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Wednesday, April 30, 2008

RNAi Therapeutics: Integrate, Collaborate, or Die

It was with a healthy dose of suspicion that I went today to a presentation by Mike Varney from Genentech on their small molecule operations which is a new addition to the Genentech and which he was spear-heading. Of course, as the pioneer of biotech the first question to be addressed was why Genentech would go into small molecules at all if that was part of the reason why Big Pharma was ailing anyway?

Hearing about the challenges of RNAi delivery ad nauseum, you’d think that relative ease of delivery such as by oral bioavailability would be a major motivation, but this was not the case. In the business of treating serious, life-threatening disease with large unmet medical needs, it becomes less relevant if you can go home and take a pill twice a day, or whether you get an intravenous infusion every other week. No, the main rationale provided was that as Genentech scientists dive deep into the biology of disease pathways, they often find themselves with promising drug targets, but which cannot be reached by antibodies. Antibodies are great for targeting cell surface proteins, but useless for intracellular targets whereas small molecules can more easily achieve that feat. Target space is the keyword here, and according to Dr. Varney target space is very limited, so limited so that as a biotech reaches the size of a Genentech or Amgen it has to think about other drug classes while at the same time Big Pharma is expanding into biologics.

You can imagine how my mind was racing wishing to tell Genentech that it was right in front of their eyes and RNAi is what they were really looking for, a technology that may address virtually any gene and also circumvent the painful lead optimization process for small molecules Mike Varney was going through (and yes, I cannot believe that they have not thought about small RNAs already even if much of the thinking was done for them by mother Roche).

But that’s not really my main point tonight. As he was describing the diverse skills it took to bring the clinic and pointing out that part of the reason that current productivity in drug development is so dismal (the latest number is $1.4B spent for every new drug developed) and Genentech was so successful is that many companies think they can get away with by just concentrating on a small part of the skill pie. And this may be very true for the current RNAi Therapeutics space as well.

While the entry barrier for target discovery using RNAi is relatively low, developing an actual RNAi Therapeutics is much more complicated than that. Just considering the scientific aspects, the development of an RNAi Therapeutic requires among other things, precise knowledge of the disease biology, basic RNAi and microRNA biology, know-how in siRNA design and chemistry, assessing off-target risk and the interplay of the RNAi trigger and/or delivery vehicle with the immune system, RNAi pharmacology, and being able to safely deliver the RNAi cargo.

I get the impression that there are a number of programs/companies out there that do not take into account e.g. the off-target profile in their design of an RNAi trigger and instead select a published sequence or one that they find can simply knock down a gene and eventually take that into the clinic, or lack the know-how required to adequately assess innate immune responses. This could be avoided if smaller companies weren’t so afraid in collaborating with each other or larger companies, partly for fear that it may negatively affect how the investment community perceives their competitive scientific prowess and IP position. Right now, I can only see very few companies that have the financial wherewithal and scientific know-how to establish an integrated RNAi Therapeutics development platform that alone could shepherd RNAi Therapeutic candidates into the clinic. Alnylam, Merck, maybe Novartis, and probably soon Roche and Pfizer may have such capabilities, and these examples may not even cover all the bases just as Genentech is collaborating with both small and large.

Smaller companies, particularly those with a focus on delivery should probably seek help in deciding which RNAi trigger to use with their particular technology. Of course, having something to offer in return will help their negotiation position. As you know, I think Tekmira/Protiva have done that quite well and obtained up to 7 InterfeRx picks from Alnylam in exchange for liposomal siRNA delivery technology. In other instances where the technological payback may not be that obvious for the established company, it may still be a good investment to help with siRNA design as part of granting a technology license, as it should also increase the odds of successful clinical development and therefore potential for royalty income further downstream. Big Pharma most likely would not be interested in such top-to-bottom technology transfer arrangements, but it may be a consideration for some of the more established pure play RNAi Therapeutics companies.

Monday, April 28, 2008

Lipidoids Expand Chemical Space for Cationic Liposome Delivery of RNAi Therapeutics

[Important update, see end of entry]

Following a long series of presentations and publications involving Protiva, Tekmira, Alnylam, and Merck/Sirna, PEG-stabilized cationic liposomes known as SNALPs have to be considered one if not THE most advanced systemic RNAi delivery technology to date. While efficacy was very potent at single-digit mg/kg doses, the major drawback of the first studies on SNALP-siRNA delivery were slight elevations in liver enzymes, an indicator of toxicity (Zimmermann et al. study). It was therefore important to expand on those studies and search for formulations with even better knockdown efficacies and inherently less toxicity, thus pushing the therapeutic index well into predictably clinically safe ranges.

The recent announcements 0.1mg/kg IC50 knockdown efficiencies for SNALP-like formulations by both Protiva and Tekmira (due to their upcoming merger from now on referred to as Tekmira for simplicity) support the notion that the exploration of new chemistries should facilitate the development of SNALP RNAi for clinical use, and although only the surface has been scratched, a first IND with realistic chances at therapeutic success may not be very far away.

To exploit the chemical space available for SNALPs though requires the ability to generate new and diverse lipid chemistries as well as the ability to manufacture and formulate these chemistries to scale. The latter has been achieved by Tekmira’s spontaneous vesicle formation by ethanol dilution method allowing for the speedy manufacture of SNALP-siRNA formulations that can support late pre-clinical and clinical studies, while the long-awaited paper by Akinc and colleagues from the MIT (Langer/Anderson lab) and Alnylam Pharmaceuticals on so called lipidoids and that has now been published in Nature Biotechnology [Akinc et al. (2008): A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.], will now faciliate the efficient exploration of novel, SNALP-compatible lipid chemistries.

Rather than laboriously synthesizing and testing one lipid after the other on a hypothesis-driven basis, Akinc and colleagues developed a synthesis method that allowed them to generate libraries of cationic lipids with quite unusual and diverse characteristics to systematically evaluate them for siRNA delivery. A first library gave an indication of which chemistries worked better than others, and a second library was generated based on the characteristics of the best performing ones in the first set.

Initial tests were based on silencing reporter genes in tissue culture. It should be noted that for these high-throughput experiments, probably for speed and ease, simple siRNA-lipoplexes were used (siRNA-lipid mix), instead liposomally formulated siRNA as for later tests in vivo (siRNA captured inside liposomes), and this may be one limitation as it could have caused them to miss even more promising in vivo silencing chemistries. In any case, the best candidates were then taken forward into rodent and non-human primate studies, this time formulating the lipids together with cholesterol and PEG-lipid into cationic liposomes, essentially based on the same principles as SNALPs (stabilized liposomes containing diffusible PEG-lipids, the latter interestingly manufactured by Alnylam itself).

