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.

SomaGenics Reports on the Use of shRNAs for the Treatment of HCV

At the end of a day packed full with fun RNA science from the San Francisco Bay Area, Brian Johnston, President and CEO of SomaGenics presented some of his company’s data on the use of shRNAs for the treatment of HCV, historically one of the early therapeutic targets for RNAi.

Most shRNA approaches are based on DNA-directed expression of RNA hairpins in the nucleus that are then fed into the RNAi pathway. Somagenics’ approach is slightly different in that they are introducing the RNA directly into cells. Concentrating more on the science than IP issues here I only want to briefly remark that this probably overlaps quite a bit with the Hannon patents (licensed to both CytRx and Alnylam) on the use of shRNAs as RNAi triggers, both transcribed and synthetic.

The scope of the experiments were limited either to in vitro culture studies or its glorified in vivo counterpart, the hydrodynamic co-transfection experiment. Here, a large volume of RNA-containing liquid is injected into the tail vein of a mouse in a brief period of time, physically forcing the RNA into hepatocytes. It is of interest, that unlike the findings of the back-to-back papers of the Hannon and Rossi labs more than two years ago, hairpins (directed to the conserved HCV IRES) with minimal stems, 19 base-pairs, were generally more effective than those with extended 25 base-pair hairpins. While it has to be said that compared to the Hannon and Rossi papers which argued that the long stems that were processed by Dicer would increase RNAi efficacy, the sample size here was quite limited, it nevertheless shows that the processing of hairpins are often not as predictable as one would wish based on microRNA biology.

Johnston further reported that the shRNAs were at least as efficient as the corresponding siRNAs with some of the IC50s in the low picomolar range. I am curious how this will translate into in vivo uses for example because shRNAs, unlike classical siRNAs and Rossi-type two-stranded Dicer-substrates, contain the double-stranded RNA within one molecule which should make this structure thermodynamically quite stable. A potential disadvantage is that it is so much more expensive to synthetically make the ~42-55 nucleotide hairpin RNAs since cost and purity of manufacturing RNAs increases more than linearly after you reach a certain size, say 25 nucleotides. While these studies mostly used RNAs generated through biochemical synthesis in the test tube using recombinant phage RNA polymerases, for the clinic they are working together with Agilent to generate the “same” RNAs to scale. I say the “same” here since the phage polymerase leaves a triphosphate 5’ end while chemical synthesis does not. This is not a trivial issue here since 5’ modifications are known to influence RNAi processing. We will therefore have to wait how the transition from in vitro transcribed to synthetic shRNAs will affect the reproducibility of their data and potential concerns from the FDA. Similarly, some mention was made that shRNAs may tolerate RNA modifications less well than siRNAs, probably because they are subject to additional processing steps.

The use of more challenging animal models is also warranted before entering the clinic. It is therefore important for them to find suitable delivery solutions which they apparently have only started to. It was good to hear though hearing him mention Protiva/Tekmira’s SNALPs and Mirus Bio’s Dynamic PolyConjugates as probably the most advanced RNAi delivery platforms to the liver, reflecting my views on what’s out there in the literature. Overall, I like the fact that multiple RNAi approaches are in the pipeline to tackle HCV, namely siRNAs: Sirna/Merck; DNA-directed shRNAs: Tacere/Benitec and Nucleonics; and now synthetic shRNAs: SomaGenics. And maybe Tekmira/Protiva should harness the power of SNALP and officially nominate HCV as a clinical development program. I hope that the next 2 years should finally see programs moving into the clinic after HCV RNAi had been delayed in the wake of company-specific issues (Benitec; Merck/Sirna vs Protiva).

