Monday, March 31, 2008

….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.

Tekmira and Protiva Reunification Forms Leading RNAi Therapeutics Delivery Company

[Corrections/clarifications following Tekmira conference call: Apparently, the new company has also exercised an InterfeRx pick for the ApoB program. In addition, the PLK-1 option by Alnylam is for a 50:50 development and commercialisation arrangement to be exercised any time before start of phase II trials.]

Over the past weekend, as the RNAi world was congregating in nearby Whistler, something very unusual happened in Vancouver, BC. For the sake of RNAi Therapeutics, personal ambition has been set aside and an equitable and very cleverly crafted outcome was found. The outcome relates to a fight for one of the leading systemic RNAi delivery platforms that has cost the Tekmira and Protiva literally almost as much in legal and other related expenses and management time as was spent on technology development. It has also put their many partners, currently Alnylam, Regulus, Merck, Roche, and others, with at least four more pharmaceutical parties evaluating the technology, in a difficult situation and created many inefficiencies.

With more than $35M in cash (Tekmira: ~$20M; Protiva: ~$15M; Roche and Alnylam equity investments: $10M; minus fees, minus severance pay), leading liposomal siRNA delivery IP and science, numerous and lucrative partnership activities, lawsuits out of the way, plus 7 InterfeRx licenses from Alnylam, the new Tekmira will emerge as the first strong public RNAi Therapeutics delivery-focused company. The $10M equity investments for $2.4 a share, $5M each by the Roche Venture Fund and Alnylam, values the combined company at over $100M, almost 3x the current price, with 48% owned by former Tekmira, 44% by formerly privately held Protiva, and 4% by Alnylam and Roche each. It is moreover more than likely that this sets the stage for further partnership arrangements, some of them potentially similar to the one involved in the exercise of the first target pick under the InterfeRx license.

Through this reunion, Tekmira has now also selected its long-awaited first target under InterfeRx. The target is polo-like kinase 1 which Protiva had been developing for the treatment of liver cancer and metastatic colorectal cancer. Perhaps related to the fact that one of Alnylam’s lead development programs is also aimed at liver cancer, Alnylam has retained the right to obtain this program prior to the start of phase II trials. The other development program in Protiva’s portfolio is/was ApoB 100, for which no InterfeRx pick was exercised. This is consistent with the observation from probably half a dozen RNAi studies on ApoB knockdown that I have heard from which have reported sequence-specific fatty liver phenotypes in pre-clinical models.

This very positive business development goes hand in hand with the recent presentations of much improved SNALP and SNALP-like formulations pushing the IC50s down to the 0.1mg/kg range, particularly important since the therapeutic index is by far the most pressing issue with liposomal RNAi delivery. Methods enabling the delivery of liposomes into the endosomes of target cells, with subsequent safe, yet efficient escape of the siRNA cargo into the cytoplasm, will be critical. I am sure the scientific group led by Ian MacLachlan will be best qualified to find such solutions.

This is also the time to congratulate the managements of the parties to a deal that until now almost seemed impossible given the complicated entanglements and heated exchanges in the past. One can only hope that this sets an example for RNAi Therapeutics to hold off on the legal battles until real products come close to reality.

Disclosure: The author currently owns Tekmira stock.

Sunday, March 30, 2008

Day 4 of the RNAi Keystone Conference

As I alluded to already in my previous post on the conference, the issue of transcriptional gene silencing (TGS) mediated by RNAi-related mechanisms in mammals has been somewhat clouded. If it existed and the rules for inducing them could be clearly laid out, it may represent an alternative means of harnessing RNAi for therapy. Presentations on the (at least in mammals) germline-specific Piwi-associated small RNAs (piRNAs; by Alexei Aravin, Cold Spring Harbor) in mice and the small RNA profile from human embryonic stem cells showing that many of them correspond to transposable elements, often regarded as the parasites of our genomes and therefore have to be contained (e.g. by RNAi), the nuclear localizations of Dicer (Witold Filipowicz, Basel) and Argonautes (poster by the Meister group in Munich), and various data on the nuclear localization, and possibly processing, of microRNA precursors into mature microRNAs, are all very suggestive that promoter-targeted TGS may be part of biologically-relevant gene regulation and have promise as a therapeutic technology platform.

In addition to the nuclear localization of Dicer, there was additional data on this central RNAi enzyme. What is often so amazing about science and was also demonstrated a number of times at this conference, different groups working on apparently different phenomena converge on the same results. John Rossi (City of Hope) for example had been studying whether DNA-directed long shRNAs could be processed such that HIV would be targeted by more than a single small RNA, thereby minimizing the likelihood of escape mutants. Like the structural biologist Jennifer Doudna (Berkeley), he finds that mutations in the helicase domain converts human Dicer from an enzyme that does not like to processively chop long dsRNAs into a string of short RNAs into one that does it with reasonable efficacy. Of course, we always hope here that findings like these may allow us to adjust therapeutic designs such that in this case long DNA-directed shRNAs may be processed more efficiently by Dicer. From a basic biology perspective, studies such as Doudna’s on the structural requirements of Dicer should also encourage us to consider endogenous RNAs as Dicer substrates even if they do not result in small RNAs.