Overall, IC50s in the low mg/kg range were routinely observed for a number of liver targets. This was achieved without significant toxicities based on careful safety analysis, and only in some cases mild elevations, less than 2-fold, of liver enzymes were observed. Similarly, the absence of negative interference with endogenous microRNA pathways was reported last year. This is a good start and may not have employed the most efficacious siRNAs, but by optimizing the formulations further, e.g. by engineering additional fusogenic lipids and other properties into these particles, sub-mg/kg doses that would be desirable in the clinic should be achievable and still be compatible with the more scalable manufacturing technologies practiced by Tekmira (although 50nm particles and high encapsulation efficiencies were achieved, the extrusion-based method as employed in the present study may limit scale).

Beyond the liver, the lipidoid formulation showed some promise for the delivery of siRNAs to the lung as demonstrated in an RSV model. Interestingly, inhibition of RSV replication was enhanced by lipidoid-siRNAs (almost 3-log knockdown at 2mg/kg) over unformulated, naked siRNAs (1-log knockdown), in contrast to previous studies on which Alnylam’s ALN-RSV01 is based that showed somewhat less viral knockdown with naked siRNAs and that suggested no enhancement of siRNA delivery to the lung by formulation with other delivery chemistries. In addition, given the propensity of such nanoparticles to be taken up by phagocytic cells of the immune system and the largely unmodified siRNAs used for targeting RSV, follow-up studies need to look at any innate immune responses elicited by such lipidoid-siRNA combinations.

In summary, this study opens up a wide chemical space for the systematic evaluation of cationic liposome-mediated delivery of drugs, particularly siRNAs, but also microRNA antagonists (demonstrated in this study) and beyond. Following some of the recent breakthroughs and due to the triangular relationship between Alnylam, Tekmira, and the MIT, complementary in terms of both know-how and IP, progress of SNALP-siRNAs into the clinic may hopefully occur within the next few months and should be followed by next-generation chemistries.

[Update May 1, 2008: According to a report by RNAiNews , an Alnylam spokesperson indicated that the company had not given guidance on the specific liposome formulations to be used for their hypercholesterolemia and liver cancer clinical programs. This is in contrast to an earlier report by RNAiNews from last year’s Beyond Genome conference in San Francisco which indicated that Alnylam had chosen “choose lipidoids over SNALPs” for these indications (also discussed in a blog entry here). It therefore seems that Protiva/Tekmira's 0.1mg/kg IC50 liposomal nanoparticle formulations may be the current frontrunners in entering the clinic (see also a recent PR).

The sometimes imprecise use of the terms SNALPs and lipidoids may be partly to blame for the confusion. SNALP refers to a liposomal formulation technique, while lipidoids are a new class of lipids generated by combinatorial chemistry which can now be evaluated for liposomal drug delivery. As such, lipidoids could be used within the context of SNALPs, and both of these approaches are therefore complementary to each other.]

ENHANCE Ripple Effects Felt at ISIS Pharmaceuticals

The waves started by the ENHANCE tsunami have finally come to engulf ISIS Pharmaceutical’s stock this Friday as the FDA indicated that it wants to see costly and more time-consuming outcome studies for all but the most at-risk cardiovascular patient population in the approval of ISIS’ lead product mipomersen, an antisense compound directed against ApoB-100 in the liver. As I had discussed earlier in the wake of two high-profile studies that failed to correlate the responses of some of the most trusted biomarkers for diabetes and cardiovascular disease with disease outcome, most notably Merck/Schering-Plough’s ENHANCE study, this was almost to be expected.

The 29% drop in the stock that day was caused by a discussion between ISIS and their partners at Genzyme with the FDA on the approval path for mipomersen. Instead of being able to gain accelerated approval by merely showing LDL-cholesterol lowering as a surrogate biomarker for a broader patient population, this will only be accepted for the small homozygous familial hypercholesterolemia (ho FH) population, and it was not clear from the conference call whether this would be a full or an accelerated approval. For all other indications, mipomersen now has to show tangible benefits with regards to cardiovascular events, which, although it would eventually increase the market value of the drug considerably, means longer and more expensive outcome trials before the real commercial value of mipomersen can be realized (in addition, ho FH will be delayed by at least one year, because the FDA requires 2 rodent carcinogenic studies, instead of the one they had planned for).

What can the RNAi Therapeutics space learn from this experience? One lesson is that it is very important to be clear early on with what the almighty FDA wants to see in approving a drug, something particularly relevant for novel technologies and novel drug targets such as will be the case with RNAi. It is probably not a coincidence that clarity has come now that Genzyme has joined the mipomersen effort, as it is probably THE company experienced with gaining regulatory approval for unique, high-margin drugs with small, but very well-defined patient populations, a category that a number of RNAi Therapeutics will fall into as part of the personalized medicine revolution.

Another lesson is that the fortunes of an entire technology platform may be subject to the woes of a single dominant development program. While the cardiovascular disease market is enormous and the potential rewards substantial, this is not without a reason and substantial investments have to be made before gaining approval. A number of RNAi Therapeutics companies such as Alnylam, Tekmira/Protiva, RXi, Mirus Bio, and Merck have all indicated an interest in targeting very promising liver targets for hypercholesterolemia by RNAi, and although it may limit the ultimate financial reward, a company like Alnylam that carries much of the hope for realizing the therapeutic promise of RNAi, may have to think twice about to what degree it wants to make itself dependent on a single development program such as ALN-PCS01.

Companies targeting ApoB by RNAi (probably including Tekmira/Protiva, RXi, and Merck) should benefit considerably by learning from ISIS’ pioneering ApoB experience, and actually should have a chance at gaining accelerated approval should ISIS be able to show that lowering LDL-cholesterol by targeting ApoB is associated with cardiovascular benefits. For companies like Alnylam though interested in other, previously untested targets, the decision may only confirm that, yes, outcome studies will be required.

