Thursday, September 27, 2007
Nature Publishes Reassuring Study by Alnylam on the in vivo Delivery of siRNAs and their Effect on the Endogenous microRNA Pathway
Indeed, competition with the microRNA pathway in vivo which in some cases caused the death of mice, were first reported in a study by Grimm and colleagues in the journal Nature last year. Ironically, rather than a demonstrating a failure of the viral delivery system used in that study, it was the extreme efficiency with which small RNAs could be expressed with the double-stranded AAV vectors that allowed the competition to be observed. In what is an often overlooked aspect of these studies, compared to non-viral delivery methods, silencing efficiencies of over 95% can be easily achieved with these vectors at doses that have no adverse effects on either the microRNA pathway or the viability of mice.
In an ideal world, the Grimm et al. studies would have been embraced as an opportunity to study the dose-limiting steps of therapeutic RNAi to inform future RNAi therapeutics strategies. Instead, as the Press lives from feeding the public the simple messages, rather than reporting complicated truths, it decided to label the studies as yet another example of the dangers of gene therapy, and - somewhat understandably- caused some companies involved in developing RNAi Therapeutics to distance themselves from DNA-directed RNAi for political reasons.
As the trusted leader of RNAi Therapeutics, Alnylam was therefore given the platform to reassure the RNAi community this week in Nature that, unlike AAV-RNAi, liposomally delivered siRNAs had no obvious adverse effects on the endogenous microRNA pathway (John et al., 2007). The study further highlighted that liposomal siRNA delivery has advanced to a point where around 80% gene silencing in hepatocytes can routinely be achieved following systemic administration of therapeutically viable doses of siRNAs, including their repeat administration.
Although I welcome Nature’s decision to document progress in the important area of RNAi therapeutics, and understand Alnylam’s desire to publish in the highest profile journals, I would like to take this opportunity to address a few misconceptions about the studies. One important misconception is that delivering RNAi with AAV per se is more toxic. To make this point, a direct comparison of the intrahepatic levels of small RNA levels following both routes of administration would have been necessary. Given the >99% transduction efficiency of double-stranded AAV in mice and the consequently extremely high gene silencing efficiencies, it is quite likely that double-strand AAV vectors are currently the most potent delivery system to the liver in terms of small RNA delivery and gene knockdown.
I would therefore not be surprised at all to see similar competition with microRNA function following administration of very high siRNA dosages. This is supported by numerous studies that have shown competition for gene silencing when very high levels of two or more siRNAs were introduced simultaneously into tissue culture cells. However, given the ability of hundreds of microRNAs to function in a given cell at any time, such observations represent only extreme cases and suggest a wide therapeutic index. Unfortunately, the relatively small range of doses used in the John et al. studies (2mg/kg to 5mg/kg) did allow for a careful evaluation of related competition in vivo and concomitant dose-limiting toxicities.
I guess the purpose of this Blog really is my plea to the field of RNAi Therapeutics to keep learning from each other, instead of letting the Press and uninformed “analysts” play on the fears of investors, through their indiscriminate use of buzzwords, thereby polarising and separating what really belongs together. In this spirit, I would like to stress that this study is yet another proof-point of the viability of RNAi for therapy leading up to the possibly first proof-of-concept gene silencing results in Man to be revealed in the coming months- once again by Alnylam.
PS: Although a combination of liposomal delivery methods were used in these studies, the details were not disclosed. Apparently, another study on lipidoid-delivered siRNAs, a technology developed by the Langer and Anderson groups at the MIT, has been submitted to Nature Biotech and is about to be published. Lipidoids differ slightly from the Tekmira-owned SNALP technology, and looks likely to be the technology used for Alnylam’s first clinical systemic RNAi program (liver cancer or hypercholesterolemia), for which an IND is expected by the end of 2007. More than knockdown efficiency, we should be looking for the toxicity profile as I regard this to be the big unknown that will determine the success of this program, particularly if it turns out to be for hypercholesterolemia.