Somewhat surprising to me, Thomas Tuschl’s (Rockefeller) presentation focused on the use of microRNAs as diagnostics. It is worth remembering that his group discovered and patented many of the first human microRNAs and that IP may have most immediate value in diagnostics. Specifically, his group is refining technologies for detecting microRNAs in situ (using LNA-spiked probes) and the quantitative profiling of microRNAs by counting. It appears to me that while first-generation microRNA diagnostics in the clinic will involve interrogating a limited number of microRNAs by qRT-PCR (one of Rosetta’s first diagnostics for squamous versus non-squamous lung cancer appears to be based on the PCR quantitation of a single microRNA), while in another 10-20 years quantitative sequencing technologies take over, possibly by-passing array-based detection methods for clinical use altogether.

Phil Sharp (MIT) reminded us that the complexity of the transcriptome may have important implications for our understanding of microRNA regulation. 3’ UTRs are the main site of microRNA action (although there were some bioinformatics talks at the meeting that strongly supported functional microRNA target sites in the open-reading frames of genes as well, though to a much lesser extent than in 3’ UTRs) and may be dynamically regulated according to the state of the cell, e.g. proliferation. Since the extent of these 3’ UTR changes (due to alternative splicing or polyadenylation) may be quite considerable, this complicates efforts to unravel the functional microRNA interactions for the identification, including safety, of candidate microRNA therapeutics.

High-throughput RNAi library screening for drug target identification (commercial examples are Cenix Biosciences, Galapagos Genomics), using synthetic siRNAs as well as plasmid and viral-based shRNA, is now well established. According to Ed Harlow (Harvard), this really has opened up mammalian biology for the type of genetic experimentation long reserved to model organisms. Focusing on the human kinome and cancer (all the predicted human kinases), he made the observation that the functional outcomes of suppressing a given gene may differ considerably according to the experimental system (e.g. depending on the cell lines). This should serve to caution us that context such as the presence of an oncogene or tumor-suppressor gene or not and redundancy can have a profound impact on the importance of a kinase in cell signaling. Another observation from his screens was that the resulting drug target candidates coming out of these screens were not biased towards the kinases that have been already subject of the majority of kinase-related studies, illustrating how RNAi may also benefit small molecule drug development. While in vitro RNAi screens are common, the costs associated with high-throughput in vivo screens would seem prohibitive. It is therefore notable that Michael Hemann’s (MIT) approach to studying the role of genes in cancer drug resistance involves the transduction of a library of shRNAs into cancer cells of the blood and administering them to mice. Changes in the relative abundance of a given shRNA prior to and after drug treatment are then monitored as a measure of the importance of the targeted gene in drug response and resistance. I could imagine that similar approaches to extend the power of RNAi screens may have near-term potential not only for cancer, but also other settings where it is possible to select for relative cell viability (viral infection, degenerative diseases etc).

John Rossi (City of Hope) reported on progress with the first DNA-directed RNAi gene therapy trial (corporate sponsor: Benitec) involving a triple RNA therapeutic against HIV, including a U6-driven shRNA, lentivirally delivered to bone marrow transplant cells in AIDS lymphoma patients that are in need of a BMT anyway. This is a limited trial with an ultimate enrolment goal of 6 patients. Given the complexity of the therapeutic and trial designs it has taken considerable efforts before the first patient could be dosed, but happily the first patient has now been treated a couple of weeks ago. It will be exciting to follow the progress of this patient. Since he/she also received untreated BMT cells for safety reasons, it should be possible to monitor the enrichment/non-enrichment of treated vs untreated T-cells as an indicator of treatment success. Since lentiviral vectors have not been associated with oncogene activation, probably my main safety concern for this trial is the use of a U6-driven shRNA. Not only did having two U6 promoters in one vector cause excision of the U6-shRNA cassette due to a recombination event in some of the genomically integrated vectors, it is known that U6-shRNAs have a tendency to be toxic due to sheer promoter strength as well as possibly its particular processing. For future DNA-directed RNAi development programs, the use of H1 promoters may therefore be even more promising.

A word here on viral escape mutants to RNAi Therapeutics as antivirals. The triple construct in the HIV trial was based on the notion that hitting the virus through three different mechanisms should limit viral escape. As such, it was reported in studies leading up to this trial that HIV likes to mutate around shRNA target sites. To my knowledge, this had not been reported for the HIV sequences targeted by the ribozyme and decoy components in this vector. Contrary to popular opinion, I am actually quite pleased to see such mutations being selected for since this is a clear indication of the efficacy of the shRNA. Moreover, with RNAi the target site for the shRNA could be chosen such that largely unfit viruses were selected for.