A new player in RNAi Therapeutics: Dicerna Pharmaceuticals

IN VIVO Blog (http://invivoblog.blogspot.com/2007/10/dicerna-crashes-rnai-party.html) reported today that a new RNAi Therapeutics company, Dicerna Pharmaceuticals, is about to debut. According to the same source, a $13M Series A financing round is expected to be announced in November. This company is aptly named after an enzyme in the RNAi pathway, as it is founded on the slightly unorthodox way to induce RNAi by providing synthetic Dicer-substrate siRNAs (D-siRNAs) of 26-30bp in length to effect gene silencing.

Dicer is this cool enzyme that digests ('dicerna' in Malay means: 'digested') long double-stranded RNAs into the shorter 21-23bp siRNAs with 3’ overhangs, the structure discovered by Tuschl and colleagues to efficiently induce gene silencing by RNAi in mammalian cells. Once delivered inside the cells, D-siRNAs are then processed by Dicer into 21-23bp effector siRNAs which then are incorporated into the RiSC complex to mediate gene silencing. Tuschl-like 21-23bp siRNAs are currently by far the most widely used method of inducing RNAi in human cells and fairly well understood.

In an elegant Nature Biotech paper in 2005 (Kim et al.: “Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy”), Drs. Kim and Rossi from the City of Hope, in collaboration with Mark Behlke from the nucleic acids synthesis company IDT, found that D-siRNAs can effect remarkably potent RNAi in human cell culture, often more potent than siRNAs of the same sequence. Importantly, in this and follow-up work they worked out some of the basic rules that would make D-siRNAs more practical inducers of RNAi such as better predicting strand incorporation and blocking one end of the dsRNA with non-RNA residues and modifications to force directional Dicer processing.

This Nature Biotech paper was accompanied by a similar paper from the Hannon group in Cold Spring Harbor which found that DNA-directed small hairpin RNAs (shRNAs) with double-stranded RNA stems longer than minimal 19-21base pairs similarly make them often more potent inducers of RNAi. Like Rossi and colleagues, it was speculated that this is due to biochemical coupling of Dicer processing to the RiSC effector complex. In addition to certain advantages in terms of potency, which I feel need further validation on a larger scale, D-siRNAs may in some instances facilitate RNAi delivery where covalent linkage of parts of the RNAi delivery system with D-siRNA is helpful as the active siRNA would be freed from the carrier by Dicer cleavage, although again it remains to be shown that the covalent attachment of e.g. peptides by itself is not inhibitory to Dicer processing.

[Erratum: The 2005 Hannon paper described the use of synthetic, not DNA-directed hairpins, with extended duplex length.]

In addition to these potential biological advantages, certainly a big part of the motivation that went into founding the company from an investors’ perspective is that Dicerna should be sufficiently distinct from the Tuschl siRNAs, a space clearly dominated both in terms of IP and know-how by Alnylam Pharmaceuticals. One can therefore expect that the new chairman and co-founder Douglas Fambrough from Oxford Bioscience Partners will do his best to make Dicerna his second Sirna Therapeutics, which he and his partners sold to Merck last year for a whopping return on their investment.

However, like with Sirna Therapeutics his claims of having freedom-to-operate will likely be clouded by uncertainty as there are a number of areas where Alnylam’s pre-dating IP will significantly overlap with Dicerna’s claims. This is not helped by comments, also cited in the IN VIVO Blog, of new CEO James Jenson stating that Tuschl’s landmark work had been conducted in flies, when Tuschl II –which by the way has issued and is exclusively licensed to Alnylam- is all about RNAi in mammalian cells, all this after laying the groundwork in work described in Tuschl I (also claimed by Sirna and CytRx, but has not issued) through amazing biochemical work in flies: Tuschl the prolific!

Importantly, Tuschl’s work as described in Tuschl II essentially discovered that RNAi operates in mammals and defined the basic rules of synthetic siRNAs. This, in my mind, should go a long way in the patent courts. In its worst case, D-siRNAs could therefore be regarded as simple pro-drugs of siRNAs. This also includes the 3’end overhangs which are thought to be beneficial for D-siRNAs since they are an important recognition element for Dicer.

