RXi Reports on Dicer-insensitive 25bp RNAi Triggers

There is little question that Alnylam enjoys considerable control over arguably the most desirable type of synthetic RNAi triggers: 19-21 base-pair dsRNAs with 3’ overhangs- some of this control, of course, being somewhat subject to the outcome of the Tuschl Tussle and the review of the Tuschl II patent in Europe. In any case, this had caused a number of competitors to try and avoid this IP estate by designing around these Tuschl-type siRNAs. One of the more popular workaround designs are (blunt-ended) 25 base-pair dsRNAs, being developed by the likes of RXi, Intradigm (now part of Silence Therapeutics), and Invitrogen (RXi has a deal with Invitrogen, most likely covering aspects of RXi’s 25mers). RXi now presents data (Salomon et al.: Modified dsRNAs that are not processed by Dicer maintain potency and are incorporated into the RISC) on the performance of these 25mer siRNAs that are consistent with the notion that it is possible to find highly potent siRNAs with such structures, and that, interestingly, modified 25bp duplexes may often not be subject to Dicer processing before being loaded into the RISC effector complex.

One concern with the use of 25mers is that, yes, they, like a lot of other structures can harness the remarkably robust RNAi gene silencing pathway, but is its potency sufficient for its therapeutic translation? Because RNAi triggers have to satisfy a few other requirements besides potency, as a platform, it is not sufficient to find one or two highly potent sequences, but maybe 5-10 to choose from. The importance of such choice arises from the need to minimize off-targeting and immunostimulatory potential, related to this the ability to modify a siRNA without compromising too much activity, and finding sequences that are conserved in pre-clinical animal models etc.

Sufficient potency to me means 50% knockdown (aka IC50) values of at least 300pM in standard tissue culture experiments (Alnylam’s siRNA in TTR01 e.g. has a single-digit pM potency). Salomon and colleagues provide examples of 2 siRNAs with IC50s of 50-150pM and add, but without showing the actual data, that they ‘have identified duplexes with potency in the picomolar range for over 10 genes and at multiple sites per gene’. So while I am convinced that highly potent 25mer siRNAs can be discovered, the question about the ease of finding those remains an open one. Support that they may not be that rare comes from the gene-specific 25mer siRNA patents filed by Intradigm/Silence Therapeutics that show silencing results from relatively detailed siRNA tiling assays.

It should be possible to increase the pool of potent 25mers by applying design rules like those by Zamore and controlled by Silence Therapeutics. Salomon and colleagues for example report that some of the modifications in RXi's particular 25mer design (unmodified guide strand, 4 5' and 3' terminal 2'-O-methyls in the passenger) affected the unwinding and loading of the passenger strand which may therefore be subject to the Zamore end-stability claims. Moreover, the value of the 3’ terminal mismatches of the guide strand for enhancing RISC turnover, as covered under the first issued Zamore patent, may be particularly high for long guide strands (25mers, see below) because such extended base-pair complementarity (=thermodynamic stability) could negatively impact RISC detachment from the cleaved message and therefore the efficiency with which it can be recycled for the next round of target cleavage. In that sense, Intradigm may have been quite clever in realizing the synergy of the 3' terminal mismatches particularly for the long duplexes as practiced by the company.

Another interesting aspect of the study, also relevant for IP purposes, is that the 25mers were loaded onto RISC without prior Dicer processing. 25mers may have been expected to be subject to Dicer processing to generate Tuschl-type siRNAs. Although this particular legal theory remains to be tested, this may not be the ideal patent for the 25mer companies because Alnylam may argue that Dicer substrates just serve as pro-drugs to the Tuschl-type siRNAs and therefore would require a license. Moreover, if they were indeed processed by Dicer, they might conflict with Dicerna which specializes on Dicer-substrate siRNAs (Dicerna typically uses 27mers). It is therefore of interest that this publication demonstrates quite convincingly that 25base-pair duplexes, whether modified or not, are not processed by Dicer prior to loading into cleavage-competent RISC. Accordingly, the 25mers may well steer clear from infringing either Alnylam’s Tuschl-type siRNAs or Dicerna's Dicer substrates.

As I stated, Alnylam’s RNAi trigger estate is scientifically the most desirable in a number of regards. Extending the length of the dsRNA for example creates additional challenges as it relates to innate immune stimulation, and reduced delivery efficiency for certain siRNA-conjugate delivery approaches. On the other hand, it is certainly possible to find therapeutic candidates for workaround designs like the 25mers and the judicial application of design rules may address some of the disadvantages in the efficiency of finding potent duplexes.

As Alnylam’s Chinese Wall in RNAi trigger is being tested and faces key challenges later this year, both in the US (Tuschl Tussle) and in Europe (oral hearing on T-II at the EPO following challenges by the likes of Merck and Silence), it is delivery more than ever that will be key to Alnylam in strengthening its position in deal negotiations. In particular, assuming that Big Pharma cannot afford to wait forever before making their moves in RNAi Therapeutics, SNALP delivery should continue to be their key for commanding the next $100M+ licensing terms. I'm not really sure what is holding them up as the present situation must have been predictable for the company and I still feel that a move coordinated with Tekmira and one Big Pharma company could be the solution.

PS: While this paper has only come to my attention this week via my pubmed alerts, some of the data contained therein has been previously presented by RXi at scientific conferences. Moreover, whereas the paper appeared in the June edition of NAR, it first appeared online already in early 2010.

