Monday, August 18, 2008

RNAi is not Antisense

From the abstract of an important paper in RNAi:

"Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.
Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA.

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts...Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference..."

RNAi is not antisense- This very finding formed the basis for awarding the 2006 Nobel Prize committee to Andy Fire and Craig Mello. Despite this authoritative decision, as the promise of the RNAi Therapeutics platform is getting closer to reality by the day, the promise also of financial fortunes has led to efforts to re-brand RNAi as yet another antisense technology. Although this in a way could be considered as some kind of validation of the competitive nature of RNAi, it has important implications for RNAi intellectual property.

In the past, ISIS Pharmaceuticals, undoubtably a leader in RNA therapeutics and with nucleic acid modification IP very relevant for RNAi Therapeutics, has made a number of statements I cannot agree with. Essentially, the company claims having discovered RNAi and has long regarded Alnylam as one of their many “satellite companies”. This, however, does not reflect reality in as much as repetitively claiming that RNAi is an antisense technology does not make it a true statement.

The latest example can be found in ISIS Pharmaceutical’s press release on the issuance of an ApoB-related antisense patent summarizing the claims as covering “the use of both single-stranded and double-stranded (siRNA) antisense drugs complementary to any site of the mRNA of human apoB regardless of their chemistry or antisense mechanism of action.” “Double-stranded antisense drug”- quite an oxymoron. But note that nowhere in the claims of the patent are duplex RNAs mentioned, instead “12 to 30
nucleobases in length targeted to a nucleic acid molecule encoding apolipoprotein B wherein said compound is 100% complementary to the nucleic acid sequence set forth in SEQ ID NO: 3” are claimed.

RNAi being antisense, having discovered RNAi first, and Alnylam being a satellite company, just does not agree with the fact that the very reason why Fire and Mello’s discovery is considered a major breakthrough, is because they found that RNAi is induced by double-stranded RNAs and not by antisense molecules as had been widely believed, and this indeed was the very working hypothesis that Fire and Mello famously disproved and for which they were awarded with the Nobel Prize. Come to think of it, RNAi as an “anti-antisense” technology may be a more apt description.

The claim why RNAi is supposed to be an antisense technology is largely based on the observation that during RNAi, the double-stranded siRNA is unwound, and the single-stranded guide RNA strand incorporated into RiSC which it then guides to target mRNA partly, but not exclusively, based on base-pair complementarity. Just because base-pairing interaction is involved, however, does not make it antisense. What is important for a biotechnology that harnesses an endogenous biological pathway are the features that make it being recognized as a bona fide substrate. In the case of RNAi, this clearly is double-stranded RNA with a couple of additional characteristics, and not single-stranded antisense RNA. This is the result of RNAi having evolved as a way to recognize and fight RNA viruses which are characterized by double-stranded RNA intermediates. Re-branding RNAi after one of the components of its inducers would be like saying the wheels of a car are the car.

While single-stranded antisense RNAs may also induce RNAi, research by ISIS itself and others has shown that they do so much efficiently, about 20- to 50-fold less efficient than double-stranded RNAs, that is about as efficient as a conventional RNaseH antisense molecule (probably another reason for ISIS’ growing interest in RNAi).

With so much “new fundamental” trigger IP being claimed, RNAi now becoming an antisense technology etc., are we about to see a different approach by the RNAi Therapeutics companies in possession of the real fundamental IP? I would not necessarily expect legal proceedings as long as it does not start to affect their business dealings. However, such rhetoric has the potential to lead to market inefficiencies, hurt smaller companies with a less active PR department (see Tekmira and their ApoB program), and lead to a situation in which as the first RNAi Therapeutics are approved and patent disputes to be resolved, the debate will have been re-framed to the disadvantage of RNAi Therapeutics companies.

PS: The RNAi-is-Antisense claims also get to the important issue of target IP. Target-specific IP should be encouraged as it incentivizes the identification of suitable RNAi Therapeutic gene targets, as long as certain minimum validation standards are satisfied. I would therefore agree that if the ISIS antisense patent had been a milestone in demonstrating the suitability of apo-B inhibition for the treatment of hypercholesterolemia (note: ribozymes targeting Apo-B were specifically exempted from the claims which already suggests that the present patent is not the seminal ApoB-hypercholesterolemia patent), this should also have consequences for drugs based on other mechanisms of actions such as RNAi, monoclonal antibodies etc. In cases where there is a proven benefit in targeting at the RNA level, the royalty rate should climb commensurately, although I do not believe that in practice this means that an antisense therapeutic will be able to stop an RNAi Therapeutics from reaching the market and vice versa.

Note: The abstract of the Fire and Mello paper has been added after the first publication of the blog entry.


fritz said...

