Tuesday, July 28, 2009

Antisense or Guide Strand? That’s the Billion Dollar Question

As somebody who has spent the better part of my last decade in academic science, which in its ideal form is guided by the self-less pursuit of the ‘truth’, I must admit that I am somewhat irked when I read a paper on Argonaute 2 enzymology and find science being used also (though not exclusively) as a tool of corporate propaganda. You may think that this is nothing new at all. However, I’d like to use the latest publication by ISIS Pharmaceuticals on RNAi as an excuse to highlight a few of the issues that come up when ISIS Pharmaceuticals, by sheer repetition, claims that RNAi Therapeutics is an antisense technology.

In the paper by Lima and colleagues in the Journal of Biological Chemistry (‘The Binding and Cleavage Specificities of Human Argonaute 2’), the investigators study the effect of sequence length and modifications of what they call the ‘antisense’ strand on Argonaute 2 binding. What the authors call here ‘antisense’ is widely referred to by the rest of the field as the ‘guide strand’, that is the small RNA of the double-stranded RNA trigger that gets loaded onto Argonaute 2, the enzyme in RNAi that carries out the target mRNA cleavage. Admittedly, the ‘antisense’ is sometimes used in the RNAi literature to operationally describe the relative polarities of the RNAs involved (guide strand, passenger strand, target mRNA). However, when ‘antisense’ comes up 243 times within a manuscript, but ‘guide’ not at all, even as a verb, I believe it is a valid hypothesis that this cannot be a coincidence, but is calculated corporate speak.

I have explained before why RNAi by its very nature is a double-strand RNA technology, most tellingly illustrated by the fact that the discovery that what had been thought was mediated by antisense was actually mediated by the double-strand RNA contaminants of antisense preparations was awarded the Nobel Prize, so I will not repeat here myself. This is also not meant to criticize the scientific merits of the paper which furthers our understanding of the chemistries that could be used for the less efficient form of RNAi, namely single-strand RNAi and must be seen in the context of the recently announced ssRNAi initiative by ISIS and Alnylam, except maybe for the observation that the finding that single-strand RNA binds purified Argonaute 2 apparently better than double-strand RNA does not reflect Argonaute 2 loading in a living cell.

The point is that whether RNAi is labeled as an antisense technology or not can have significant consequences for the interpretation of the scope of patents, some of which date from before the discovery of RNAi in worms. It is all good for Alnylam for example to implicitly subscribe to ISIS notion as long as they have exclusive access to that IP as it helps constrain the freedom-to-operate of competitors such as Merck and Silence Therapeutics. However, when these early patents are ultimately not deemed as critical, can this leave Alnylam with patents that were pursued less rigorously so as not to conflict with the ISIS’ IP? One of the Tuschl patents comes to mind, I believe dubbed ‘Tuschl IV’, which for the first time explicitly described the use of single-strand RNAs for the induction of RNAi, but, to my initial surprise, had received very little attention among all the ssRNAi talk.

Aside from this political minefield, labeling RNAi Therapeutics as ‘antisense’ just does not do justice to the many ground-breaking discoveries this field has seen, and the people and institutions that made them and that you would think deserved their rightful place in RNAi history and a larger piece of the royalty pie. Specific modifications when used within an siRNA yes, but not the fundamental mechanism of action.

I leave it up to the reader whether this makes ISIS Pharmaceuticals an even better investment, and I must say they clearly deserve credit for their pioneering role in oligonucleotide chemistry and clinical application, and are now harvesting the financial fruits not only from their own drug development pipeline, but also from licenses for all types of therapeutic RNA approaches, including microRNAs (progress so far have exceeded my expectations, and according to a just published comments by Regulus’ CEO on HCV-miR122 this looks set to continue for the foreseeable future) and aptamers. Regardless, 243 times antisense does not make RNAi such.


Anonymous said...

At the risk of sounding weak by not posting under my name, but I do not have tome to go through tis useless process.

YOU ARE EXTREMELY BIASED TOWARDS RNAi, making your blog clearly a self-marketing device to obtain a job in the RNAi industry.

A corporation has strategies. These strategies are for the benefit of many: employees, shareholders, customers (in this instance, patients). What are your motivations? Based on your portfolio, you must be kicking yourself in the non-'guide' part of your brain for not investing a bigger chunk in ISIS. As the year draws to an end, and based on all the guidance and signs, you will be asking others to kick you as well, and maybe that will bring some sense or guide to your ideas.

Reading through your blogs, I could not resist to finally comment. You have demonstrated a very weak capacity of scientific understanding. Either that, or you have allowed yourself to be compromised somehow, as you take weak data published and make a shrine out of it, and take some of the strongest data to be presented and belittle its meaning, implications and the efforts that have gone into unraveling and advancing this technology.

In future blogs, try to be fair, unbiased and honest. Otherwise, I think you are headed in the wrong direction for securing any sort of future in the antisense or biotech/pharma industry. At your level, your critical thinking and analytical abilities should be much more advanced, and certainly, even with a small amount of ethics, have to be able to judge right from wrong.

Good luck.

Anonymous said...

spoken like a true ISIS investor.