Kreutzer-Limmer is another important cornerstone of Alnylam’s IP strategy, indeed important enough for them to buy the company (Ribopharma AG) that owned it very early on. Kreutzer-Limmer pertains to dsRNA-mediated gene silencing in mammalian cells, including predicted Dicer substrates, and although less well known in the scientific community due to lack of scientific publication, it is actually thought to have been the first demonstration of such gene silencing. Scientifically, my heart is with Tuschl’s detailed work, but Alnylam played it safe by just removing the uncertainty.

In addition, I would not be surprised if there wasn’t a note-book entry or publication that made use of long siRNAs either by design or accident. This would not be unlike early in the shRNA arena where scientists have made use of shRNAs with minimal and relatively long dsRNA stems alike.

Practically, the relatively small field of D-siRNAs will have to achieve what thousands of researchers around the world have done for siRNAs, namely coming up with siRNA design rules that consider all of potency, off-targeting potential, and the induction cytokines, as Rossi’s work has shown that these rules may differ from that of siRNAs. For these and other reason, I expect the complexity of developing D-siRNA therapeutics to be probably increased.

Nevertheless, I am curious to see more data come out that carefully characterize and compare the potencies of siRNAs and D-siRNAs. Comparative gene tiling studies would be an obvious experiment. This reminds me of the finding of hyperfunctional siRNAs, i.e. the odd siRNA that will be active in the low to mid picomolar range, and I could imagine a situation where efforts to find such siRNAs prove difficult for certain genes, while a D-siRNA is hyperfunctional, and vice versa.

I certainly look forward to Dicerna as a new member of the RNAi Therapeutics community. The science is certainly sound and innovative, and should be tested for use as a human therapy, which we all know would not happen without patent protection.

PS: This new development makes me wonder where that leaves Nastech Pharmaceuticals which has built so much of their RNAi program on Dicer substrates and is about to spin out mdRNA as their pure play RNAi Therapeutics subsidiary. It is clear that COH granted them 5 exclusive targets, but I am less sure about the other rights to Dicer substrates they had obtained.

3rd Day of the OTS Annual Meeting: Novartis Demonstrates Anticancer Activity of Orally-administered siRNAs

The third and last day of the conference was another action-packed day with topics ranging from the basic science of RNAi to its clinical applications.

To better understand endogenous microRNA function and siRNA off-targeting, transcript analysis such as high-content microarrays has been widely used. This, however, captures only the changes in RNA levels, which is a problem since microRNAs and off-targeting have been thought to frequently act at the translational level with little changes in transcript abundance. Transcriptional analysis should therefore miss important microRNA targets and siRNA off-targets. Nikolaus Rajewski introduced an intriguing solution to this problem by harnessing a labelling technique coupled to mass-spectrometric analysis that allows changes in protein levels as a consequence of small RNA over-expression or inhibition to be measured in high-throughput. Preliminary results confirm that this technique indeed retrieves those targets that have been down-regulated both on the RNA and protein levels, but also discovers those genes where only the final protein output has been altered. If sufficiently robust, this technology should ultimately facilitate the development of, most importantly, safer, but also more potent RNAi medicines.

Arndt Borkhardt from Duesseldorf, one of the first to publish applications of RNAi in humans in 2002, presented an intriguing correlation between trisomy 21 (Down Syndrome)-related childhood leukaemia and over-expression of microRNAs encoded on chromosome 21. Importantly, only leukaemic cells, but not normal tissues over-express these microRNAs. Using zebrafish as a model system, he was able to show that the inhibition of these microRNAs indeed caused haematological abnormalities, corroborating a causal contribution of these microRNAs to leukaemia. It not strongly supports the rational of using microRNAs as cancer diagnostics, but also suggest these microRNAs to be valid targets for the treatment of childhood leukaemia. Continuing along the lines of microRNAs as therapeutic targets, Hermona Soreq from Israel made a compelling case that miR-132 and miR-182* (star) are important feedback regulators of the cholinergic anti-inflammatory response and may be targeted for modulating inflammatory responses in disease. Her geographical proximity to Rosetta Genomics makes me wonder whether this company will or has already become involved in thinking about clinical applications centered around miR-132 and 182*.