Isis does have an impressive catalog of drug candidates in their pipeline; however, the fact that RNAi can be much more powerful than antisense in addition to big pharma's interest in RNAi as a platform, whereas antisense was the bastard child of genomics for many years, seems to have bothered some folks at Isis. Obviously, Isis has spent a vary long time developing and optimizing RNA drugs (my hat's off to them as they have forged through some tough issues) while nascent Alnylam has grabbed the spotlight in a few short years. Clearly, one can see why there might be such a sibling rivalry developing at Isis HQ. As you point out, there is a difference between RNAi and antisense. Alnylam owns the IP, and even the IP from Isis for double stranded molecules for transcription inhibition. Personally, I can't see the issue going very far, as if it really comes down to a pissing contest ( if the IP really could block RNAi or that Alnylam could block Isis on their targets) for gene targets, Alnylam through their collaborators at Roche, Novartis, Takeda, and importantly many research institutions, will be able to lock Isis out of some of the most valuable targets to come out of the genomics revolution.

Even if such IP stands up, I don't think either company has much to gain by pissing the other off...

bion4maticist said...

Please document your statement that Isis claims to have "discovered RNAi first". Where, when, and by whom, please.

Is RNAi an antisense technology? It depends on how you define the word. One common understanding is, in fact, anything that uses Watson-Crick pairing for target selection. For example, the Wikipedia (which I'm quoting here not as an erudite authority, but to document what's commonly understood) says:
"Antisense molecules interact with complementary strands of nucleic acids, modifying expression of genes....Many forms of antisense have been developed and can be broadly categorized into enzyme-dependent antisense or steric blocking antisense....Double stranded RNA acts as enzyme-dependent antisense through the RNAi/siRNA pathway...".

Another commonly understood meaning of the word narrows antisense to mean the type of RNAseH-mediated therapeutics that Isis has developed.

So, yes, RNAi operates by an antisense mechanism (if you're thinking of the broader sense of the word), and yes, RNAi is not "traditional" RNAseH-mediated antisense (if you're thinking of the narrower sense of the word).

But of course the real issue is not the meaning of the word, but whether Isis' patent on APOB applies to RISC-mediated antisense. Isis thinks it does. You think it doesn't. I think it'll have to be decided in court someday soon.

Anonymous said...

Thank you to the respondents for understanding that Isis is spelled Isis not ISIS! Maybe someday Dirk will catch on, or maybe he will start writing about ALNYLAM :)

GS said...

Good points all around. In terms of developing something clinically and commercially useful, the multiplied effect of RNAi is a key difference over antisense IMO.

Reminds me Dirk... from your recent RNAa thread, is that effect also amplified as with RNAi, or 1:1 with the triggers as with antisense?

bion4maticist said...


It's true that some antisense mechanisms are not catalytic and are therefore 1:1 with target and lack a "multiplied effect". These are the steric blocking mechanisms such as morpholinos.

But the RNAseH-mediated antisense mechanism, like the RISC-mediated (RNAi) antisense mechanism, is catalytic. RNAseH cleaves the target mRNA, but leaves the DNA-like antisense oligo (the drug) intact. So one molecule of the drug can result in the destruction of many mRNA target molecules. Just like siRNA.

Anonymous said...

From the PharmExec article titled RNA Revolutionaries

Accordingly, Crooke has undertaken a subtle rebranding. RNAi, he says, is merely a subset of antisense. "We've pioneered all the major mechanisms in antisense, including, of course, RNAi."

- James

GS said...

Thanks bion4maticist

Any idea whether currently used miRNA, shRNA and aRNA triggers also remain intact & thus potentially catalyse also?

Anonymous said...

Interesting comments and viewpoints, everyone.

To me Isis' logic sounds a bit like an inventor of a steam engine claiming credit for a combustion engine on the argument that combustion engines are really only a subset of steam engines inasmuch as, at the end of the process, both make a wheel turn around.

But what if the waterwheel inventor joined that debate...?

Having said the above, I appreciate that there may be arguments to be made for both sides of the debate, which means that it could get quite ugly if it ended up in court.

Let us hope that that will not be the case but it appears to me that Isis is being more and more assertive in their view of RNAi as a subset of antisense. Alny should avoid making the same mistake that the West made with Russia in believing that repeated claims will go away if you ignore them (even as such claims are repeated with greater force and confidence each time the previous claim goes unchallenged).

The advantage of the corporate world is that you always have the merger option. Although I am not a big fan of Alny/Isis merger proposition, it is clearly a better prospect than an ugly fight (Crooke could retire and be the chairman and let JM run the show ;o)


bion4maticist said...

Thanks for the Crooke quote, anonymous.

It's meaning is obscure. What does it mean to pioneer a mechanism? My guess is that Crooke was not claiming to have discovered RNAi--he didn't, and that's obvious to everyone. I'm guessing he meant, "We've pioneered [in figuring out how to exploit as therapeutics] all the major mechanisms in antisense, including, of course, RNAi."