I welcome the trend towards evidence-based medicine and addressing some of the problems associated with direct-to-consumerism, but again, the FDA and politicians should not blindly destroy the drug development industry by forgetting that at the same time there need to be clear rewards for innovative (patent protection!) and efficacious (reimbursement!) drugs. This way the pie could stay around the same size, and yet everybody including patients, payers, and the industry would be winners.

Also this week: In a catastrophic financing, Nastech raised just shy of $8M in a registered direct offering at a probably historically low $1.73 a share, and may raise another $3M at $2.17 a share. One has to wonder why this relatively small fund-raising round was done at all at these so unfavorable conditions- unfavorable at least for the present shareholders. In any case, this may be the best time to cut their losses and fully commit to developing RNAi Therapeutics while monetizing what is left over from TJT nasal delivery. Their experience in drug delivery and peptide technology in particular may position them to be a respectable player in peptide-facilitated RNAi delivery (direct conjugation of RNAi trigger to membrane-penetrating peptides and/or using peptides as ligands for targeted delivery). But there is one advice that I would give them: please do not waste any more of your shareholder money and your own credibility on senseless and blatant patent workaround efforts such as three-stranded siRNAs. As the 36% drop on Friday illustrates, investors are sophisticated enough to see through this and rather than claiming to own everything under the RNAi sun, or universe, it is more credible to specialize in an area of your expertise and be good at it. Develop clinically relevant delivery for example, and the market will more than generously reward you for it.

Sunday, April 27, 2008

Small RNAs with First Clinical Impact

Small RNAs are about to make their clinical debut. This week, Asuragen, a leading microRNA diagnostics and therapeutics company, announced that it is about to launch the first microRNA-based diagnostic test for the differentiation between chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC). PDAC is the most prevalent pancreatic cancer type and known for its late clinical presentation and poor prognosis. Because chronic pancreatitis that has a roughly comparable incidence and presents with often similar symptoms such as abdominal pain, digestive problems, and biliary obstruction resulting in mis-diagnosis rates of up to 25%, diagnostics that help to differentiate between them are invaluable for ensuring optimal treatment.

The test will be made available on May 2, 2008, from Asuragen’s own CLIA-certified test laboratory. It is a PCR-based test of apparently a single microRNA that is specific for pancreatic cancer cells and was originally identified as part of a collaboration between Asuragen and a clinical group from a hospital in Bochum, Germany [Szafranska et al. MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene 26:4442-52 (2007)].

Other tests by Asuragen, but also notably Rosetta Genomics and Exiqon in the field, are about to enter the market in the coming months. Like the present one, they are PCR-based diagnostics of one or a few microRNAs. Despite the 25% mis-diagnosis rate, it seems that the need to obtain biopsies from the pancreas is not trivial and may therefore limit the use of this particular test. Tests which may diagnose a cancer based on microRNAs isolated from blood samples, and are currently under development, should have wider applicability, although this may be at a rather late stage of cancer.

Asuragen is a private company and it has been therefore more difficult for me to follow their progress. The fact that they beat the publicly held Rosetta Genomics and Exiqon in bringing the first microRNA diagnostic to market is likely based on their heritage of having been spun-out from the RNA research reagents company Ambion (now an Applied Biosystems subsidiary), therefore giving them prime access to very relevant technologies and know-how. The fact that they operate a CLIA-certified laboratory, of course, may be another significant efficiency giving them an advantage over its competitors. Ultimately, however, it would appear that the commercial success will depend on their ability to make the test widely available through larger diagnostics distribution partners. Alternatively, microRNA diagnostics that are tied to certain drugs, such as Rosetta’s squamous versus non-squamous non-small cell lung cancer (NSCLC) Dx that ensures that Genentech’s Avastin is not used for squamous NSCLC, could benefit from the marketing reach of large therapeutics partners.

At any rate, the fact small RNAs are about to have a real clinical impact less than a decade after their widespread occurrence in biology was first recognized, is to be celebrated and indicates that their small size and stability, and their biological involvement in disease makes them a very promising class for molecular diagnostics.

Friday, April 25, 2008

The Business of RNAi Therapeutics

I’m often asked about explaining RNAi and its potential as a therapeutic modality and investment in more layman terms. I hope my review, The Business of RNAi Therapeutics, that came out today in Human Gene Therapy will be able to address at least some of these questions. Although originally written with the scientist in mind asking him/herself about what the development of RNAi Therapeutics may mean as an investment or business opportunity, the science should be pretty digestible and accessible to the scientifically minded lay public. Here’s the abstract, the rest is freely accessible on the Human Gene Therapy website:

Business for RNAi Therapeutics has never been better. The confluence of the genome revolution, a pharmaceutical industry greatly challenged by generic competition and starved for innovation, and pre-existing nucleic acid drug development know-how that can now be directly applied to RNAi Therapeutics, means that RNAi, less than 10 years after its discovery in worms, has every chance of becoming the next major drug development platform for improving healthcare and generating profits at multiple levels. From the perspective of a molecular biologist interested in both basic and applied aspects of RNAi and related investments, I would like to first put RNAi into the context of the drug development enterprise and then discuss various strategies for translating the science of RNAi into commercially viable drugs as well as some of the risks the industry faces. While the discussion will focus on RNAi Therapeutics and the major US and European markets, it should be kept in mind that RNAi is a global effort and holds further commercial promise in agriculture, the research reagent and services markets, and the related area of microRNA-based therapeutics and diagnostics. Read on…here

Sunday, April 20, 2008

Lost in Translation? Financial Express Report Says Multiple Merck RNAi Therapeutics Candidates in Clinical Development

[Update 23 April, 2008: added "multiple" to the title to emphasize potential clinical candidates in addition to the widely known wet AMD RNAi trial by Merck/Allergan]

This week I escaped from the lab to step into the warm and sunny California spring weather to see what was going on at Stanford’s Biotechnology job fair on the lawn of the Medical School. The first booth that I encountered was that by Sirna Therapeutics, the RNAi Therapeutics subsidiary of Merck. Strangely enough, when I started asking what kind of jobs they were offering, they suggested me to sign a non-disclosure agreement before saying anything more. Go figure, how do you intend to recruit the right people if you can’t properly tell them what jobs you are trying to fill?

I think this is somewhat symptomatic of Merck’s secretive attitude towards their RNAi Therapeutics development efforts. Following the Sirna acquisition, essentially nothing specifically has been disclosed about the stage of their development programs, one for wet AMD already in the clinic and others that were were close to IND filings as of before the Sirna takeover.

