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.