Thursday, July 8, 2010

Silence Therapeutics Issued Important Zamore siRNA Design Rule Patent

Silence Therapeutics announced yesterday the issuance of a United States patent related to the design of gene silencing siRNAs. This is the second such patent issuance in little more than a month for which Intradigm, Silence’s merger partner earlier this year, had exclusively licensed from the University of Massachusetts and that is based on fundamental work by the Zamore lab on the enzymology of RNAi. These and other recent patent issuances related to sequence/gene-specific siRNAs address some of my concerns that I had with regard to Silence’s RNAi trigger strategy that I felt was at the risk of being too narrow and, as a consequence, becoming outdated. They should complement Old Silence's lipoplex delivery technology for vascular endothelia with valuable siRNAs in the fields of oncology and retinal disorders. Together, these developments provide evidence that the merger is starting to prove synergistic, with Silence providing the technical know-how and Intradigm complementary IP.


The two Zamore patents that were recently issued in the US relate to guidelines on how to design siRNAs with increased efficacy and selectivity. The first one, announced last month (US patent 7732593) teaches the use of mismatches between the 3’ end of the siRNA guide strand with the target mRNA so as to enhance the ability of the RNAi gene silencing complex RISC to detach from a target mRNA once cleaved and seek out new target mRNAs (note: one of the reasons RNAi is so potent is because one siRNA can destroy many mRNAs). The biochemical experiments on which these claims are based have well stood the test of time. The value of this particular patent for Silence Therapeutics is that it gives them and potential partners optionality for eventually replacing the somewhat restrictive Atu-siRNA design with next-generation siRNA structures based on solid scientific evidence. It has to be said, however, that while many current siRNAs have in fact two such mismatches, or ‘non Watson-Crick base pairs’, namely the classical Tuschl dTdT overhang that was originally conceived as stabilizing the siRNA from exonucleolytic degradation, the claims specify at least three such mismatches in the last five nucleotides of the guide strand. Moreover, the benefit of such mismatches in living cells remains to be determined, as some of the helicases and nucleases that may facilitate RISC detachment from a cleaved target mRNA in living cells may have been missing in the test tube experiments by the Zamore lab.


So while US7732592 is certainly a quite useful patent to have control over, it is the Zamore patent US7750144 of which the issuance was announced yesterday (‘Methods and Compositions for Enhancing Efficacy and Specificity of RNA Silencing’) that should provide for some interesting discussions. This patent is based on one of the classic findings in RNAi molecular biology, namely that the efficacy and selectivity of small duplex RNAs, both siRNAs and miRNAs, is critically determined by the relative thermodynamic stabilities of the two ends of an siRNA (Schwarz et al., 2003: Asymmetry in the assembly of the RNAi enzyme complex). Accordingly, it is the strand of which the 5’ end is less stably base-paired that is preferentially loaded into the RISC gene silencinng complex to become the guide strand, while the non-incorporated strand is discarded as the 'passenger strand'.


This has a few implications for siRNA design. First of all, it increases the absolute loading and consequently silencing efficacy of an siRNA. Secondly, by preferentially loading just the desired strand to become the guide, the potential undesired/off-target activity of the passenger strand can be virtually eliminated. These asymmetry rules have also been extensively validated in living cells and are part and parcel of most siRNA design strategies and algorithms. They are also consistent with structural X-ray crystallographic findings that show that the 5’ end of a guide strand within RISC is unpaired. As a result, it is very likely that a number of siRNA therapeutic candidates currently in the clinic could be interpreted to fall within the scope of this patent. This should also encourage some of the research & reagent companies that sell synthetic siRNAs to take a license from Silence Therapeutics.


The actual issued claims relate to a method of decreasing passenger strand activity by introducing one or more changes in the interaction between the 5’ end of the guide strand with the passenger strand such that the loading of the guide strand is enhanced. The broadest interpretation, and the one that I would expect Silence will take, is that any siRNA containing such a modified nucleotide or mismatch in the specified region (the first 5 nucleotides of a guide strand) would infringe. Most siRNAs should fall within this definition. In some cases, such modifications may have in fact been motivated by other things like stabilizing an siRNA against degradation or reducing innate immune responses, but this remains to be tested, unless the companies involved choose to settle without resorting to yet more exhausting and extremely tedious legal battles. I should note here that when one follows the patent prosecutions especially in Europe, there does not seem to be a single patent that gets issued that will not be opposed by the other party, no matter the scientific merits (my personal opinion). It is enough that some in Big Pharma routinely abuse the patent system to bully smaller competitors into bankruptcy, essentially a way of doing business, and pure-play RNAi Therapeutics companies should be smart enough to know that by adopting these practices they only destroy the size of the shared pie they all depend on: patent-protected innovation. But I digress...