Sunday, September 23, 2007
Merck was an early entrant into the emerging RNAi therapeutics field, and acknowledged Alnylam’s fundamental technology and IP leadership when they signed a broad co-development deal in 2003. It was clear, however, that Merck’s ambition has always been to play a more prominent role in RNAi therapeutics, and has been instrumental in energising the effort through a number of initiatives such as their support for this year’s RNAi Therapeutics meeting in Keystone, an RNAi delivery challenge, and bringing leading bioinformatics to bear on addressing off-targeting through their Rosetta Inpharmatics subsidiary.
One year ago, Merck woke up the investment community by announcing the acquisition of Alnylam’s chief rival, Sirna Therapeutics, for a then whopping $1.1B. Following the millennium technology bubble and stalled progress with developing ribozyme therapeutics, Sirna transformed itself into an RNAi company by leveraging its existing RNA capabilities to rapidly screen many genes with numerous siRNAs and claim them in their patent applications as exclusive RNAi therapeutics targets. Based on this achievement, Sirna Therapeutics promoted itself as the leader in RNAi Therapeutics, although it has always been questionable whether such a brute-force approach would eventually stand up to the novel and non-obvious criteria required of a patent.
So why did Merck end up buying Sirna, not Alnylam which they chose to partner with in the first place? One can almost be certain that Merck approached Alnylam. However, unlike management at Sirna Therapeutics, schooled in the art of turning around flagging businesses and probably a little bit focussed on maximising short-term shareholder value, Alnylam always had full confidence in the strength of their IP position and the prospect for RNAi therapeutics and consequently put in place mechanisms that would make an unsolicited takeover almost impossible
Ironically, the events that followed the Sirna acquisition by Merck further underlined Alnylam’s dominant RNAi therapeutics IP position. It seems odd that for a company that touted itself as the leader, many of its former staff have now left the company, and it appears that except that Sirna’s proprietary development pipeline, except for its AMD program that it had licensed to Allergan, had been completely overhauled.
From Alnylam’s perspective, the advantage of terminating their partnership with Merck appears clear. As an early deal that largely benefited Alnylam in terms of credibility rather than financially, it was far less attractive compared to the more recent deals with Roche and Novartis (up to $120M compared billions in future milestones plus royalties), so that the freed up time, resources, and RNAi therapeutics targets (nine following an amended 2006 agreement in addition to Nogo for spinal cord injury) can now be focussed on more rewarding partnerships and in-house development programs. It also means that Alnylam would not be fostering a Merck increasingly positioning itself as a rival rather than partner by denying them access to their considerable know-how.
It also becomes more and more apparent that Merck is suffering from the consequences of having inherited Sirna’s aggressive business practices. As part of an ongoing dispute with the Canadian biotech company Protiva Biotherapeutics which accused Sirna of having misappropriated trade-secrets relating to liposomal RNAi delivery, a recent court order is likely to have adversely impacted Sirna’s systemic RNAi delivery efforts with potentially further financial liabilities down the road. In Europe, Sirna was also engaged in opposing Alnylam’s Kreutzer-Limmer patents relating to the use of short double-stranded RNAs for gene silencing in mammalian cells, the very same IP to which Merck gained access through their Alnylam collaboration. After the Sirna takeover, however, Merck appears to have made the crucial mistake of continuing to oppose those patents. I suspect, although I am not familiar with the details of the Merck-Alnylam agreement, that this alone should have given Alnylam the right to unilaterally terminate the type of relationship that Alnylam, in the wake of the Alnylam-Roche alliance, repeatedly said it would never pursue again.
To be sure, the value of Sirna may have been higher to Merck than to other pharmaceuticals since Sirna’s technical RNA capabilities should have nicely complemented Merck’s existing RNAi therapeutics development and bioinformatics initiatives. However, given the strength of Alnylam’s patent portfolio, the Tuschl and Kreutzer-Limmer series to name just two, it is difficult to imagine that Merck is really convinced that it has the freedom to bring RNAi therapeutics to market without a license from Alnylam. It will therefore be interesting to follow whether Merck will now pursue a late, but more costly licensing strategy, or whether it will respond to the new situation in unusual ways. In any case, I hope that the new rivalry between Alnylam and Merck will be a friendly albeit competitive one for the benefit of the entire field of RNAi therapeutics.