Jost Seibler represented Artemis, an Exelixis subsidiary specialising on generating mouse RNAi transgenic disease models. They have developed technologies that allows them to use constitutive and conditional (tet technology) RNAi transgenics in a matter of around 4 months, which far exceeds timelines for producing knockout animals. This makes these animal attractive target validation tools. Importantly, a number of examples demonstrated that essentially knockout phenotypes can thus be generated with U6 and H1 promoters driving shRNAs. It was also comforting to see that the shRNA hairpins worked in essentially all cell types and tissues with weaker knockdown only in B-cells and the spleen in general. It will be interesting to see whether other RNAi inducers work more efficiently in these cells and whether this is the consequence of some type of selective disadvantage on cells expressing high levels of small RNAs. 34 constitutive, and 48 conditional knockout models have already been successfully generated.

Volker Patzel definitely raised some eye-brows when he reported that specifically siRNA, and not other nucleic acid types, triggered gene silencing in a variety of prokaryotes. Complicating things was that the mechanism of action ranged from transient to persistent knockdown and even gene knockout effects, and that a variety of parameters such as cell transfection competency and host factors made the technique quite tricky. John Rossi certainly made a good point suggesting that bacterial genetics is most likely to uncover the mechanism of the observed silencing effects.

John Rossi himself was up next providing an update on the lentiviral RNA trials for HIV/AIDS that got under way earlier this year at the City of Hope (a collaboration with Benitec). This trial involves a lentivirus expressing 3 types of RNA therapeutics, an RNAi shRNA hairpin against an HIV gene, an HIV-TAR decoy, and a ribozyme directed against the host factor CCR5. It is a particularly important trial since this is the first time in gene therapy that not only a lentivirus is used as a vector, but also DNA-directed shRNAs are employed. Preclinical parameters such as safety and efficacy as measured by vector persistence, expression, HIV knockdown, and the ability of the pluripotent precursor blood cells to normally differentiate after lentiviral transduction were very encouraging. Unfortunately, his hopes to present data at this meeting from treating the first of three enrolled phase I HIV leukaemia patients were dashed due to 65% cell viability after a thawing step in the protocol, when at least 70% was the target. The FDA would have still allowed treatment to continue, but the clinical investigators felt they should not move the goal posts, and it is hoped that the 2nd and 3rd patient cells will fare better, so that they can be re-infused with the HIV-immunised blood progenitor cells. In the second half of his talk, Rossi detailed experiments inspired by a recent report from Bruce Sullenger’s group on successfully using RNA aptamers for targeting siRNAs in vivo. This is an interesting targeting approach since both the targeting agent as well as the therapeutic moiety derive from the same chemical class. As an alternative to the above lentiviral strategy, he would now like to evaluate such aptamer-siRNA RNA hybrids to target anti-HIV siRNAs systemically to infected cells with an gp120 aptamer developed by William James from Oxford University.

