Final Day of Keystone RNAi Conference

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

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

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

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

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

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

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

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

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

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

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

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

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

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