Although all three components of the triple construct are RNA-based therapeutics, it illustrates the potential of combining RNAi Therapeutics with other mechanisms of actions into one drug. Similarly, Rossi’s group is now developing an aptamer designed to both neutralize circulating HIV virions as well as target attached anti-HIV siRNAs to HIV infected cells. Another example may be liposomally delivered siRNAs, which in the case of a cancer therapy could be designed such that not an oncogene would be targeted, but also such that the RNA would stimulate the immune response against the cancer. Interestingly, a poster presented by Nigel McMillan (Brisbane, Australia) reported on the maybe somewhat surprising finding that the targeting of an mRNA led to the production of a truncated protein (presumably by translation from the cleaved mRNA) which stimulated an immune response against the oncogene with, if it holds up, some interesting implications for microRNA evolution (cleavage vs non-cleavage) and RNAi Therapeutics.

The aptamer-siRNA combination involved the Dicer-substrate design. The rationale being that such a combination necessitates the covalent attachment of the RNAi trigger to the carrier so that Dicer cleavage has to liberate the active small RNA from the carrier. This is reasonable, but one should note that smaller siRNAs covalently attached to cholesterol on the passenger strand, and probably also the 3’ end of the guide, are also known to be bioactive (although I am not sure whether some efficacy was compromised by such designs). Also, I would be curious to see whether attachment via disulfide bonds that would break in the reductive cytoplasm would liberate even larger amounts of bioactive dsRNAs, both long and small, thus further illustrating that covalent attachment to the delivery vehicle does not represent a fundamental disadvantage for the delivery of short siRNAs.

Of course, this gets us right into the IP issue. A poster by RXi was instructive of how companies aim to get around Alnylam’s perceived dominance in this arena (almost like watching a virus mutate around an shRNA target site). As many of you know already, the focus is on the use of dsRNAs longer than 23bp as well as blunt-end versus overhang dsRNAs. Without discussing the relative scientific merits of the different approaches, it is notable that RXi’s poster put great emphasis that their 25bp Stealth siRNAs were not processed by Dicer into smaller siRNAs as would have been expected for equally long unmodified dsRNAs, yet were still active in gene silencing. Unlike Dicer-substrate oriented approaches, RXi’s intention is to circumvent any claims by Alnylam that Dicer-substrates are nothing more than pro-drugs to the classical Tuschl design, certainly novel and non-obvious from an argumentation point of view. Scientifically, I would be interested in whether these dsRNAs work as efficiently in Dicer knockdown or knockout cells, in other words whether Dicer would still recognize these particular dsRNAs and facilitate RNAi by handing them over to RiSC, independent of whether Dicer had processed or not, and whether this rather than dicing is how Dicer facilitates gene silencing for a range of non-diceable RNAi triggers. In other words, since Dicer is known to aid in loading the RNAi effector complex, RiSC, aren’t all dsRNAs, including siRNAs, Dicer substrates and would the size of microRNAs/siRNAs be a consequence of Dicer processing, but not necessarily a requirement for RNAi activity? Tuschl’s early work on single-stranded RNAi, showing that ssRNAs of various length, i.e. not only 19-21mers, but also 29mers and others, were equally efficient in triggering RNAi suggests just that [Martinez et al. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563].

Local Tekmira had a poster on the 10-fold improvement in efficacy of some of the new liposomal nanoparticles down to 0.1mg/kg, similar to what had been reported on Protiva's website and at Alnylam's R&D day. But rather than discussing here more about SNALP-like technology which I have done extensively in the past, and before I will summarize the conference’s last day, I will have to first read up on the Tekmira news that came out today, an apparent victory of reason and impressive management skill.

Thursday, March 27, 2008

Day 2 and 3 of Keystone RNAi Conference

The Keystone conference kept me kind of busy, so I did not find the time to summarize what’s been happening here in the mountains up Vancouver, so here only a general impression on where the field seems to be heading and some highlights from the abstracts.

David Bartel (MIT) started off the second day of the conference. Well known for his groundbreaking work on microRNA discovery and microRNA target prediction work, he usually has two or three biological short stories to share that are well done and insightful. Same this time. One had to do with the technical challenge of assessing the genome-wide impact on protein expression by a given microRNA. While transcriptional profiling is a well-established and affordable method to measure changes in mRNA levels (RNA that gets translated into proteins), because microRNAs were initially believed to regulate gene expression largely by inhibiting translation without changing RNA levels, a lot of the real targets (according to Bartel’s own studies about 100-200 per microRNA) may have been missed by just looking at transcript levels (note: siRNAs largely regulate their genes by destroying the mRNA, so mRNA quantitation is a reasonable way of measuring target knockdown).

Like Klaus Rajewski (Berlin) who presented tonight, Bartel’s group compared the transcriptional impact of a microRNA by gene expression microarray and compared that to a less well known method of quantitatively measuring genome-wide changes in protein levels, SILAC. SILAC involves the incubation of cells in the presence of light (e.g. control) versus heavy (e.g. treatment) isotope media. When added at the time of the microRNA addition, newly synthesized proteins will incorporate these isotopes. If an mRNA for a given protein had been targeted by a microRNA less of that protein would be made. Cell lysates are then prepared and mixed (control and experiment) and the entire protein content analyzed by mass-spectrometry. Although the peptide signature of the mass-spec for a given protein is essentially unchanged, the incorporation of the heavy isotope would cause the profile to slightly shift for the sample grown in the heavy media, so both treatments can be compared with the relative intensity of the corresponding peptide signals indicative of microRNA regulation (say for a microRNA target the peak would be only 60% of control, meaning a 40% knockdown).