Tuesday, September 18, 2007
Journal Club: Alnylam and Collaborators Make Progress in Understanding and Optimising siRNA Uptake In Vivo
The study by Wolfrum and colleagues follows another high-profile publication 3 years ago (Soutschek et al.) where Alnylam scientists demonstrated gene silencing in mice following systemic administration of cholesterol-conjugated siRNAs. That study showed that although such siRNAs could silence genes particularly in the liver and gut, quite high amounts of siRNAs were needed (50mg/kg). By studying the uptake of the siRNA conjugates in these tissues, the authors not only hoped to understand why they functioned at all, but also to optimise their potency.
Efficient in vivo drug delivery requires favourable pharmacokinetics. Particularly, a drug has to be present in the blood for sufficient length of time so that it has a chance to accumulate in its target tissue. One reason for example why many experimental drugs fail is because they are rapidly excreted through the kidneys. This may often be prevented if the drug could interact with components of the blood such as the abundant lipoprotein particles.
Indeed, the authors find that siRNAs conjugated to cholesterol or other lipophilic molecules associated with the similarly greasy HDL and LDL lipoprotein particles. These would ferry them around in circulation and bring them into the proximity of cells that carry on their surface receptors for either HDL and/or LDL. Strikingly, pre-assembling the siRNA with purified HDL and LDL particles quite significantly increased the potency of the siRNAs. Furthermore, mice lacking either of the receptors for the lipoproteins were much less prone to gene silencing by the same pre-formulated siRNA particles.
In a further interesting twist, it was shown that siRNAs were not taken up by the cells as part of internalising lipoproteins, but that the siRNAs would take advantage of their proximity to the cell membrane during the docking, release, and re-docking process of their lipoprotein carriers with their receptors. Amazingly, through a combination of gene knockdown experimentation and blockage by antibodies, at least one of the actual entry routes for the siRNA was inferred to be the human homologue of the SID-1 gene that had earlier been shown to mediate systemic RNAi in the worm C. elegans.
Systemic RNAi describes the spread of an siRNA from one cell to another cell in the same or even different tissues. Systemic RNAi in worms and plants is associated with the amplification of RNAi, and both systemic RNAi as well as RNAi amplification were thought to have been lost during human evolution. It is therefore a surprise that SID-1 would still function in siRNA uptake, with demonstrated selectivity for siRNAs relative to other types of nucleic acids. This also raises the intriguing possibility that some sort of natural siRNA uptake should occur in humans.
Of more immediate importance, the present paper opens the door for the systematic screening of new lipophile-siRNA conjugates with improved association kinetics with lipoprotein particles, or even pre-formulation of such conjugates with lipoproteins or other natural or synthetic carriers of the blood. I look forward to what this line of investigation will yield next.
Sunday, September 16, 2007
I would therefore like to take a closer look now at Rosetta Genomics, next to Regulus arguably the only other major pure-play microRNA-focussed company. Rosetta has pleasantly surprised me by assembling a strong IP portfolio, which it has then followed up with a series of well-designed corporate and academic partnerships. This is complemented by a growing tool-box allowing for clinically-relevant extraction, detection and measurement of microRNAs. Like other players in this field, Rosetta believes that given the emerging importance of microRNAs in gene regulation, these molecules would also be involved in human disease so that they could be both harnessed for clinical diagnostics and therapeutics.
Rosetta is an Israel-based company, founded on the discovery and patenting of human microRNAs using high-throughput computing and bio-technologies (2005 Nature Genetics study). In the wake of the Human Genome and other sequencing projects, the founders of Rosetta hypothesised that the key to human complexity was not due to an increased number of genes, but at least partly due to the emergence of primate- and even human-specific microRNAs, and their search for new microRNAs consequently accommodated that notion. This was against the mainstream of most microRNA discovery efforts then which heavily relied on the notion of biological conservation, and Rosetta would be able to detect a number of microRNAs that had been missed.