Dirk Haussecker said...
This comment has been removed by the author.
Dirk Haussecker said...

I wished your comments were more specific. Taking my last blog entry as an example, you seem to confuse my scientific, investment, and moral views on e.g. this specific paper and ISIS' general claim of RNAi being an antisense technology.

Scientific: I think I made it clear that the study had some value (human Argonaute 2 enzymology is really under-studied given its importance!), and this is why I spent a good part of yesterday to actually read it.

Investment: I suggested in the concluding paragraph that this situation may make ISIS an even better investment.

Moral: It is against scientific etiquette to re-label established scientific terms, esp. when it is an obvious effort to claim ownership to discovery and technology.

It seems that since we obviously disagree on the latter point, it clouded your appreciation of my scientific and investment views, as well as my acknowledgement of the remarkable turnaround by ISIS.

And finally...if my blog was an attempt to land a job in industry, as you suggest in your ad hominem, then this blog would be as spineless and useless as some of the commercial content you find on the internet.

Good luck with your investment in antisense. We should see exciting progress in this area, especially for the inhibition of microRNAs, but also splice modulation and other masking-type mechanisms of actions.

Anonymous said...

Dirk, I am a bit confused as to what is so nonconventional with the term 'antisense' strand. There's an early review of Phil Sharp's (Nature Reviews Genetics 2002) that uses the term (as just one example), and many other reviews do, too. Anecdotally, I've found it to be more common than 'guide' strand terminology, although I'd have to say that the term 'guide' is much clearer than 'antisense'.

Dirk Haussecker said...

I agree with you that the term 'antisense' also has its rightful place in RNAi terminology so as to describe oligonucleotide polarities. But this does not make RNAi an 'antisense technology'.

I therefore do not believe that I am over-interpreting when I believe, especially in the context of various remarks by ISIS in the past, that using 243 times 'antisense' in an article, and no mention of 'guide' at all, is no coincidence but meant to re-inforce the notion of RNAi being antisense.

bion4maticist said...

You know who started it all: It's those dang Fire & Mello guys. In their paper "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans" they mention antisense 27 times, and guide or guide strand not even once!

Dirk Haussecker said...

How many times did Fire and Mello use the term 'antisense' to state that it was not the single-stranded antisense that induced the gene silencing, but the double-stranded RNA?

I would suggest that you either read the paper yourself or have it read by somebody in the field and that is familiar with the RNAi biotech landscape. Then come back and let us know how how antisense was used in the article. As somebody who has worked with Argonautes in the past, reading this paper has an unnatural feel, and since Fire and Mello and the discovery of Argonautes the terminology has certainly taken shape.

It is no secret that ISIS would want RNAi to be labeled an antisense technology. As you can see from the abstract below, another main point the authors make is that purified Argonaute 2 has higher binding affinity for single-strand RNA than double-strand RNA, as if single-strand RNA were the natural substrate for Ago2 and dsRNA would even interfere with its function. As we all know, however, this is not the case in a living cell where dsRNA has been shown over and over again to be at least 10-100 more potent on a molecule-to-molecule basis.

I do not have a problem if you disagree with my analysis and choose to discard it as an over-reaction. Others, however, may use it when considering the complex relationship between ISIS and RNAi Therapeutics. As long as 'antisense' is used in good faith and in the correct context, that's fine, but I would submit that ISIS would not mind a slippery slope interpretation of the term antisense in RNAi.

Here's the abstract:
"The endonuclease Agonaute2 (Ago2) mediates the degradation of the target mRNA within the RNA-induced silencing complex. We determined the binding and cleavage properties of recombinant human Ago2. Human Ago2 was unable to cleave preformed RNA duplexes and exhibited weaker binding affinity for RNA duplexes compared to the single-strand RNA. The enzyme exhibited greater RNase H activity in the presence of Mn2+ compared to Mg2+. Human Ago2 exhibited weaker binding affinities and reduced cleavage activities for antisense RNAs with either a 5’-terminal hydroxyl or abasic nucleotide. Binding kinetics suggest that the 5’-terminal heterocycle base nucleates the interaction between the enzyme and the antisense RNA and the 5’-phosphate stabilizes the interaction. Mn2+ ameliorated the effects of the 5’-terminal hydroxyl or abasic nucleotide on Ago2 cleavage activity and binding affinity. Nucleotide substitutions at the 3’-terminus of the antisense RNA had no affect on human Ago2 cleavage activity whereas 2’-methoxyethyl substitutions at position 2 reduced binding and cleavage activity and 12-14 reduced the cleavage activity. RNase protection assays indicated that human Ago2 interacts with the first 14 nucleotides at the 5’-pole of the antisense RNA. Human Ago2 preloaded with the antisense RNA exhibited greater binding affinities for longer sense RNAs suggesting that the enzyme interacts with regions in the sense RNA outside the site for antisense hybridization. Finally, transiently expressed human Ago2 immunoprecipitated from HeLa cells contained the double-strand RNA binding protein HIV-1 trans-activating response RNA-binding protein (TRBP) and deletion mutants of Ago2 showed that TRBP interacts with the PIWI domain of the enzyme."

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