Arguably the highlight of the day was a presentation by Francois Watt from Novartis demonstrating for the first time sequence-specific anticancer activity of ORALLY administered siRNAs. These experiments were motivated by preceding mouse cancer model results using local and introperitoneal administration of siRNAs targeting the angiogenesis factors VEGF-R2 and Tie-2. These siRNAs were stabilised with an undisclosed modification at the 3’end for exonucleolytic stabilisation of the siRNA, which was confirmed by incubating them in gastric acids and intestinal lavage as a prelude to the oral delivery experiments. I wonder whether the 3’ end modification also facilitated the presumable uptake of the siRNAs. Clearly, it will be important to quantify the bioavailability of the otherwise unformulated siRNAs. Relatively large amounts of siRNAs had to be administered (100mg/kg), but I expect this to come down with more experience on oral delivery. Since Novartis had been involved in antisense development before their interest in RNAi, it can be speculated that their 3’modification had been informed by previous antisense results and may be covered by relevant modification IP. For all you Alnylam watchers, such positive data augurs well for Novartis exercising the Alnylam adoption license. Overall, my impression is that while companies can be very secretive with their corporate strategies, scientific data are often more believable since the pressure for them to publish glowing results in leading academic journals is less than for academic researchers. Also, they should have little motivation to further pour money into corporate partnerships if the science did not support it.

Francois Watt was a difficult act to follow, but Jeremy Heidel from Calando did a good job by detailing their development of cyclodextrin-based nanoparticles for delivery of siRNAs in cancer applications. Much of the data had already been published, but it was still a useful summary of their preclinical efficacy and tox studies for their first clinical program targeting RRM2 with transferrin-decorated nanoparticles. While transferrin did not affect the biodistribution of these particles, it enhanced siRNA uptake presumably simply by bringing the siRNA into proximity of the target cell which may then take up the siRNAs by transferring-independent mechanisms, a theme similar to the findings by Stoffel and Alnylam (recent Nature Biotech paper) and others. Importantly, these nanoparticles are bioactive at low mg/kg concentrations and can be safely repeat administered up to 27mg/kg. The only immune response observed was directed against the human transferrin component of the nanoparticles in mice, and importantly not the PEG component. This response against the increasingly popular transferrin should therefore not be observed in humans. Everything appears to be now in place for Calando to start their first systemic RNAi therapeutics trial.

Of course, one has to be aware that in the past many promising preclinical results in mouse models of cancer were not held up in human studies. This, however, is more a problem of choosing appropriate model systems and marrying them to the right targets, less a problem of the therapeutic platform per se. I am hopeful that, once efficient targeting is achieved, RNAi therapeutics will have a higher success rate in cancer than other treatment modalities since the choice of the target is not limited by the molecular structure of the protein.

Posters- an interesting poster on flu (influenza A) RNAi was presented by the infectious disease group at the MPI in Berlin showing that LNA-stabilised, but not unmodified siRNAs, when formulated with PEI caused potent knockdown of flu viral titers in a mouse model following intravenous administration. Santaris, the LNA antisense company had a poster dedicated to their ApoB100 antisense program, showing that 12mer LNA gapmers are better at suppressing ApoB100 than longer gapmers. However, I was puzzled by their findings that not only was LDL potently reduced, but that HDL was also suppressed.

Lastly, I wanted to find out more about the rationale for BMS to partner with ISIS for targeting PCSK9 by antisense, when their poster presentation on targeting PCSK9 by RNAi was so positive. I was taught that Big Pharma may use different techniques for their target validation and that the ultimate choice of the targeting technology was determined by other factors. Apparently, the greater experience with systemically administered antisense compounds in the clinic compared to RNAi and the need for formulating siRNAs for efficient delivery to the liver, trumped the need for considerably higher doses of antisense oligos to achieve equivalent knockdown potencies. Ultimately, I came away with the impression that while BMS saw the promise of PCSK9 in the treatment of hypercholesterolemia, Alnylam was just not willing to partner their PCSK9 program at this early stage. In any case, I wish BMS and ISIS well on developing PCSK9-targeted therapies and it would not be unprecedented if two different technologies targeting the same gene will ultimately come to market. I expect, however, that the therapy with the better safety profile will ultimately dominate in the market. While much is being made about the off-targeting potential of siRNAs, it is remarkable that 3 days of observing RNAi next to other nucleic acid-based therapies, demonstrated that while the RNAi field tackles all sorts of potential safety considerations head-on, off-target profiling and the study on the induction of cytokine responses were essentially missing in the presentations on other RNA-targeting technologies.