Indeed, their hypothesis was supported by their 2005 Nature Genetics paper, almost doubling the number of sequenced human microRNAs at that time (adding 89 microRNAs), a number of them not conserved beyond primates. Based on partly theoretical considerations, predictions as to the total number of microRNAs were also revised upwards from initial estimates in the field of around 250 to well over 800. These efforts have resulted in patent applications exceeding 500,000 pages, probably using the same computing power used for predicting microRNAs.
I should add, however, that most of these non-conserved microRNAs were restricted to 2 clusters in the genome and should therefore be of less diagnostic value as would be expected for an equal number of more randomly distributed microRNAs. Furthermore, most of the previously cloned microRNA, particularly those by Thomas Tuschl, licensed exclusively for therapeutics use to the parent companies of Regulus, Alnylam and ISIS, should be amongst the biologically most important microRNAs simply based on their higher expression levels (the reason why they were detected by cloning in the first place).
At that point, I thought just another publication based on bioinformatics that was showing that the complexity of microRNAs may be higher than initially thought. Also, their theoretical approach and computer-driven technologies made me wonder whether this would ever develop into a meaningful hands-on biotechnology operation.
Rosetta took a number of steps to change this perception. First, it has gained access, at least for diagnostic use, to the large majority of human microRNAs through licensing agreements, most importantly with the Max-Planck Institutes and Rockefeller. Next, similar to what Alnylam has done, they have come out with a number of high-quality, peer-reviewed publications, ranging from microRNA detection technologies to the functional elucidation of certain disease-associated microRNAs. Partly, this was done through academic collaborations which allows them to stay product focussed and capitalise on opportunities should they arise from discoveries in microRNA research. Other collaborations with corporate and clinical partners have given them access to relevant technologies such as one with ISIS for the therapeutic targeting of microRNAs using antisense technology, and clinical specimens from hospitals which will be used to test their diagnostics.
How they were able to orchestrate this transformation is not clear to me and quite impressive, but looking at the line-up of illustrious early investors and SAB (scientific advisory board), populated with Nobel Laureates and the likes of Robert Langer (also on Alnylam’s SAB), suggests that they have enough influence to get the attention of key audiences. The expansion of their activities in the US should further nurture current and future partnerships and attract new investors.
These investors may be attracted by Rosetta’s first issuances of microRNA patents and its strategy to use early revenues from their more mature microRNA diagnostics efforts to fund the potentially more lucrative area of microRNA-based therapeutics on quite attractive financial terms. It is their aggressive goal to have 3 microRNA diagnostics products on the markets by the end of next year, with their most advanced program being for the classification of Cancer of Unknown Primary (CUP) where the goal is to identify the original tissue from which a cancer has spread. A recent presentation at the AACR cancer meeting suggests that this can be achieved with 85% accuracy by profiling 19 microRNAs. Their initial therapeutic pipeline, meanwhile, focuses, similar to Regulus, on diseases of the liver, such as liver cancer and HCV infection. This is done in collaboration with ISIS Pharmaceuticals, and it will be interesting to see how the recent formation of Regulus will affect this relationship.
After a difficult IPO and little attention from Wall Street, the time is ripe for Rosetta and Regulus to lead the charge in translating the important biology of microRNAs into medical use.
Sunday, September 9, 2007
Forming this joint venture not only consolidates and complements much of the IP in microRNA-based therapeutics, it also allows the respective companies to focus on their core operations in RNAi and antisense while giving the new company a distinct identity and independence that should incentivise their employees and help attract 3rd party funding, particularly in the form of alliances with larger partners. The joint venture also acknowledges the fact that microRNA therapeutics sets a new paradigm for treating disease with its unique set of challenges and risks.
