (how soon?). The transition of antisense for gene knockdown to gain-of-function applications is most noticeable in the case of AVI Biopharma. After many years of attempting gene knockdown with their steric-block morpholinos, the company finds success in applying its technology for the modulation of gene splicing. It is considered the closest competitor to Prosensa’s Duchenne Muscular Dystrophy exon skipping program which has only recently entered an attractive $25M upfront plus multimillion bio$$$ milestone and royalty deal with GSK after a series of high-profile publications on DMD-related exon skipping. The partnership potential of AVI’s DMD program then as well as its recent capital raising should provide the needed capital cushion for the combined company to invest in RNAi Therapeutics for gene knockdown and some new splice modulation opportunities. mdRNA, of course, is scheduled to run out of cash early next year and it would be a miracle if the two companies had not contemplated such a merger, particularly after AVI Biopharma moved in as mdRNA’s neighbor not too long along up from Oregon. With AVI’s cash reserve, the combined company may then find it easier to attract platform partners for mdRNA’s technology further bolstering the financials.

3) Silence Therapeutics and Archemix (not really an antisense company, but close enough). It is almost two months now that Silence announced to be in reverse merger talks. Since then, however, not a word except for so-so news on the results of opposition proceedings at the EPO related to their core patent. What Silence needs is cash (who doesn’t?), and what it can offer is a phase I cancer program, RNAi Therapeutics drug development expertise, and an siRNA structure that is not without use, although facing very serious patent challenges. What Archemix needs with its growing pipeline is access to public markets as evidenced by a previously failed reverse takeover attempt with cash-rich NitroMed, as does by the way Quark Pharmaceuticals which should also be counted as a possible Silence Therapeutics merger candidate. The fact that Silence Therapeutics has some aptamer in its blood line and the increased investment by Archemix into aptamer-mediated delivery of RNAi (Dicerna) and microRNA (miRagen) Therapeutics should help a combined company find a common language. Until any such deal is announced, however, Archemix would have to be prepared though that yet another potential partner will walk away from it last minute as Silence Therapeutics should also be receptive to other offers.

Which one is the most likely combination? Add your vote on the right.