The 3rd Annual Meeting of the Oligonucleotide Society was a full success. Many thanks to the organisers and presenters for this!!! This is a time when the right ingredients to making oligonculeotide therapies are clearly coming together, and this should become even more evident at the 4th Meeting to be held next year in Boston.

RNAi by another Means: Dicer-substrate RNAs

The most widely known and used triggers of RNAi are small interfering RNAs (siRNAs) and DNA-directed hairpin structures. Whereas siRNAs are channelled into the downstream steps of the RNAi pathway, the RiSC complex, hairpin RNAs in humans first have to undergo processing by the endogenous microRNA pathway to yield the active small silencing RNAs. There is, however, a third way to induce RNAi, and these are so called Dicer-substrate RNAs. Their potential utility for gene silencing was recognised following the finding by the Cleary/Hannon [Siolas et al. (2005) Nat. Biotechnol. 23:181] and Rossi [Kim et al. (2005) Nat. Biotechnol. 23: 222] groups that longer RNA duplexes of around 25-27 bp that undergo initial processing by the RNAi enzyme Dicer to yield the active small RNA are in some cases more efficient in gene than the equivalent siRNA. This is thought to be the consequence of Dicer actually forming part of the RiSC loading complex, thereby ensuring efficient hand-off of the small RNA product into the RiSC silencing complex.

There are a number of reasons why Dicer-substrate RNAs have not become a mainstream tool for inducing RNAi yet. Among these were the difficulty of manufacturing Dicer-substrate RNAs that would yield predictable small RNA effectors, non-specific perturbations of gene expression due to cytokine induction by the dsRNA, and the lack of reliable Dicer-substrate RNA design rules. Finally, RNAi inducers upstream of siRNAs may compete with more elements of the microRNA pathway than necessary and the longer length of RNAs will add to the cost and complexity of synthesis.

Some of these challenges, however, are being met mostly as a result of a collaboration between the Rossi lab of the City of Hope, California, and the nucleic acid synthesis company IDT which licensed Dicer-substrate RNAs for use in non-therapeutic applications. Creating dsRNA with one blunt end that contains DNA nucleotides on one strand and 2 nucleotide 3’ overhangs on the other end introduced directionality into Dicer processing. Moreover, it appears that the same modifications that can be introduced into standard siRNAs to avoid cytokine induction also work well for Dicer-substrate RNAs. One weakness that remains, however, is the lack of efficient Dicer-substrate RNA design rules. However, in collaboration with Bio-Rad, IDT is screening and developing sets of validated Dicer-substrate RNAs that have greater than 85% knockdown efficiencies.

It remains to be seen how well accepted Dicer-substrate RNAs will eventually become. With IDT, possibly the world’s largest synthetic nucleic acids supplier for research purposes, behind the technology, Dicer-substrate RNAs should be able to reach most researchers in the field. It will therefore be their hands-on experience, publications showing the benefits of Dicer-substrate RNAs and word-of-mouth that will determine the success of Dicer-substrate RNAs. As siRNAs have shown, a biotechnology that works predictably does not need much advertisement. Some interest meanwhile is demonstrated by the fact that Novartis is apparently testing a small library of Dicer-substrate RNAs for target validation purposes, and Nastech Pharmaceutical Company has obtained an exclusive license from the City of Hope for developing Dicer-substrate RNAs as therapeutics against a handful of gene targets.

With regards to IP issues, I would expect that their therapeutic use would require some kind of licensing agreement from the beneficiaries of the Tuschl I and II patents partly because of the 2nt 3’ overhang structures and the fact that Dicer-substrate RNAs are the immediate precursors of siRNAs. A precedent for this kind of licensing agreement has been set before by Benitec, which in 2005 has taken a license from Alnylam for the “targeted gene silencing mediated by short interfering RNAs (siRNAs) generated from DNA constructs introduced into cells”.