RNAa- Alnylam Continues to Gobble Up Fundamental RNAi-related IP

Earlier this week, Alnylam announced the consolidation of intellectual property related to RNA activation (RNAa). RNAa is a method of sequence-specifically activating genes by the introduction of cognate double-stranded small RNAs. It is similar to siRNAs, but in this case the dsRNA corresponds to certain gene promoter elements instead of the mRNA in the case of siRNAs. In addition to once again positioning Alnylam up to be the gate-keeper of a potentially new class of small RNA therapeutics that harness natural gene regulatory mechanisms, as RNAa touches upon RNAi biology, this development has also to be seen as further cementing Alnylam’s grip on fundamental RNAi and siRNA IP thereby creating immediate value.

RNAa quietly emerged over the last 3-4 years in the shadows of the RNAi revolution. When it was discovered that RNAi-related mechanisms function in transcriptional gene silencing (TGS) in fission yeast (explanation: in TGS, siRNAs mediate chromatin modifications that prevent a gene to be transcribed in the first place, rather than being targeted for degradation after having been transcribed as in RNAi), researchers around the world, including myself, started to look at whether a similar mechanism operated in mammalian cells. While initially met with some skepticism and controversy, the overwhelming evidence now strongly supports that TGS indeed exists in mammalian cells.

One branch of TGS research introduced siRNAs targeted to gene promoters with the aim of silencing that gene by recruiting chromatin silencing machinery to the targeted DNA elements. As was subsequently reported, in some cases they do. But to the surprise of many, some of these siRNAs had just the opposite effect, namely they activated expression from the corresponding gene. As detailed in Alnylam's press release, this does not appear to be an isolated effect, but was shown by a handful of labs to be applicable to a number of genes looked at.

While the mechanism of action of these small RNAs, referred to as ‘antigene RNAs’ (agRNAs) to distinguish them from their better known cousin, remains spotty, it appears that low-level non-coding transcription around the targeted promoters are recognized by the agRNA in a process that involves Argonaute proteins and leads to activating chromatin modifications. In addition to sharing with RNAi the Argonaute proteins, good agRNAs appear to follow similar design rules as siRNAs: 19-21 base-pair dsRNAs with seed-match dependency. 3' overhangs have also been used, although the utility of this structural feature has not been explicitly demonstrated. Consistent with an epigenetic mechanism, the effect can be quite long-lasting (~10-14 days of gene activation following agRNA introduction in tissue culture cells, and likely to be even longer lasting in primary cells).

Moreover, agRNAs appear to tap into a natural gene regulatory pathway as endogenous small dsRNAs had previously been shown to switch on the activity of cognate promoters. MicroRNAs, some of which are known to be localized to the nucleus, have also been implicated here.

From Alnylam’s press release and the Q2 '08 conference call, the consolidation of RNAa IP that is based on the first reports of RNAa in the literature, was explained as representing yet another small RNA-related business opportunity for the company, similar to how it views microRNAs. Due to the early nature of RNAa, it is likely that while Alnylam will pay for IP-related expenses, it will be the academic labs that licensed the technology that will conduct much of the initial research. It also remains to be seen when we will see a Regulus-like RNAa spin-off.

I would argue that if RNAa and RNAi had little overlap, Alnylam would have been better advised to let others invest instead of diverting focus and resources from RNAi Therapeutics that should yield a much higher return on investment. RNAa is still at an comparably early development stage, and the number of therapeutic opportunities should be much restricted compared to RNAi Therapeutics. Initially, I would expect the re-activation of tumor-suppressor genes for cancer therapy to be the major attraction.

One factor, however, that could have tipped the economic scales in favor of an investment in RNAa is the fact that RNAa and RNAi seem to intersect both with respect to biology and technology. One paper suggests that Argonaute 2, the Slicer of RNAi, is indispensable for RNAa. As the functions of the 4 human Argonautes slowly emerge from the research labs around the world, this adds to the notion that siRNAs, once introduced into cells, could function in more than one way in regulating gene expression. As such, RNAa broadens the methods of using siRNA structures protected by Alnylam IP.

This synergy in IP may also at least partly explain why Alnylam and not ISIS Pharmaceuticals, its Regulus partner, embraced RNAa in such a comprehensive manner. This is despite of ISIS’ contribution of research reagents to one of the underlying papers and a RNA therapeutics business strategy that has been broader than that of Alnylam’s. Still, with Alnylam securing fundamental IP related to harnessing natural small RNA gene regulatory pathways and ISIS in possession of an impressive and complementary nucleic acid IP portfolio and a near-term drug opportunity in mipomersen, I would not count out a future involvement of the Carlsbad company. I also wonder at which point it may even make sense to just combine the two companies.

PS: Should RNAa turn out to be widely applicable and if RNAi Therapeutics history is any guide, odds are that competing companies will come up with Atu- and RXagRNA designs that vary slightly in structure, claiming to have superior activity and enjoying freedom-to-operate. Nucleonics and MDRNA, however, should serve as stark reminders that this may not be the best strategy of creating shareholder value. The so very demonstrative nature of the RNAa IP consolidation this week should serve as a useful reference against which competing claims will have to be measured.

RNAi-related Phenomena and their Relevance for RNAi Therapeutics

Following the discovery of small RNAs as part of RNAi and a growing range of other classes of small RNAs, it soon became clear that RNAi is just one part of a network of small RNA-directed cellular processes. MicroRNAs which are involved in a wide range of post-transcriptional gene regulatory processes are probably best known, but small RNAs have also been found to play roles from chromatin silencing to chromosome rearrangement. The existence and importance of these functions can vary greatly between different organisms. Nevertheless within a given organisms these functions are interconnected by partly making use of overlapping sets of proteins or proteins from the same gene families.

One concern in the development of RNAi Therapeutics for human use would therefore be that small RNAs may engage unwanted small RNA-directed processes. In humans, the microRNA pathway and RNAi-related chromatin silencing are probably the main concerns. The microRNA pathway is actually the main reason for off-targeting in RNAi experiments, and I have outlined potential strategies about how to selectively engage RNAi and not the microRNA pathway in earlier posts. Silencing chromatin through the introduction of small RNAs in human cells, however, is much more controversial. Even if we assume that this pathway does exist, then according to published papers you would probably need more than one small RNA targeting the same gene. Moreover, one would likely have to introduce small RNAs targeted to the promoter region and not the transcribed gene as siRNAs do in RNAi. A possibly related interesting phenomena that was reported recently is RNA activation, or RNAa. In these examples small RNAs targeted to certain promoters actually upregulated gene activity. It is clear, however, that not any small RNA will have this effect, and again siRNAs targeted towards the transcribed genes are not likely to result in promoter activation.

Alone from the fact that there are 4 Argonaute genes and additional related Piwi genes, and new classes of small RNAs being discovered on almost a monthly basis, one can expect the RNAi-related universe to expand ever more. However, largely based on microarray studies, it seems that exogenous small RNAs almost exclusively engage the RNAi machinery and the related microRNA pathway. It is therefore a focus on the microRNA pathway that is most likely to ultimately improve the specificity of RNAi Therapeutics.