A February 7 interview of the Financial Express of India with Sirna Therapeutics’ Bharatt Chowrira, responsible for Merck’s Asian licensing and collaboration activities ex-Japan, could shed astounding light on Merck’s recent RNAi Therapeutics activities and have significant repercussions for the competitive landscape of RNAi Therapeutics.

Not only is it notable that while the interview was predicated on Merck’s general collaboration activities in India, most of Dr. Chowrira’s comments were geared towards RNAi Therapeutics, even more tantalizing, intended or not, was the following section:

“''We are serious about the R&D relationships in Asia in areas such as contract research and clincical trials and will be embracing partnerships,'' he said adding that Merck is building a virtual laboratory by mounting best scientific programs and there is more of external research conducted by partners. The company is searching for partners with a true collaboration mindset and the ability to deliver compounds against novel targets.

On the research front, the company is working towards providing personalized medicine. It has screened over six lakh RNAi, out of which two to three are in clinical trials and 10-20 are candidates for in pre-clinical trials. For starters, RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. RNAi targets include RNA from virus [DH: HCV?] and play a role in regulating development and genome maintenance [DH: cancer?].”

It is known that Allergan is responsible for the development of one Sirna Therapeutics-derived product candidate, but is it really true that Merck has another one or two RNAi Therapeutics programs in the clinic (and 10-20! in pre-clinical studies)? Unlike development-stage biotechs for which making scientific progress public is an essential element in attracting investor interest, Big Pharma often likes to be secretive about their research programs, particularly in areas that here highly competitive.

If true, this would cast events in the past several months going back to Merck’s acquisition of Sirna in a completely different light. Much has been made of the $1.1billion price-tag, particularly as the impression was growing that Merck’s efforts to moving RNAi into the clinic were stalling. However, this equation would change dramatically from a discounted cash flow perspective if they were actually conducting RNAi Therapeutics trials right now in India. As such, Merck could gain valuable early insights into the safety and efficacy of SNALP-RNAi in man, for example by targeting ApoB or PCSK9 and measuring protein levels in the blood early on in phase I. That information could then either be used to progress these drug candidates further or to optimize the formulations. In addition to the competitive advantage, the secretive nature of these studies would also spare them the public humiliation that any failure would bring with it.

As the involvement of Tekmira, Protiva, Roche, Merck, Alnylam, and four as yet undisclosed Big Pharma/biotech companies document, the interest and potential competition for gene targets that can be addressed by SNALP-RNAi could be considerable. Could therefore say a conflict between Merck and Alnylam or one of Alnylam’s partners (e.g. Novartis) for the same gene targets have contributed to the break-up of Alnylam and Merck in the fall of last year? Did the court injunction forbidding Merck to further develop SNALP-based programs force them into the Protiva settlement, because otherwise it would have imperiled their clinical development activities? Does the possibility that Merck may have more than one additional RNAi clinical trial on top of wet AMD ongoing mean that the initial experience was positive so that they could rapidly expand the technology towards targeting multiple genes as is the promise of RNAi?

Maybe everything here is a misunderstanding and the truth was lost in translation, but in any case such a scenario certainly makes for an interesting exercise in game theory.

Thursday, April 17, 2008

Regulus Deal Makes GSK Clear Force in MicroRNA Therapeutics

Only months after closing a similar agreement for the development of antiviral microRNA therapeutics with LNA company Santaris (Santaris PR), GSK today agreed to a co-development partnership with the Alnylam-ISIS microRNA therapeutics joint venture Regulus Therapeutics in the inflammatory disease area (Regulus PR).

In both deals, GSK has the option to select up to 4 microRNA therapeutics candidates developed by Santaris or Regulus in the respective disease areas for comparable financial terms that gives the microRNA therapeutics companies important and largely undilutive upfront cash, development milestones, and royalties (for exact terms, please refer to the Santaris and Reguls PRs).

It is also a vote of confidence by Big Pharma that it sees microRNA therapeutics as a significant new emerging treatment modality. While the antiviral programs with Santaris should be based on the straight-forward targeting of either viral or host microRNAs directly implicated in viral infection, the anti-inflammatory programs with Regulus may be based on more complex microRNA pathway biology.

Interestingly, in both cases the emphasis is on microRNAs as targets for antisense antagonists and much of the discussion in today’s conference call on Regulus’ general microRNA therapeutics activities centered on such antagonist approaches, although I previously was under the impression that microRNA agonist approaches would also fall within the scope of Regulus.

Actually, when I woke up this morning and saw the headlines, I thought that the GSK-Regulus alliance would relate to a mystery surrounding microRNA-122 in HCV replication that is currently being developed by both Santaris and Regulus. Since miR-122 is by far the most advanced antiviral microRNA therapeutics candidate program, it is no stretch of the imagination that GSK will exercise their rights to Santaris’ HCV program that, in collaboration with the Sarnow lab, was subject to a recent high-profile publication in Nature. The issue here is that the Sarnow patents for targeting miR-122 in HCV from Stanford were exclusively licensed to Alnylam (now part of Regulus IP) and GSK-Santaris would therefore ultimately need access to this important patent. A Regulus-Santaris collaboration, however, is complicated by the fact that this program pits ISIS’ against Santaris’ LNA antisense chemistries. It is hoped that miR-122 will eventually aid in synergizing microRNA therapeutics efforts rather than leading to a Merck-like situation where GSK would end up in mutually exclusive collaborations. This issue probably should be addressed early on before it may cause harm down the line.

Wednesday, April 16, 2008

Update on AAV-based RNAi for Huntington’s Disease: Time to Move on from U6-shRNAs?

Huntington’s disease (HD) is a dominant genetic disease caused by a triplett expansion in one of the two copies of the huntingtin (htt) gene. So far there is no treatment that appropriately addresses this disease. As with other dominant diseases, suppressing the insulting gene copy by RNAi should bring a therapeutic benefit, hopefully even delay its progression. A recent paper from Beverly Davidson’s group in Iowa reports on their latest experience in developing an DNA-directed RNAi Therapeutic for the treatment of Huntington’s disease [HD; Mc Bride et al (2008): PNAS 105:5868).