After showing that most of the changes can be bioinformatically accounted for by microRNA regulation, thus validating the technique, both Bartel and Rajewski essentially came to the same conclusions. One is that most regulated genes change both in mRNA and protein content, with the translational inhibition only accounting for a small part of microRNA regulation. This is important since this adds to the validity of assessing off-targeting by transcriptional profiling. The other good news is that most of the changes by microRNAs, the mechanism that largely accounts for RNAi Therapeutic off-targeting, are rather subtle, less than 2-fold in most cases, thereby lowering the side-effect risk through off-targeting.

Unlike previous RNAi Keystone conferences that placed more emphasis on basic principles and mechanisms, the first 3 days were dominated by high-throughput technologies. Even more so than SILAC, deep-sequencing of small RNAs cannot be avoided. It is now possible to obtain millions of sequence reads from a single sequencing reaction, mostly to discover new populations of small RNAs, but also with a trend towards quantitating them by counting the sequence reads.

Other presentations made use of more ancient transcript analysis method, the microarray, but that does not mean with less biological insight. Tom Gingeras from Affymetrix, e.g. looked at the complexity of RNAs found in human cells by microarray and cloning, and one of his observations was that a given gene locus, including those coding for proteins, often harbor multiple overlapping transcripts. That poses the challenge that even though a gene may have been well validated as an RNAi Therapeutic target, one has to make sure that the right mRNA(s) is targeted to avoid unpleasant surprises. This may be even more so important when screening the genome with siRNAs for gene discovery purposes.

Dinshaw Patel (New York) reported on the molecular structure of an Argonaute protein in archaebacteria. This work shows how the guide strand nucleic acid is bound and also suggests how it may recognize and bind its targets. This may add to our understanding of what makes for an efficient siRNA and offers the prospect for thermodynamically optimized siRNA designs. It may also inform us what types of siRNA modifications may be optimal. For example, many of the protein-guide strand interactions are mediated by Argonaute amino acid side-chains making contact in a non-sequence specific manner to the phosphate backbone of the guide, and one would probably want to avoid disturbing this interaction too much. The structure also suggests that there is quite some spatial flexibility in the 3’ end of the guide RNA which may explain why microRNAs normally tolerate 3’ mismatches with their target RNA. How about then siRNAs which are bulky at their 3’ ends such that the spatial flexibility was reduced to enforce target specificity? It certainly will be exciting to watch the impact of RNAi structural biology on the design of future siRNAs.

Similar improvements in efficacy and specificity may come from studying the function of Argonautes in humans (Argonautes are the effector proteins of RNAi). There are 4 human Argonaute proteins, with Ago-2 being the one that efficiently cleaves target mRNAs using siRNAs, whereas the function of Ago1, 3, and 4 is less well known. Some believe that all four Agos may get loaded with a given siRNA and this may very well affect knockdown efficiency and influence off-targeting. Joel Belasco e.g. proposed that the differential expression pattern of the 4 Agos in the various tissues may affect just that. Consistent with that, Dirk Grimm (Heidelberg) found that the Argonautes compete with each other for hairpin-derived small RNAs with the relative level of Ago2 determining knockdown efficiency. One idea therefore would be to design siRNAs that specifically get incorporated into Ago2 while avoiding the other Agos. The molecular structure of all four human Agos would certainly be invaluable in that effort.

Some of you may be wondering whether transcriptional gene silencing (TGS, adding an siRNA to change the chromatin structure so that the resident gene is silenced at the transcriptional level, that is less RNA produced rather than destroying it by RNAi once produced) occurs in mammals and whether it may have therapeutic utility. One problem this aspect of RNAi has suffered from was poor reproducibility and controversy around some of the studies. A particularly interesting talk was therefore given by Danesh Moazed from Harvard as his studies offered the possibility that these discrepancies may have been due to differences in the chromatin context of the targeted genes, and some human gene promoters may therefore be more amenable to TGS than others. I probably would not rush investing into therapeutic TGS just yet (there is no company based on TGS that I am aware of), but I would certainly not want to write it off either. Adding to the credibility of siRNA-induced TGS in humans, a poster by New England Biolabs, a well respected vendor of reliable molecular biology reagents, reported on the identification of efficient promoter-silencing dsRNAs.

The program of the next two days of the meeting will have more on RNAi Therapeutics, but there have been some abstracts readers here may find of interest (I had to miss those, so just the highlights here from the abstract book). One was a study by Alnylam and USAMRIID where they targeted the Ebola virus (Protiva had a similar publication with the US Army in 2006) using liposomally (probably SNALP) formulated siRNAs. Encouragingly, this formulation was able to protect both mice and guinea pigs from an otherwise lethal dose of virus- in an apparently target sequence-specific manner. Moreover, viral titers were reduced when RNAi was administered not only before viral challenge, but also after. Of course, I would have loved to ask about the toxicity profile of these treatments, but hopefully this will be addressed in one of the upcoming oral presentations of this meeting.