Based on pioneering work by Regulus SAB member David Bartel and his group at the MIT, it is now believed that the approximately 1000 human microRNAs regulate around one third of our genes, “that is one third of our genes” (Stanley Crooke). It follows that each microRNA targets multiple genes. Targeting a microRNA for therapy is therefore based on the premise that the functions of these genes are linked to a common biological process hence limiting the potential for unwanted side-effects by disturbing unrelated pathways. In this sense, microRNA- and RNAi-based therapies are opposite philosophies where siRNAs seek to surgically target single disease-associated genes, whereas microRNAs are aimed at whole networks. Genetic research supports both approaches, and a number of microRNAs have been shown in animal models and human cells to specifically affect discrete regulatory pathways, a fact that is arguably best understood by two key SAB members of Regulus Rx, namely David Bartel and David Baltimore. A recent catalogue of microRNAs established by yet another Regulus SAB member, Thomas Tuschl from the Rockefeller, however, suggests that many microRNAs are constitutively expressed in a number of tissues, thereby slightly questioning whether this is the case for most microRNAs. Although a truly exploding field of research, compared to RNAi Therapeutics where there are already an abundance of well-defined targets, the development of Regulus Rx will heavily depend on progress made in elucidating the exact role of specific microRNAs.
Regulus Rx’s first development program, targeting miR-122 for the treatment of Hepatitis C Virus (HCV) infection is a particularly interesting case. This program has been licensed from Stanford University where Catherine Jopling from Peter Sarnow’s group has shown that miR-122 specifically interacts with part of the HCV genome and thereby stimulates HCV replication. MiR-122 is by far the most abundant microRNA in the liver, and it is surprising that no obvious toxicities have been associated with its inhibition in vitro or in vivo, as demonstrated by work from both Alnylam and ISIS scientists. It is even thought that inhibiting it may have applications in managing cardiovascular disease. Earlier this year, Alnylam and ISIS were issued a patent covering miR-122 as a therapeutic target. I am quite curious to learn whether last week’s announcement of HCV miR-122 as the first development program of Regulus is also based on results targeting HCV replication by miR-122 inhibition in vivo. In this regard, it is notable that a leading group in HCV biology, the Rice group at Rockefeller only recently confirmed miR-122’s importance in HCV replication in a paper jointly published with Thomas Tuschl from the same institute.
Regulus Rx will be based in Carlsbad, CA, not only because of the better weather, but also because this is where ISIS Pharmaceuticals is located. This acknowledges the fact that antisense technology, pioneered and long dominated by ISIS, is the key enabling technology for antagonising mature microRNAs many of which are processed out of introns of RNA Polymerase II transcripts and therefore not a target for RNAi. This underscores the importance Alnylam has attributed to ISIS’ patent estate, also demonstrated by their exclusive licensing ISIS’ oligonucleotides modification patents for the use in double-stranded RNAi Therapeutics. It also sends out the subtle message that as Alnylam respects other parties’ IP it expects anybody interested in RNAi Therapeutics to follow this example by licensing from Alnylam.
The 50:50 joint venture also speaks volumes to the negotiating leverage Alnylam has gained in recent years, made possible in large part by its solid financial position. Only 3 months ago, the financial strength of Alnylam has allowed it to revise a partnership agreement with Medtronic that has given Alnylam a larger stake in the financial success of any drugs coming out of that collaboration in return for increased funding by Alnylam. Unlike ISIS which not long ago had to repeatedly issue debt to fund their operations and is biting its nails to gain financial freedom by partnering its ApoB100 antisense program, Alnylam could easily pay the $10M to equal the companies’ stake in the new venture. In addition to its financial muscle and Tuschl III which gives Regulus exclusive access to some of the most important microRNA targets, Alnylam further brings to the table its invaluable access to leading scientists in academia and Big Pharma alike.