Previous studies from the Davidson lab showed that AAV-delivered shRNAs were able to knockdown target genes in mouse brains and subsequently improve the behavioral deficits and pathology of polyglutamine-repeat diseases such as Huntington’s. These promising results led to a collaboration with the AAV gene therapy company Targeted Genetics and Sirna Therapeutics (pre-Merck acquisition) with the goal of advancing an AAV-based RNAi Therapeutic for HD into the clinic.

Their prior work on Huntington’s was based on a transgenic mouse model which expressed a human fragment of the huntingtin gene containing a triplet expansion. However, as the shRNAs in those studies were directed specifically against the human htt gene, but left the mice’s own huntingtin gene copies untargeted, the present studies were designed to evaluate the safety of knocking down all the huntingtin gene copies in a mouse. This is important for the development of an RNAi Therapeutic for HD in man since targeting a small RNA towards the highly structured triplet expansion is unlikely to be effective, and since the mutant htt allele is not associated with a common polymorphism in the htt mRNA which would facilitate a discrimination between mutant and wildtype huntingtin. This issue is also of particular concern since the huntingtin gene is considered to be essential during early mammalian development and little is known about its requirement in the adult brain.

For that reason, McBride and coworkers chose a different mouse model where a triplet expansion had been inserted into one of the existing mouse htt alleles which has the added advantage that it also preserves the natural expression pattern of htt.

Encouragingly, a 50-60% knockdown of mouse htt was observed over a period of 4 months without any obvious target-specific side-effects. Transduction of this particular AAV administered to the striatum, a region in the brain particularly affected in HD, was fairly broad and specific for neurons, the cell type critically affected during HD. Given that not every neuron was transduced and that non-neuronal cells may also express some htt, this 50-60% knockdown implies that very efficient knockdown of htt can be achieved in a subset of neurons and (based on previous studies) should be therapeutic and safe. Longer term studies are ongoing.

The unpleasant surprise, however, was that as previously reported by a number of other groups, some of the tested U6-driven shRNAs were toxic. This is likely caused by the very high levels of small hairpin RNAs from the strong U6 promoter which may jam up the endogenous microRNA gene regulation which is harnessed by RNAi. Whether it is just the level of the hairpins or also the specific biogenesis of the shRNAs, which only resemble microRNA precursors, but actually undergo a slightly different biogenesis involving an unusual set of enzymes and RNA structures, is an open question. In any case, to make a long story short, it is probably advisable that future DNA-directed RNAi Therapeutics programs avoid U6-driven shRNAs altogether and instead use other Pol III-driven systems or Pol II promoters. The latter have the added safety advantage as they can be chosen to be tissue specific so as to minimize side-effects in non-target cells. Since efficient RNAi does not require high small RNA levels, vector transduction and long-term expression should in many applications be more important considerations than mere promoter strength.

Whether it is advisable to go ahead with the particular U6-shRNA that was found to be non-toxic and efficient in knockdown, or instead go back and re-design all of the vectors and repeat 2-3 years of pre-clinical studies, must be a tough decision to make, a situation similar to that faced by other RNAi Therapeutics programs currently in development given the steep learning curve the field is still undergoing.

It is maybe for these reasons that Merck’s subsidiary Sirna Therapeutics decided not to continue with this program and fully hand over the commercial rights to Targeted Genetics. In some way, the AAV-RNAi program always stood out as being an awkward fit for Sirna Therapeutics as it seemed at odds with their siRNA modification focus. Maybe at the time there were not that many advanced RNAi Therapeutics programs to chose from so that Dr. Davidson’s studies seemed like an opportunity to fill the early pipeline. Given the unpredictability of early studies and the number of RNAi Therapeutics programs under evaluation, it is best to let pipeline decisions be driven not by history, but by where the science is progressing swiftest. AAV RNAi for HD still has much promise and the path forward fairly obvious, but as many things in drug development it will be a long and arduous one.

Thursday, April 10, 2008

RNAi Therapeutics and the Indiscriminate Press

I was taken aback by the crude reporting on the back of a recent Nature paper about the potential immunostimulatory potential of siRNAs which may be responsible for any apparent efficacy seen with RNAi drug candidates currently in clinical development.

Needless to mention, the potential of nucleic acids in general, and double-stranded RNAs in particular, to elicit innate immune responses has long been known. In fact, the very discovery of siRNAs as triggers of gene-specific silencers in vertebrate cells was partly based on the hypothesis that such dsRNAs should not trigger an interferon response. That early RNAi Therapeutics programs, may not have sufficiently taken into account all the potential innate immune surveillance mechanisms present in the body, as at the time the field was still on a steep learning curve, should also not be too surprising, and I have pointed out before the risks of rushing RNAi Therapeutics into the clinic . But since two of RNAi’s most advanced, first generation candidates are nearing the crossing line, one probably understands why the press and critics of RNAi Therapeutics caught onto the findings the way they did.

A deceptively balanced New York Times article left us with the impression that the Nature paper marks a serious setback for the development of RNAi Therapeutics, instead of emphasizing that while RNAi is a relatively easily accessible tool in the laboratory, the study shows that the success of RNAi Therapeutics depends on companies and collaborations with the means and commitment of establishing a safe and efficacious RNAi drug development paradigm. I would also predict that a number of current clinical RNAi candidates will only poorly knock down their actual target in vivo and may elicit additional responses, although this does not exclude that as long as they are safe and show therapeutic efficacy they may still be considered approvable.

Scientists did their part in confusing the public to the extent that the outsider may be forgiven if he/she started to doubt the very existence of RNAi:

“The discovery is "paradigm shifting," said Dr. Charis Eng, chairwoman of the Genomic Medicine Institute at the Cleveland Clinic who works with siRNAs in cancer research. "Up until now, we all believed it's absolutely specific for gene X, so it prevents gene X from doing its job," she said.”
“Paradigm shifting?” “Absolutely”? Or how about the following passage:

"RNA interference does, of course, exist," said Ambati, a University Research Professor and the Dr. E. Vernon Smith & Eloise C. Smith Endowed Chair in Macular Degeneration Research. "It is just that siRNA functions differently than commonly believed -- not via RNA interference."

Huh? siRNA functions, but not via RNAi? I don’t think he referred to the microRNA pathway here, and, to be generous, it may have just been an awkward attempt to explain his findings to a lay person, but wouldn’t it be the responsibility of the reporter, interested in educating the public about an emerging biotechnology, to avoid such ambiguous and factually wrong statements by confirming with Dr. Ambati the scientific correctness of his statements?