Afterword

When I started this blog, it was initially from the viewpoint of an academic scientist with a keen interest in RNAi drug development. As such I had a strong bias towards patents built on ground-breaking scientific discoveries and somewhat sneered at workaround technologies. As the years have passed, I have however come to the realize that while an academic scientist might be embarrassed to report to peers that a workaround may come close to the real thing, workarounds are a well accepted, even encouraged business practice and Big Pharma will take this into account in their RNAi trigger deal-making. Moreover, because publishing cutting-edge research as quickly as possible is the objective of academic research, in many cases, despite the ‘more worthy’ nature of such science, it has often proven difficult to build strong patents on many of these discoveries (issues such a pre-mature disclosures etc). Biotech companies, on the other hand, can conduct research in a way to maximize its IP value.

Structure-guided Evolution of Ever More Potent and Specific RNAi Triggers

As the RNAi pathway is being rapidly dissected structurally, RNAi triggers continue to evolve rapidly both in terms of safety and efficacy. The 5’ end of the guide strand of an siRNA has received particular interest in that regard, and a paper in Nature now provides structural support for why human Argonaute 2, the important Slicer enzyme that cuts target mRNAs, preferentially accommodates ‘Us’ (uracil) and ‘As’ (adenine) of the genetic alphabet in its 5’ guide binding pocket (Frank et al.: Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2).

Humans contain 4 related Argonaute proteins, Ago1-4, but only Ago2 can function in RNAi gene silencing. Not only will good incorporation efficiencies of RNAi triggers in Ago2 enhance gene silencing, loading less into Ago1, 3, 4 which only contribute to microRNA off-targeting without adding to RNAi efficacy should also improve the safety of RNAi Therapeutics. In fact, they may even compete with loaded Ago2 for target mRNAs and thus inhibit RNAi gene silencing. Similarly, because the RNAi function is pretty much dispensable in most cell types and developmental stages, burdening only Ago2 with the RNAi trigger should also reduce the risk of saturating the microRNA gene silencing functions of cells as Ago1, 3, and 4 should be able to cover for Ago2’s contribution in microRNA gene silencing through genetic redundancy.

Because of the structural relatedness of Argonautes, however, many RNAi triggers typically end up to some degree in all 4. In theory, however, Ago1-4 will differ in molecular detail and this provides the rationale for attempting to design RNAi triggers in a way (for example through chemistry, the nature of the double-strand RNA structure, or directing them into certain processing pathway) that the guide strand of an RNAi trigger gets preferentially loaded into Ago2. The 5’ end is of particular interest here because this is where the guide strand exhibits most ‘personality’ by exposing not only the relatively anonymous sugar phosphate backbone, but the entire nucleotide including its 5’ modification, typically a phosphate, to the Argonaute proteins.

It is therefore not surprising that, as indicated also by the patent literature, companies in the space are working on 5’ strategies that at the moment are particularly aimed at increasing the affinity of the guide strand towards Ago2. The message from the present Nature paper is apparently quite simple in that it suggests uracils and, a little bit behind, adenines to give you the best affinities. This is also consistent with the observation that the majority of microRNAs, the naturally occurring small silencing RNAs, also bear a uracil at their 5’ ends. An important technical advance in that paper is that these conclusions were drawn from studying the relevant domain (‘MID domain’) of human Ago2, including its crystal structure, while most of the earlier structure-based analyses have made use of more distantly related bacterial Argonaute proteins.

Related observations have been reported last year, when it was found that replacing the 5’ uracil of the microRNA let-7 with another nucleotide dramatically reduced its RNAi activity (Felice et al: The 5' terminal uracil of let-7a is critical for the recruitment of mRNA to Argonaute 2). Interestingly, the effect did not seem to be so much on the small RNA by Ago2, but rather at the target mRNA recognition step. It should be noted here that the present Nature studies only looked at the structure and affinities of single nucleotides, not the entire guide strand, with Ago2.

For the future, I would hope, for the noted potential for enhanced efficacy and especially specificity to see more of these studies, particularly those that systematically investigate the effect of the 5’ nucleotide on the relative incorporation efficiencies of silencing RNAs into the 4 human Argonautes. These would complement other studies, including one to which I contributed, that look at the secondary structure of the RNAi trigger with respect to the various Argonaute incorporations. A nice precedence of differential Argonaute loading based on the nature of the 5' nucleotide already exists in plants where it has been found that the mere identity of the 5’ nucleotide determines the sorting of a small silencing RNA into the various Argonautes.

Whether a 5’ uracil has discrimination effects in addition to the affinity benefit remains to be seen. However, the results already suggest that since the 5’ end of a small silencing RNA does not directly interact with the target mRNA anyway, it might be worth thinking about siRNA screening libraries that all start with a uracil. Of potentially even more immediate importance, DNA-directed RNAi promoter strategies ought to be re-considered as many of these still employ minimal hairpin RNAs driven from Pol-III promoters in which the guide strand is found at the 5’ end of the hairpin and that initiate with a non-uracil residue.

If you are a non-scientist and still reading this, you are doing pretty well. The take-home message is that while very potent, picomolar RNAi triggers can already been routinely discovered, RNAi trigger design is still an area with lots of potential for further improvements. In 5-10 years I can imagine RNAi Therapeutics to have matured to an extent where structurally highly adapted RNAi triggers effect potent gene silencing with minimal off-targeting activity.