In summary, the recent events have lessened my concerns that it was largely Intradigm that was the beneficiary of the merger as their historical focus on IP is now bearing fruit in the form of some quite fundamental patent issuances and should nicely complement Silence’s strength in the science and clinical translation of RNAi which I had considered to be the stronger of the two. So while I still await data that similarly support the company’s claim that the merger also brought synergies in siRNA delivery, the combined company appears to be quite undervalued here with a market cap of around $20M. The patent issuances should also bode well for the extension of the RNAi trigger-focused part of Silence’s relationship with AstraZeneca. Because of this and because the markets appear to be on summer vacation with the stock virtually unchanged following such strategically important patent news for the company, I’ve decided to buy a few shares of Silence here.


PS: For the various reasons that you can also find in Silence’s regulatory filings, investments in RNAi Therapeutics in general and Silence Therapeutics in particular have to be considered very high risk. Moreover, although I consider myself fairly familiar with the molecular biology of RNAi, I am not trained in intellectual property and my interpretations of the Zamore patents have to be read in this light. Please also read the disclaimer at the bottom of the page.

8 comments:

Anonymous said...

Hi Dirk,

Please dicuss the pending merger of MRNA and Cequence. The market seems to have discounted the share price of MRNA since the announcement. Is the combined technology and patent library of the combined company fairly valued? Are the shares currently attractively priced in your opinion? Thanks for any update, joe

Dirk Haussecker said...

Hi Joe...I understand your concern about mdRNA's share price. Let me only say as much that I feel that valuations in the RNAi Therapeutics space are generally quite depressed, and a number of companies have significantly more value than the market cap would suggest.

Anonymous said...

Hi Dirk,
What do you think about ALNY's publication this week of their new rules on more potent siRNAs?
Also, can you comment on Santarus publication of their 1-2mg/kg LNA results...is lack of toxicity good for Tekmira too?
thanks

Dirk Haussecker said...

The Alnylam publication on enhancing siRNA potency by destabilizing a central position in the siRNA duplex is actually quite timely given the Zamore patent issuance. The work by Addepalli et al. confirmed the Zamore differential end-stability rule, but then went on to show that you could make an siRNA even more potent through other select chemistry. Generally, the more potent, the better for RNAi Therapeutics, also as it somewhat decreases the delivery hurdle and should also be safer.

Dirk Haussecker said...

The Santaris study is certainly an interesting one. It supports once again that LNAs are amongst the most, if not THE most potent antisense chemistry. About 50% knockdown of LDL-cholesterol was achieved with weekly dosing of 2mg/kg in non-human primates, about 10-20 fold more potent than mipo, but about 20-50 fold less potent than SNALP-siRNA (comparing non-human primate data). Also positive is the fact that it had a surprisingly quick onset of action.

There were, however, a few confusing aspects of the study that may have implications for the safety of this chemistry. With respect to ApoB knockdown as an approach to treating hypercholesterolemia, it is encouraging that no fatty liver phenotype was observed early on despite the quick onset of action (this is in contrast to Mirus Bio’s RNAi study). There was, however, a ‘mild’ increase in neutral lipids (6-7% of normal liver weights) with prolonged dosing which they said was accompanied by similarly mild increases in liver enzymes to about 3x ALT. The study authors suggest that this might be a target-dependent phenotype. This, however, appears to be incompatible with what other groups have reported, namely acute fatty liver phenotypes early on that then normalize through feedback mechanisms (main proponent ISIS). It is similarly strange that the ApoB/LDL-c reduction was accompanied by a quite significant reduction in HDL-cholesterol (about 30% reduction when LDL-cholesterol was reduced by 60%; this was observed both in mice and primates). This would appear to be contrary to the genetically expected outcome. So while LNA antisense is quite powerful compared to other antisense knockdown methods, more probably needs to be learned about its toxicology. At the end of the day, no technology will ever be without side-effects and the real risk-benefit needs to be studies in carefully designed clinical trials. LNAs are certainly at that point. A lot of patients would benefit from new treatment options for hypercholesterolemia, and let the best drug reap the commercial benefit.

Anonymous said...

Dirk,

What happened to the RNAi litigation blog?

Anonymous said...

Dirk,

What happened to the RNAi litigation blog?

Dirk Haussecker said...

I temporarily removed the link to the RNAi Litigation blog to allow for the link to the Alnylam review on the State of RNAi Therapeutics. I guess I will have to reorganize and add a blog roll column to the blog.

By Dirk Haussecker. All rights reserved.

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