Or how about the following headline from RNAi News (which btw I otherwise think does a fantastic job on reporting on RNAi developments- though not for free, of course):

“Study Shows All siRNAs Have Anti-Angiogenic Property Associated with Immune Response”

Sounds pretty dire and does not leave much to be hoped for.

Having criticized the press for careless reporting, it would be, however, equally wrong to brush the whole RNAi-immunostimulation issue aside as being made up by the press and shorts. That would be making it too easy. In an excellent and unusually candid review article on the potential innate immune response to siRNAs, Ian MacLachlan (CSO of Protiva, soon to be Tekmira) predicted to the T the Nature results and made the somewhat unsettling observation that when his group investigated siRNAs from a number of the early preclinical RNAi studies for AMD, cancer, and infection, they found a disturbing bias in the inherently immunostimulatory potential of “active” vs “control” siRNAs. Fortunately science does not stand still and it is encouraging to see that it has become common practice for RNAi Therapeutics studies to look at the potential non-specific immune responses.

A few words on the scientific and IP implications of the interplay between RNAi and innate immunity. At least in the case of synthetic dsRNA-triggered RNAi, it appears that, a priori, being appropriately modified, short and overhung is safest. In the cytoplasm, long dsRNAs may trigger interferon responses and blunt ends may be recognized by sensors of foreign RNA. TLR7 is encountered by dsRNAs (but also ssRNAs) delivered via endosomal uptake, and as the Protiva group has so elegantly shown this may be abrogated by simple 2’-O-methyl modifications. TLR3 was reported in the Nature study to be activated by siRNAs on the cell surface and thus would be invisible to siRNAs delivered within “fat globules” according to Merck’s RNA Therapeutics VP Alan Sachs referring to liposomal delivery technology. SiRNAs, however, would have to be over 20 nucleotides long to facilitate TLR3 dimerization necessary for signaling. It is therefore likely that dimerization would be particularly susceptible to modifications at the ends of the dsRNA, which should also be compatible with silencing activity. I’m sure that as I speak, the systematic analysis of TLR3 activation by siRNAs and the effect of modifications is ongoing, if not already largely completed by some groups.

One has to also keep in mind that the Nature findings were made in mice and the rules of innate immunity are notoriously difficult to translate into primates. Based on my own over-the-weekend literature research, it appears to me that TLR3 activation in humans may well occur in the endosome, and based on structural studies, 21 base-pair dsRNAs should hardly trigger TLR3 responses in human cells. In agreement with this, Dharmacon scientists have shown before that 27mer Dicer-substrates, but not classical Tuschl siRNAs trigger most likely a TLR3-related immune response. That does not mean that modifications to Dicer substrates should not be able to abrogate TLR3 activation, but shows that by choosing non-standard, less studied designs, often as a means to circumvent IP, new obstacles may emerge for which there will be relatively little support by the general research community.

Finally, since tonight is the night to challenge some of the little scientific inaccuracies propagated in the press and companies in the space, I would like to take a look at the press release issued by Silence Therapeutics that publications such as the TLR3 in Nature “SUPPORT SILENCE THERAPEUTICS COMBINED DEVELOPMENT APPROACH”. While it is true that in the two studies by Silence Therapeutics that were cited in the press release, immune responses were not found to be triggered by the siRNAs used therein, referring to Alan Sachs’ “fat globule” comment to support the rationale behind Silence’s lipoplex technology is somewhat misleading since according to the formulation method and drawings in the papers, siRNAs should be externally associated with the liposomes and therefore in theory be exposed to cell surface TLR3, and are not internally captured as is the case with the liposome technology used by Merck and others. Another little fact not mentioned in the press release is that the absence of cytokines was determined for 19mer dsRNAs and are therefore different from Silence’s claimed, and notably larger 23mer “AtuRNAi” molecules.

Monday, April 7, 2008

Journal Club: Therapeutic RNAi Delivered via Vitamin A-Coupled Cationic Liposomes Reverses Liver Cirrhosis in Rats

Illustrating how the basic concept of liposomal RNAi delivery can be modified to target specific cell types at low dosages, a study by Sato and colleagues published last week in the online version of Nature Biotech shows how the addition of vitamin A to cationic liposomes carrying siRNAs against Hsp47 can not only efficiently knock down the gene in hepatic stellate (HS) cells and block disease progression, but that this can actually reverse cirrhosis in a number of rat models for this disease [Sato et al. (2008). Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nature Biotech doi:10.1038/nbt1396].

It is known that by adjusting their stability and therefore pharmacokinetics, liposomes can be adjusted to shift their relative accumulation in tissues such as liver, tumour, or lung. For example, recent improvements in formulation methods now allow for the delivery of around 90% of the injected dose to the liver. While for liver applications, this already minimizes the exposure of non-target organs to the RNAi therapeutic, the ability to knock down genes in specific cell types at low dosages should further improve its therapeutic index.

Adding small conjugates such as sugars (e.g. Mirus Bio’s DynamicPolyConjugates to target RNAi to either hepatocytes or Kupffer cells) to a basic delivery formulation appears to be particularly promising. The authors of the present paper reasoned that since the HS cell which is central to liver cirrhosis efficiently takes up vitamin A, adding this vitamin to liposomally formulated siRNAs may facilitate their uptake in HS cells, in addition to endowing the nanoparticle with favorable pharmacokinetics by binding to retinol binding protein in circulation.

Indeed, while some non-specific uptake was observed into phagocytic cells, the modified A-liposomes efficiently entered HS cells in a vitamin A-dependent manner while largely avoiding other cell types of the liver, including hepatocytes. Actually, delivery was truly targeted since overall drug uptake was greatly enhanced in cirrhotic rats versus non-cirrhotic rats, likely a reflection of the HS cell activation/proliferation state.

Importantly, knockdown of Hsp47, required for collagen production and therefore a potential target for treating cirrhosis, was achieved at the clinically relevant low dosages of 0.1mg/kg to 0.75mg/kg, resulting in the survival of rats concomitant with resolution of the cirrhosis both by histology and normalization of liver enzymes.