Finally, it is notable that the otherwise wide-ranging technology access licenses that Alnylam has granted Novartis and Roche did not include microRNAs. It would therefore be appropriate that following last week’s development the newco be put on a solid financial footing through a significant partnership. But let’s give them some time for this as although the day may never end on Alnylam, on earth their day still has only 24 hours.
(For more on the scientific rationale for microRNA therapeutics, please read my Blog from 26 May, 2007: “MicroRNAs as Therapeutic Targets”)
PS: Once the dust has settled, we should learn more about the exact scope of Regulus Rx. Specifically, it is not clear to me yet whether in addition to antagonising microRNAs by antisense, the scope of Regulus will encompass the microRNA agonist approach as well where microRNAs mimics are introduced for therapeutic purposes. While this would strengthen Regulus’ portfolio, it would be a significant contribution of Alnylam IP since such mimics would essentially be based on siRNA technology.
Thursday, September 6, 2007
Regulus Therapeutics, located in Carlsbad, California, near ISIS’ headquarters, combines Alnylam’s leading microRNA target IP estate (Tuschl III and siRNAs and microRNA mimics) with ISIS’ antisense and nucleic acid modification capabilities. This clearly creates the industry’s leading effort in microRNA-based drug development guided by a stunning line-up of leading scientists in microRNA research and gene regulation. As a pure play, Regulus Therapeutics should be ideally positioned to build on its existing assets in microRNAs. It further allows Alnylam management to concentrate on the development of RNAi Therapeutics while capitalising on their early IP in microRNAs.
Rosetta Genomics and Asuragen are the only two other notable microRNA-based companies left with ambitions both in diagnostics and therapeutics. It will be interesting how today’s announcement affects these two companies, particularly Rosetta’s existing significant relationship with ISIS. At the very least, it will help microRNAs get the attention of Wall Street and Big Pharma.
Wednesday, September 5, 2007
Ideally, the next development cycle will yield siRNAs with higher specificity and potency. This should allow for the use of lower amounts of drugs in the clinic for obvious reasons of safety, but also cost. At the moment, algorithms can pretty well predict siRNA sequences that will give a decent knockdown in tissue culture experiments in the low nanomolar range. However, once in a while, we stumble across those “super-silencers” that have IC50s in the mid-to-low picomolar range, yet we do not understand what makes them so good.
I expect that the intense study of the RNAi-related pathways in both model organisms and human cells will ultimately explain their behaviour and reveal rules for designing better and better siRNAs. Exemplary are recent studies by the Zamore group in the fruit fly system that showed that small RNAs are partitioned into separate RNAi effector complexes based on their structure as double-stranded precursors prior to loading into the activated RNAi effector complex. Similar to flies and most other multicellular eukaryotes, there are also a number of related RNAi effector complexes in human cells. It is, however, still unclear how much they differ from each other or what their functional overlap is. It is therefore intriguing to speculate that it were possible, similar to what has just been demonstrated for flies, to introduce small RNAs that would specifically harness the RNAi-cleavage pathway, while remaining invisible to the complexes responsible for the non-cleavage silencing pathways. This is because the microRNA-like non-cleavage pathways are responsible for most RNAi off-target effects, and it would further minimise competition with the endogenous microRNA pathway.
The use of different RNAi triggers (PolII::sh-miR; PolIII::shRNA; Dicer-substrate; Tuschl siRNA; 3-stranded siRNA) or, possibly even more exciting from a drug development perspective, chemical modifications and structural variations to the siRNAs may allow us to introduce the desired bias into which effector complex the small RNA will be incorporated. Along these lines, Dharmacon reported not long ago the use of chemical modification at the 2nd nucleotide position of the guide RNA that would still allow for on-target cleavage activity, but almost eliminated microRNA-like off-target silencing by the siRNA in tissue culture. Although a recent abstract by Alnylam scientists for the Annual Meeting of the Society for Neuroscience suggests that this particular modification may not always be neutral to on-target activity, a combination of chemical modification guided by a deepening understanding of RNAi pathways in humans should yield next-generation RNAi molecules with higher clinical success rates.
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