I haven’t had a chance to read the latest miR-122 inhibition study in Nature using Santaris’ LNA technology, but a poster abstract by Santaris described the long-lasting (up to 90 days) inhibition of miR-122 with the expected LDL cholesterol lowering effects, all of this at reasonable doses of around 3mg/kg. LNAs are certainly a promising microRNA-inhibition technology and an IPO should not be too far off. Not to be outdone, Regulus Therapeutics described an improvement of their original antisense chemistry for miR-122 inhibition with an apparently 8-fold increase in potency. I assume this must have been through a phenotypic read-out (e.g. cholesterol lowering) since they now find that this chemistry inhibits, but does not destroy the microRNA.

A lesser known method of inducing RNAi developed by Cequent Pharmaceuticals in Boston, hairpins expressed in E. coli (transkingdom RNAi/tkRNAi), is slowly getting ready to enter the clinic within the next year. In their initial program, they will orally administer tkRNAi bacteria targeting beta-catenin for the treatment of familial adenomatous polyposis. IND-enabling studies are under way.

Lastly, a poster from Rosetta Genomics illustrated the potential of microRNA diagnostics beyond cancer by detecting changes of microRNAs in the body fluid that reflected a disease state.

Journal Club: Study Shows TLR3 Induction by siRNAs with Anti-angiogenic Effects, Questioning Ongoing RNAi Clinical Trials for Wet AMD

RNAi is a relatively young therapeutic platform and we are rapidly learning about the gene-specific and non-specific effects of RNAi triggers in vivo. Ultimately, the value of RNAi Therapeutics lies in the gene-specific knockdown of therapeutic target genes, but it is clear that non-specific class effects may confound the analysis of pre-clinical and clinical results. This is especially true for a number of the early RNAi Therapeutics candidates that entered the clinic at a time when less was known about issues such as off-targeting, cytokine induction via TLR-7, and now also TLR3.

The study by Kleinman et al. from the University of Kentucky that appeared yesterday in the high-profile journal Nature (Nature doi 1038/Nature 06765), investigated siRNA therapy for wet age-related macular degeneration (AMD). Of note, there are at least 3 RNAi clinical trials ongoing for AMD: a phase III candidate by Opko Health, bevasiranib, involving intravitreal injection of an unmodified siRNA against VEGF; a phase II candidate by Allergan/Merck(Sirna Therapeutics) involving a chemically modified siRNA against VEGF-R1, also intravitreally injected; and last but not least a phase I, likely intravitreally injected “AtuRNAi” compound targeting a novel, non-VEGF pathway gene by Pfizer/Quark Biotech. Kleinman and colleagues showed that pretty much all of the siRNAs they injected, 2’O-methyl modified (Allergan drug) or not (Opko), suppressed laser-induced choroidal neovascularisation (CNV) in mice, a commonly used model for wet AMD, independent of whether their sequence was directed against an angiogenesis gene or not. Furthermore, such siRNAs were not taken up by the cells in the back of the eye, consistent with a lack of target gene knockdown. Through a series of elegant experiments, they showed that this non-specific antiangiogenic effect was mediated by binding of the dsRNA to the TLR3 receptor on the cell-surface of endothelial cells and the subsequent induction of IL-12 and interferon gamma, both of which alone could account for the observed CNV suppression.

At the moment, I cannot explain the discrepancy between these data and studies by the Opko (formerly Acuity) and Sirna Therapeutics groups that showed sequence-specific down-regulation of target-genes and cellular uptake of siRNAs using the same methods employed by the Kentucky group. Be that as it may, since TLR3 receptors are known to bind dsRNA and upregulate the IL-12 cytokine and interferon-gamma and it always amazed me how unformulated siRNAs may so efficiently be taken up in the eye, the conclusions of the studies appear credible. This receptor is different from the cytokine induction potential via TLR-7 which can be abrogated by chemical modifications, particularly 2’O-methyl.

Well, that’s the bad news. But there is also reason to be optimistic. Importantly, the authors showed that by conjugating a VEGF siRNA to cholesterol, the siRNAs were taken up into the cells and were able to knockdown its target gene and reduce CNV, even in mice lacking TLR3. All of this was achieved at the remarkably low amount of only 1ug administered siRNA per eye. This shows that RNAi could still be used very efficiently in a gene-specific manner to address AMD. Interestingly, a cholesterol-conjugated siRNA for VEGF-R1, the target for the Allergan/Merck drug, failed to ameliorate CNV, casting a doubt on the viability of this gene target that’s been relatively little characterized in the context of wet AMD. However, one should add that only one siRNA was tested for this and at the low 1ug dosage.

Cholesterol conjugation for siRNA delivery was first pioneered by Alnylam, and from recent presentations it appears that this is becoming an increasingly important technology, particularly with the elucidation of its uptake pathway in a recent Nature Biotech paper.

