Monday, November 19, 2007

The Confusing World of AtuRNAi, Stealth siRNAs and mdRNAs (Part I)

“Next-generation” RNAi is all the rage in the world of the ambitious RNAi start-up. The definition (note that everything is allowed under the guise of the satire): “An RNAi-inducing molecule derived from the classical Tuschl siRNA design, however with a magical pattern of modifications and variations in the exact length of the RNA duplex, sometimes an NA duplex, with overhang or not. As important as the chemistry that may sound impressively inventive to the lay (investing) public is that a proper name is chosen to further accentuate its apparent uniqueness. This is intended to suggest freedom-to-operate with the ultimate aim of attracting investments from people hoping the company will eventually catch up to the market cap of Alnylam (why would you invest in any of their direct rivals otherwise?).”

Considering that it has become commonplace to hear CEOs talk about their RNAi being so unique and advanced that they are now operating in parallel universes, the staid Tuschl siRNA must have really lost its relevance for the development of RNAi Therapeutics. While I think that some select siRNA derivatives given names such as StealthTM RNAi or Dicer-substrate definitely warrant further investigation, as it is yet unclear how well they will perform relative to the simple, but fundamental siRNA design, what I would like to do is to cut through the marketing fog and provide a brief overview of the types of RNAi inducers currently being used at the bench or in the clinic and how I think they relate to each other in terms of IP. In this post I will lay the foundation by giving a summary account of the history of RNAi as a tool, including some of the fundamental patents (and applications), before dissecting some of the Next-generation siRNA designs in a follow-up posting.

Studies on RNAi-related gene silencing really started in the early 90’s in plants with the observation of co-suppression whereby genes that share sequence similarity inhibited each others’ expression. Usually, this was triggered by the inappropriate processing of one of the gene products, typically from an introduced designer gene that is recognized as aberrant and therefore as a threat by the plant RNAi surveillance system. While the mechanism by which this occurs is a scientifically very interesting question, it cannot be used for gene silencing in humans and therefore has little or no relevance to RNAi Therapeutics IP. Parallel work on gene silencing in worms by Fire and Mello, of course, discovered that it was long double-stranded RNA (dsRNA) that was central to inducing RNAi and patents were filed covering dsRNAs longer than 25 base-pairs for gene silencing. This patent can be licensed non-exclusively by almost anybody that wants it, and despite it being based on work in worms and the long dsRNA nature in the stated claims, it is nevertheless considered to be a license that you should add to your IP portfolio anyway, I guess just because it has proven so fundamental to the understanding of RNAi in general and nobody would want to argue that. I also think this highlights the fact that real fundamental scientific insight will be credited by the patent courts even if the exact length of the duplex or modification pattern was not spelt out in the claims letter by letter.

Shortly after Fire and Mello published their research, Kreutzer and Limmer from the University of Bayreuth in Germany reasoned that short dsRNAs may have similar gene silencing effects in mammalian cells. This prediction, as we know, turned out to be true and now forms the basis of the Kreutzer-Limmer patents claiming short dsRNA of around 15-49 base-pairs for the induction of gene silencing in mammalian cells, although the exact length is the subject of patent challenges, including Merck’s opposition in Europe. This early work was considered important enough by Alnylam for them to acquire Ribopharma AG, the company founded on the Kreutzer-Limmer patents. Although I consider Tuschl’s subsequent work to be quite a bit more fundamental to the use of RNAi in mammals, Alnylam understood that it was important to remove any uncertainty as to the dominance of their RNAi IP position given the relative timing and overlapping content of Kreutzer-Limmer and Tuschl.

Around the same time, Hamilton and Baulcombe discovered that small RNAs were generated during plant RNAi. While they were prescient in predicting that these may mediate RNAi, they did not formally prove it and the structure of the siRNA that was detected in those experiments remained unknown. A world away, in Australia, DNA-directed hairpin vectors for reliably inducing RNAi were being described by Waterhouse and colleagues from the CSIRO. Based on the utility and impact of these vectors on plant research, the patents derived from these studies should give the CSIRO a strong position in the agricultural uses of RNAi. In many ways, the commercial development of plant RNAi is more progressed than therapeutic RNAi as traits can now be altered relatively quickly without having to resort to lengthy breeding and selection. I guess the most important question will be how uniform these knockdown phenotypes will be across a field of crops. The CSIRO patents also form partly the basis for Benitec’s claims to the therapeutic uses of DNA-directed hairpin RNAs. The Graham patents form the other pillar of Benitec’s contested patent estate describing the use of DNA cassettes driving the expression of various forms of dsRNAs, although I find these patents to be quite theoretical in nature and wonder whether most of the described non-Pol III expression cassettes would actually work for gene silencing in most mammalian cell types (to be continued…).


Two noteworthy developments last week that I would briefly like to comment on:

1) ISIS released further phase II data for their ApoB-targeting antisense compound mipomersen. The 200mg/week dose reduced by about half the level of bad cholesterol in patients already on stable statin therapy. This looks quite impressive and if no safety issues come up in the larger phase III trials, then this has the potential to become a blockbuster. I’ve been quite critical about mipomersen in the past, particularly due concerns about fatty liver which many scientists in the field would have expected to observe following ApoB knockdown. Safety data for the 200mg dose, based on liver enzyme measurements, however do not indicate this to be a problem. Ultimately, the proof is in the pudding and I would be happy to ultimately have to admit to have been wrong on this issue. ISIS explains the absence of fatty liver due to transcriptional compensatory changes in fat metabolism. Overall, these data augur well for the development of all RNA-targeting platform technologies, including RNAi Therapeutics, as it suggests that minor off-targeting should be well tolerated in many cases.

2) Pfizer announced the acquisition of Coley Pharmaceuticals for almost triple of Coley’s market cap before the offer. Coley Pharmaceuticals is an oligonucleotide therapeutics company that exploits the immunostimulatory properties of oligonucleotides for applications such as boosting vaccines or in the fight against cancer. Actually, I’ve been quite impressed by their OTS presentation in Berlin, particularly their vaccine program. This comes only days after a blog this month where I asked the question when Pfizer will make its big move in RNAi Therapeutics (11 Nov 07 Blog: “When Will Pfizer Finally Make its Big Move in RNAi Therapeutics?”). It is notable that Coley is a Massachussetts company and I would like to think that the proximity to Alnylam will not be an impediment to Pfizer’s new biotech initiative that also appears to more and more focus on oligonucleotide therapeutics. With the new oligo expertise in-house (note that Alnylam does not have another subsidiary to throw into the next deal) and plans to add more staff to a research facility in Cambridge (the headquarters of Alnylam) in addition to a possible biotech incubator near Boston, the plot thickens.

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By Dirk Haussecker. All rights reserved.

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