The paper also shows that as we are learning more about the molecular biology and pharmacological properties of synthetic RNAs, the scientific standards for publishing papers on therapeutic RNAi have risen. Consequently, the sequence-specific nature of the therapeutic effect was demonstrated by the use of two additional Hsp47-targeted siRNAs. Cytokines and interferon-alpha were also looked at, including IL-12 which has recently gained notoriety
as a TLR3-related pro-inflammatory cytokine. Reassuringly, no IFN-alpha, TNF-alpha, or IL-12 induction was detected at the reasonably early 7 hour time-point with this unmodified Dicer-substrate (Dicer substrates are longer and therefore should be more prone to immune recognition). However, some non-specific elevation in the apoptosis of rat liver HS cells was observed following treatment with VA-liposomes carrying a control siRNA. Albeit small, it remains to be determined whether this was related to sequence-specific cytokine induction, or liposomally or vitamine A-induced cytotoxicity.

It is likely that the systematic screening for improved VA-liposome chemistries and optimized siRNAs should considerably improve the therapeutic index of this interesting variation on the cationic liposome for siRNA delivery. Similarly, vitamin A derivatives that are still taken up by HS cells, but do not cause potential vitamin A-related toxicities may be warranted for actual clinical use.

Tuesday, April 1, 2008

Final Day of Keystone RNAi Conference

There were quite a number of RNAi Therapeutics related talks on the last day of the conference. Muthiah Manoharan from Alnylam Pharmaceuticals gave an overview of some of the important chemistries and formulations the company is developing.

The liposomal delivery section of his presentation, Alnylam develops liposomes both for dsRNA as well as single-stranded antagomir delivery, proved a bit hard to follow (or maybe it was just me having difficulties concentrating towards the end of the conference) since some data related to Protiva SNALP technology, then Tekmira developed novel cationic liposome formulations, and then the next moment to lipid-like particles. It is befitting that given the overlap and complementarity in terms of biodistribution and pharmacokinetics of these technologies much of this has now been consolidated into the new Tekmira. If I have it right, the previously shown successful repeat-administration data for sustained gene knockdown in the liver made use of MIT’s lipidoid technology, while the impressive mouse liver cancer data were the result of a Protiva collaboration.

Conjugation of siRNAs to cholesterol is well known for the systemic delivery to the liver and jejunum. Less well known in the context of siRNAs is the PEG-conjugation. Since according to Dr. Manoharan this conjugation facilitates equally efficient delivery to the jejunum as cholesterol does, but largely avoids the liver, PEGylation strategies may lessen the risk for liver toxities in targeted therapies of the jejunum.

With respect to modification technology, in contrast to Merck’s (notably absent from the speaker roster) emphasis on siRNA modification (‘siNAs’), Alnylam follows the natural trend and applies modifications as little as possible. It is interesting that they are still using the classical Tuschl dTdT 3’ overhang structure, albeit substituting a thioate for a phosphate in the last linkage. Their favorite modification, however, appears to be the 2’-fluoro modification, long a workhorse for many RNA technology platforms before RNAi, which has now also proven to be very promising for RNAi Therapeutics. This modification imparts not only stability on the siRNA, but also helps in avoiding immune recognition and favors a molecular conformation that may aid in target recognition. In addition to the associated higher lipophilicity, all of this appears to result in significantly improved knockdown efficiencies.

Next on the list was the session chair of the afternoon RNAi Therapeutics workshop, Frank Bennett from the antisense company ISIS Pharmaceuticals. ISIS is developing single-stranded RNAs for the induction of therapeutic RNAi. This may be due to their IP estate in single-stranded antisense as well as the notion that single-stranded molecules may have cost and delivery advantages. The flexibility of single-stranded molecules plus its amphipathic nature, particularly when extensive phosphortioate backbone modifications are applied, should make it easier for ssRNAs to cross the cell membrane (note, however, that naked antisense delivery may not always be sufficient as their own Regulus venture is looking at systemically formulated, conjugated or liposomal, antisense for the inhibition of microRNAs). Maybe not surprising, ISIS, too, has found 2’fluoro modifications to be “magic” as this has significantly improved single-strand RNAi potency, with the IC50s in a number of cases in the low or even subnanomolar range. But his own comparisons show that since 2’fluoro also improved the potency of dsRNAi, the fold-difference in potency between single-stranded vs dsRNA RNAi has not really changed, with ssRNAi being 10-fold less efficient at best (please correct me if I am wrong here, but numbers probably based on lipoplexed oligo delivery in tissue culture). For example, the most potent ssRNAi shown molecule had an IC50 of 0.43nM, while the corresponding 2’fluoro siRNA came in at 0.01nM.

Part in me believes that ISIS has a sincere interest in developing ssRNAi for their future pipeline, but another part makes me believe that ISIS’ work is supposed to widen their claim onto IP in RNAi. Accordingly, Bennett noted that in was in 1998 that ISIS evaluated the use of single-stranded RNAs to modulate gene regulation, and as it turns out, these RNAs worked via RNAi. In any case, this was a fact-based presentation and not meant as an advertisement of single-strand RNAi or antisense technology and it will be interesting to follow just how efficiently single-stranded RNAs can be designed so that they are recognized by RNAi.

David Lewis (Mirus Bio) then gave us an update on his company’s progress with Dynamic Polyconjugates (DPC), a non-liposomal technology for the systemic delivery of RNAi to the liver. As I had
noted here before , DPCs may be particularly promising in terms of safety, as depending on the attached sugar, Kupffer cells and hepatocytes can be selectively targeted. This was corroborated by the presentation of extensive safety data, a topic that unfortunately was not really addressed in the previous two speakers’ presentations. Knockdown was remarkably sustained, and particles repeat-administrable in mice for over 100 days without loss of silencing activity, at relatively low 1.25mg/kg doses. Moreover, sustained silencing was also demonstrated for non-human primates. It is notable that by targeting ApoB in monkeys, they, like many other groups, also observe the fatty liver phenotype. The small size of DPCs should make the technology also a candidate for more difficult-to-penetrate tissues.

Beverly Davidson (Iowa) summarized her group’s experience with AAV-shRNAs for the treatment of Huntington’s Disease. Like many other laboratories have observed, they also find toxicity following U6-driven shRNA expression. Albeit very potent, it is probably recommended to stay away from the U6 promoter for future development programs, especially when high copy-numbers of expression cassettes may get into individual cells, and there are more than enough alternative potent hairpin expression technologies that do not cause toxicity. According to Davidson, incorporating the RNAi trigger within a microRNA context for example was safe. AAV delivery to the striatum, the anatomical site that matters most in HD, was “phenomenal”, particularly with a viral preparation from their collaborators at Targeted Genetics (Seattle). Before RNAi gene therapy for HD can progress into the clinic though, it will be important to determine whether knocking down both the wild-type and mutant huntingtin alleles is safe or whether allele-specific knockdown is required. Studies that address this issue are now ongoing. Overall, I am quite optimistic about the future of AAV- and lentivirally mediated RNAi gene therapy.