The authors further found that TLR induction was dependent on the length of the siRNA. DsRNAs of 21bp or longer induced this response, smaller ones did not. Also, it is very likely that this response to 21bp dsRNAs and longer could be abrogated by chemical modifications. Moreover, it would be interesting to speculate that since TLR3 is a dsRNA-specific binding protein on the cell surface, one may even harness this binding property for siRNA delivery if binding could be separated from TLR3 activation and demonstrated in the article for small dsRNAs.

Finally, as we learn more about these potential non-specific class effects of RNAi triggers (in this case synthetic dsRNAs, not DNA-directed RNAi), strategies can be designed to either avoid them by the judicious design of siRNAs (chemical modifications, length and structure of RNAi trigger) or even harnessed for a synergistic therapeutic effect. Although the number of supplemental figures of this paper (!) would indicate that there had been considerable resistance to the publication of this study, studies like this are extremely valuable in informing future RNAi development programs. This information can also be used to better monitor the safety of RNAi drug candidates already in clinical trials that may very well depend on such non-specific effects for therapeutic efficacy. Even for these drugs, not all hope is lost as firstly it remains to be seen whether and how these mouse studies would translate into the human setting. It is also true that many approved drugs work, but not through their anticipated mechanism of action, and some of the future RNAi drugs may be no exception to this.

Tuesday, March 25, 2008

Day 1 of Keystone RNAi Conference: New Small RNAs and Oral RNAi Delivery

More recently discovered types of small RNAs were the focus of the keynote session on the first night of the influential annual gathering of the RNAi community, the Keystone conference on RNAi, this year held in Whistler resort near Vancouver, Canada.

Following introductions by the scientific organizers Judy Lieberman (Harvard) and Phil Sharp (MIT), Craig Mello, Nobel laureate and faculty at the University of Massachusetts, gave an account of a variety of relatively recently discovered small RNA populations that are enriched in the worm germline and are characterized by specific structural features, such as 5’ triphosphorylations or 3’ methylations, and the Argonaute proteins (worms have over 20 of those, whereas humans only have 4) with which they are associated.

For many of these small RNAs, the biological function is still somewhat unclear and the field, as was also echoed by the second keynote address by Greg Hannon (Cold Spring Harbor) is busy using high-throughput sequencing techniques and bioinformatics to catalogue them. As an aside, with the speed sequencing technology is progressing, it could very well be that the future of microRNA diagnostics will entail the quantitative high-throughput sequencing of microRNAs rather than interrogating a limited number of defined microRNAs by microarray or PCR.

A particularly interesting small RNA population is one that is marked by 5’ triphosphorylation, unlike siRNAs that are 5’ monophosphorylated. It appears that in their biogenesis, a primary small RNA (siRNA or microRNA) cuts a target mRNA which then becomes the template for RNA-dependent RNA polymerases (RdRPs) that generate the 5’ triphosphorylated RNAs (aka secondary siRNAs). Surprisingly, the secondary small RNAs are the ones that carry out the majority of the gene silencing, while the primary small RNAs are present only in very small amounts and appear to function only in triggering the amplification of gene silencing.

It is, of course, now interesting to speculate whether synthetic small RNAs with various 5’ modifications could likewise function in human cells, possibly with different biological activities from siRNAs and microRNAs. However, in the case of the RdRP-dependent 5’ triphosphorylated small RNAs, one has to keep in mind that a dedicated RdRP appears to be absent in the human genome and that 5’ triphosphorylated RNAs in the cytoplasm may trigger unwanted cytokine responses. But maybe there will be some other type of modification around which one could build a therapeutic platform.

At the start of his presentation, Mello noted that one way to induce gene silencing in worms is by feeding them with bacteria expressing double-stranded RNAs (somewhat reminiscent of Cequent Pharmaceutical’s transkingdom RNAi approach). He finally came full circle at the end of his talk, when he showed some intriguing slides from colleagues at the University of Massachusetts, Michael Czech and Gary Ostroff, on the oral delivery of therapeutic RNAi. Starting with yeast ghosts consisting of essentially a shell of beta-glucans that they filled layer by layer with RNAs, including siRNAs, to create nanoparticles that would be taken up by the Peyer’s patches in the gut. Unfortunately this part of the presentation was quite brief, but it appears that they have succeeded in ameliorating inflammation in a mouse model by targeting TNF-alpha. It will now be important to demonstrate the generality of this phenomenon by targeting a number of other genes unrelated to immune responses. As this approach appears to target immune cells, it may at least initially be applicable for orally delivering RNAi for a number of immune related disorders.

This development could be particularly interesting for RXi given the close relationship between Mello and Czech and this new public company. Also taking into account that UMass RNAi efforts will be a major beneficiary of the Massachusetts life science initiative, it seems that RXi is finally showing some signs of scientific life.

Saturday, March 15, 2008

A Framework for Progressing RNAi Therapeutics into the Clinic

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

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

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

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

Tuesday, March 11, 2008

Dharmacon’s Accell Delivery Technology Signals Increasing Focus on non-Endosomal siRNA Uptake Strategies

[Update May 8, 2008: Based on a recent patent application by Dharmacon, WO 2008/036825, it appears that Accell siRNAs are in fact very similar in principle to Alnylam's 2004 cholesterol-conjugated siRNA paper in Nature and for which the Australian patent office has now accepted a related patent application (Australian Patent Application No. 2004206255)].

Dharmacon (now part of Thermo Fisher Scientific), the company that rose to prominence as one of the first and trusted siRNA suppliers that understood to maintain their leading position by staying at the cutting-edge of RNAi research, just announced the introduction of Accell siRNAs.

Accell siRNAs may not be just yet another type of modified siRNA, but appear to be inherently cell membrane permeable allowing for straight-forward in vitro gene knockdown in all cell types tested, including the notoriously difficult-to-transfect primary cells, without the need for prior formulation with special transfection reagents. This is in contrast to conventional siRNAs that, due to charge and size, are not cell permeable and are therefore require formulation with delivery reagents such that they may gain access to the cytoplasm (the site of Ago2-mediated RNAi) following endosomal uptake and lysis of that compartment. This can be toxic, either because of the chemistry of the specific formulation or the triggering of cytokine responses by engaging TLR receptors in the endosomes.

It is also clear that although potent gene silencing may be achieved following endosomal uptake, typically only a fraction of the siRNA that had been delivered to the cell is actually active in gene silencing. In fact, the rate-limiting step in vivo in many instances may not be getting the siRNA to the target cell, but getting it into the target cells once in close proximity. In addition to better understanding the endosomal uptake-release mechanism, research into alternative technologies may therefore prove highly rewarding.

Although technical details were not disclosed, it is reasonable to assume that Accell siRNAs have been rendered more cell permeable either by the addition of a small lipophile to the siRNA duplex or by modifying the highly charged siRNA backbone without compromising siRNA silencing activity. Stanley Crooke, the CEO of ISIS, has e.g. noted before that it is the amphiphilic nature of single-stranded antisense nucleic acids facilitates their cellular uptake, so that it is conceivable that Accell siRNAs may have been engineered to be similarly amphiphilic.

The current Accell siRNA technology, however, has some drawbacks in that a special tissue culture media is required and therefore does not allow them to be directly translated to in vivo applications, although formulating them in ways that get them to their target cells should not represent an insurmountable challenge, with small conjugates such as cholesterol being likely candidates. The recommended concentration of 1 micromolar siRNA (about 50-fold higher of what is typically used in vitro) further indicates that the process needs to be optimized before Accell siRNAs will be widely used for large-scale and routine tissue culture experimentation. The immediate market potential should therefore largely come from work with primary cells.

Accell siRNAs reminds us that systemic RNAi delivery is a multi-step process, starting with extravasation of the siRNAs from the blood stream into the tissues to cell attachment and cellular entry, and the potential for improving each step is significant. It is fascinating to see how intensely each of these tasks are being addressed and the many creative solutions coming out of these efforts.

Thursday, March 6, 2008

Liposomal RNAi Delivery to the Liver Making Progress

[Disclosure: I chose to own both Tekmira and Alnylam stock, of which particularly Tekmira should benefit considerably from progress on liposomal RNAi delivery due to their IP position on cationic liposomes and relationship with Alnylam.]

Following my blog on the RSV-01 experimental infection results, I received a number of emails noticing my apparently less-than-usual bullish assessment of this Alnylam result, a concern heightened by a falling Alnylam share price.

The problem with calling the results a definite proof-of-concept for RNAi in humans is that this program targets a virus, and it is well documented that particularly unmodified siRNAs can have profound antiviral effects independent of their gene silencing activity, i.e. by inducing cytokine responses. However, I acknowledge that Alnylam and their clinical collaborators have done almost all humanly possible to (statistically) exclude the influence of various parameters such as inflammatory cytokines on the antiviral efficacy, although it is not clear e.g. when and where these markers were measured.

I guess I am just too much of a trained scientific skeptic here when I can agree with proof-of-concept for a clinical effect of an RNAi Therapeutic in man, but remain cautious on proof-of-concept for gene silencing in man. Maybe proof-of-concept should not be seen as a one-time event, but something that will emerge over time.

Especially with some of the other early clinical RNAi trials, one has to be aware that these were entered into the clinic before the entire scope of non-silencing activities of unmodified siRNAs were known (and some of the trials lacking supporting sound scientific data, btw). For those that would like to learn about this topic, I would warmly recommend the freely available, and very critical review article by Protiva’s Adam Judge and Ian MacLachlan from Protiva on the immune-stimulatory potential of siRNAs, including their non-specific antiviral and antiangionic (e.g. relevant for wet AMD applications) potentials. It is important to note that critics, also called “shorts” in stock market lingo, may exploit these issues to portray them as fatal short-comings of the RNAi Therapeutics platform, but like delivery, there is every indication that these will be overcome in a timely manner and the field should not shy away from openly addressing them.