Judy Lieberman (Harvard), together with Phil Sharp a co-organizer of the conference, presented more data on targeting cancer stem cells by RNAi, the underlying rationale being that in order to get rid of a tumour, you really need to get rid of the cancer stem cells from which the tumour mass derives. While previous work centered on antibody-protamine siRNAs targeted to stem cell-specific surface receptors, her latest work makes use of targeted liposomes, which can be potentially more consistently manufactured as well as have the added advantage that many siRNAs can be delivered with just one liposome. Using this to deliver let-7 mimics, a microRNA that is becoming more and more clear to serve as a key differentiation factor, her group showed that breast cancer stem cells dramatically lose their ability to self-renew as well as metastasize to the lung and liver.

Equally interesting was Markus Stoffel’s presentation (ETH, Zurich) on the mechanism of cholesterol-conjugated siRNA uptake. In a study previously published in Nature Biotech and discussed here , Stoffel and his collaborators at Alnylam found that the favorable pharmacokinetics and liver uptake of cholesterol-conjugates is due to their association into lipoprotein particles of the blood, and that it is cholesterol-siRNA bound by LDL that carries the RNAi to the LDL-receptor on the hepatocytes, thereby causing gene silencing in the liver. Moreover, cholesterol-siRNA pre-formulated with LDL in vitro and then administered into mice, proved useful in enhancing the hepatic delivery of RNAi.

Extending this work, his group has now found that cholesterol-conjugated siRNAs can also be consistently formulated with Intralipid , a clinically tested fat emulsion used for nutritional purposes as well as a drug carrier for certain anaesthetics, especially for indications of the heart. Quite excitingly, and in retrospect maybe not that surprising, it turns out that cholesterol-siRNAs are not delivered to the liver (a potential safety advantage when targeting other tissues), but instead go to the lung and even better, the muscle, including heart. At reasonable 10mg/kg, they observe an 80% ApoB knockdown in the lung, but this needs to be repeated now targeting genes more highly expressed in muscle/lung. Developments such as this are all the more important, since the ability to target new tissues opens up RNAi Therapeutics to a whole new spectrum of disease (note that this is an siRNA method and gene therapy methods such as AAV-mediated RNAi may also be useful for the systemic treatment of the heart).

Represented in the poster session was work by Nastech on meroduplexes. Maybe I don’t get it, but the suggested advantages of meroduplexes are so tenuous while creating all the additional challenges in developing tri-partite siRNAs. If Nastech was really making progress on delivery as has been suggested, it would make more sense focusing on those efforts which should be sufficient to create partnering interest and financing at favorable terms. I’m sure a company like Alnylam would be more than happy to then cross-license siRNA and delivery IP.

Finally, a poster by Takanori Yokota (Tokyo) reported on the delivery of alpha-tocopherol conjugated Dicer-substrate siRNAs to the liver. Effective silencing of ApoB in mice was achieved at low mg/kg dosages and was apparently well tolerated, except for the- you hear it once again- ApoB knockdown-specific fatty liver phenotype.

Overall, this year’s Keystone has proven a must-go for those that can only go to only a limited number of scientific meetings, giving them a chance to catch up on the important developments in the field over the past year and getting a glimpse into emerging trends. Next year, the RNAi community will be split between three RNAi-related Keystone meetings, one on the basic mechanisms of RNAi, another one centered on RNAi Therapeutics, and finally one on microRNAs and cancer just illustrating how much this field has grown and diversified.

….and the Winner Is: Pfizer and Coley Pharmaceuticals

While at the Keystone meeting, I was reminded that we may already have a winner for the RNAi Therapeutics Deal contest highlighted on this blog. As this contest as to who will be involved in the next large transaction of the RNAi Therapeutics space was still ongoing, Pfizer quietly bought Coley Pharmaceuticals. Although I noted this in one of my entries
and wildly speculated that this $164M acquisition may serve as a stepping stone towards grander RNAi Therapeutics ambitions, it was the 2008 Pfizer Analyst Day that made it clear that the major focus of Coley together with the RTC in Boston would be the development of RNAi Therapeutics.

Given the importance of considering TLR receptors for the design of RNA therapeutics, only highlighted by the recent Nature paper
on the class-related antiangiogenic potential of dsRNAs for the treatment of age-related macular degeneration, it now appears to have been a particularly shrewd acquisition bringing in TLR know-how as well as an established nucleic acids operation. For those hoping another Pfizer-related deal may be imminent, I would rather be skeptical as Pfizer probably has now their hands full organizing and evaluating the potential of this new platform.

RNAi Therapeutics Investment Tracker Update

Following today’s announcement by Protiva and Tekmira, I was finally able to face the reality of the plunging stock market, and updated the investment tracker. Due to the spin-out of RXi out of CytRx, I have liquidated as of today the CytRx part of the deal and reinvested the proceeds into RXi so that there are now 40.85 shares in the portfolio purchased at an adjusted share price of $12.24. Other positions were left unchanged, although progress was noted for Nastech and Rosetta Genomics.

Nastech has successfully appointed a scientific advisory board for mdRNA, collecting Nobel laureates as if they were stamps and suited for their RNAi delivery efforts. It is now critical to build on this with a successful fund-raising round and hopefully a financial separation between the nasal delivery and RNAi Therapeutics businesses. I do understand that there are overlaps, but experience has shown that exposing RNAi Therapeutics to the risk of an unrelated technology has hurt RNAi Therapeutics development efforts rather than helped it. Also, the meroduplex poster at the Keystone meeting did not make much sense to me as the whole idea is based on the premise of circumventing Alnylam’s IP no matter whether it makes sense scientifically or not. I’d rather see a company focus on delivery and license the target rights accordingly, than a company trying to re-invent the wheel and consequently making drug development for themselves even more difficult than it already is.

Rosetta Genomics has impressed me at the Keystone conference by their presence and breadth of science. The RNAi/microRNA world is watching them as they prepare the launch of their first microRNA diagnostics this year.
By Dirk Haussecker. All rights reserved.

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