As I indicated in my 2008: Year of the Liver , I believe that the demonstration of RNAi gene silencing in the human liver may very well steal the thunder from the RSV program this year. It was gratifying therefore to hear at today’s Alnylam’s R&D Day that by working together with Tekmira and applying a systematic, industrial-type evaluation of different liposomal nanoparticle formulations (LNPs, aka SNALPs), the potency of these particles has been improved such that only 0.1mg/kg dosages can effect over 50% knock downs. This is very important since these are now concentrations that should be well below the concentrations previously associated with toxicities by these cationic liposomes and likely the cause for delays in filing for the much anticipated liver INDs. Not only was it then confirmed that an siRNA administration led to silencing over an entire month, but equally important was the demonstration that repeat-administration of these particles over 3 months was able to cause sustained gene silencing, meaning that neutralizing antibodies, typically facilitated by concomitant inflammatory cytokine induction, are not generated to blunt repeat administration.

As a result, Alnylam today gave the best indication thus far that they are now ready to enter the clinic with such a systemic RNAi formulation, most likely for treating hypercholesterolemia. In addition to demonstrating safety, this phase I study may indeed provide the most impressive evidence for RNAi gene silencing activity in humans simply by measuring the level of circulating PCSK9, the gene target of this program.

Btw, comparable 0.1mg/kg efficacy and repeat-administration data with the essentially same underlying SNALP technology was also reported on Protiva’s website which I give an ‘A’ for being scientifically engaging, and possibly an ‘A+’ when Protiva and Tekmira can finally come to the realization that it does not make sense to have two companies operating in the same city on the same RNAi delivery technology, while diverting money and attention on lawsuits fighting each other.

As you can see, despite the broad progress in delivering RNAi to many other organs and tissues, based on the available data, RNAi delivery to the liver (not only by SNALPs, but also an increasing number of competing technologies) appears to be the most advanced in terms of efficacy and should be one of the major drivers of the valuation of the RNAi (and also microRNA) Therapeutics space in the next 1-4 years by filling the clinical pipelines with high-quality, promising drug candidates.

Wednesday, March 5, 2008

MicroRNA Mimicry: Not All Triggers are Created Equal

In the online version of Science, Viswanathan and colleagues report on the post-transcriptionally regulated processing of the let-7 microRNA during embryonic stem cell differentiation (Viswanathan et al.: “Selective Blockade of microRNA Processing by Lin-28.” 10.1126/science.1154040). Here, they show that the RNA-binding protein Lin-28 specifically delays the maturation of the let-7 microRNA family until later stages of differentiation. This not only provides an intriguing link between the microRNA and stem cell research areas, it also further supports the role of let-7 in cell differentiation and an additional rationalization for the use of let-7 mimicry as a cancer therapeutic.

With regards to microRNA mimicry as a therapeutic in general, this and an increasing number of other studies reporting on the post-transcriptional regulation of microRNA/small RNA function, including subcellular localization, the sorting into different small RNA effector complexes, and microRNA/small RNA maturation, however also raise the question about the critical role of the choice of the right microRNA mimic in order to achieve the desired therapeutic effect.

This may be a real problem if one assumed that in order to obtain the therapeutic benefit such a microRNA mimick would have to regulate more or less the same set of target genes as its endogenous counterpart. The introduction of a synthetic siRNA-like microRNA duplex e.g. may not sufficiently recapitulate the normal sorting mechanism if this was linked to its biogenesis. Moreover, such a synthetic small RNA would likely have to be modified for pharmacological reasons, something we know may profoundly affect, and ideally reduce the off-target spectrum of an siRNA, but in the case of a microRNA mimick may be undesirable. It is also becoming increasingly clear that even a small difference in the level of a microRNA may have profound effects on its biological output.

While this represents significant scientific challenges and calls for the use of optimal models of human disease as part of the pre-clinical validation process, it also represents IP opportunities for the increasing number of microRNA therapeutics companies. It will be interesting to see whether such companies will soon try to differentiate themselves based on the specific chemistry of microRNA mimicry or a particular gene therapy approach. More so than for RNAi Therapeutics, gene therapy may enjoy here a number of unique advantages, and it would make sense for companies like Benitec, Oxford Biomedia, or Nucleonics to consider a microRNA therapeutics program.

[see also my blog on microRNA sponges for a gene therapy approach to inhibiting microRNA function]
By Dirk Haussecker. All rights reserved.

Disclaimer: This blog is not intended for distribution to or use by any person or entity who is a citizen or resident of, or located in any locality, state, country or other jurisdiction where such distribution, publication, availability or use would be contrary to law or regulation or which would subject the author or any of his collaborators and contributors to any registration or licensing requirement within such jurisdiction. This blog expresses only my opinions, they may be flawed and are for entertainment purposes only. Opinions expressed are a direct result of information which may or may not be accurate, and I do not assume any responsibility for material errors or to provide updates should circumstances change. Opinions expressed in this blog may have been disseminated before to others. This blog should not be taken as investment, legal or tax advice. The investments referred to herein may not be suitable for you. Investments particularly in the field of RNAi Therapeutics and biotechnology carry a high risk of total loss. You, the reader must make your own investment decisions in consultation with your professional advisors in light of your specific circumstances. I reserve the right to buy, sell, or short any security including those that may or may not be discussed on my blog.