That's plenty arrogant, but not transparently false.

Send an email to my handle at hotmail dot com and we can chat about your questions. I'm not an expert, but will share what I know.

Dirk Haussecker said...

Great comments. In addition to Crooke’s repeated highly suggestive statements in investor presentations about their pioneering role in RNAi Therapeutics, similar comments can be heard at conferences. At the Keystone e.g. it was noted in no uncertain terms by somebody from ISIS that some of the single-stranded oligos they tested in late 90s for gene knockdown (on the premise of working through ‘antisense’) might have worked via single-strand induced RNAi. I wouldn’t be surprised if they are now going back to the lab and try to get more evidence for this- not because it’s scientifically useful, but for making IP claims. Maybe this explains why ALNY lets ISIS pursue single-stranded RNAi as part of the original ISIS-ALNY deal, well knowing however, that ssRNA are not the biological inducers of RNAi and accordingly ssRNAi-induced knockdown is about 20-50 fold less efficient than dsRNA-induced RNAi- hardly useful for therapeutic applications.

“To me Isis' logic sounds a bit like an inventor of a steam engine claiming credit for a combustion engine on the argument that combustion engines are really only a subset of steam engines inasmuch as, at the end of the process, both make a wheel turn around.”
Great analogy. Soon, base-pairing will fall under their patents, too.

Martin and Fritz- I agree none of the two companies will be winners if they square off in court. For the conspiracy theorist, publicly supporting some of ISIS’ claims may even be part of a combined ISIS-ALNY effort of making the RNAi patent wall even higher for competitors.

GS- the main reason why RNAi is so much more efficient as a natural silencing mechanism compared to forced RNaseH antisense is less the fact that the actual mRNA cleavage is enzymatically catalyzed, but that the siRNA gets recognized and loaded into the RiSC complex which, as a complex, is very efficient in seeking out multiple target mRNAs for destruction. RNaseH-antisense, however, does not form such a complex. Antisense recognition of target mRNA is comparatively inefficient under physiological conditions if not mediated by a dedicated complex like RiSC and requires high steady-state concentrations of the antisense. This antisense-mRNA complex then needs to be recognized by RNase H in a non-physiological context (a tri-molecular reaction essentially, versus an energetically directed two-molecular mechanism).

To clarify my statement that target recognition is not entirely based on complementarity, it is based on the observation that high base-pairing energies do not make for a potent siRNA and today’s siRNA design algorithms take into account the multiple interactions of an siRNA during RNAi, e.g. RiSC loading and release from the target mRNA after cleavage. There also appears to be an advantage of the first nucleotide of the guide strand being mismatched as a result of how Argonaute-2 holds onto the 5’ end of the guide RNA.

GS said...

Thanks Dirk.
Do you know off hand whether shRNA, miRNA, aRNA are also RiSC/ other similar complex mediated (& remain intact) and therefore amplified, or is this a particular advantage of siRNA etc?

bion4maticist, I'll be in touch...

bion4maticist said...

Dear Dr. Haussecker:

Please don't try to weasel out of this. You said, "Essentially, the company claims having discovered RNAi...", but your comment only asserts, again without documentation, the claim of a "pioneering role" in investigating RNAi therapeutics.

They have done research. They consider it pioneering. You do not. If you can't document the more extravagant claim, perhaps you'd like to edit your article. You can find plenty of examples of arrogant behavior with which to tar them without making stuff up.

I don't find the car metaphors very apt. If you really examine the issue with an open mind, I think you'll find that they are asserting something more like: "Just because cars have traditionally had gasoline engines, a car with a diesel engine is no less a car." In their view. Using the broad idea of antisense therapeutics as drugs that use W-C pairing -- with one enzyme system or another (gas or diesel), or without any enzyme at all -- as their mechanism. It isn't at all an outrageous assertion. It's one perfectly reasonable, and commonly understood, definition of an antisense therapeutic.

With regard to efficiency: It is true that siRNAs can achieve efficient knockdown of target with intracellular concentrations 1 or 2 orders of magnitude less than phosphorothioates. But, of course, that's measuring intracellular concentration. You have to get it into the cell, eh? The more advanced gapmer designs seem to be closing this potency advantage pretty rapidly.

With regard to complementarity: It is true that there are considerations for drug design that go beyond mere complementarity. This is as true for Isis' oligos as it is for Alnylam's. Does that mean Isis' drugs, too, are not based on an antisense mechanism? Or is it the single-base mismatch that's the defining characteristic?

And now some completely gratuitous opining: I hope both companies are fabulously successful. I think that there is plenty of room for both to operate. The identical dream of both companies is to develop a robust platform of genetically informed, rationally designed drugs that will allow medicine to target a vast array of previously-undruggable targets. Whether you consider these to be one platform or two, let's hope they succeed and wish them both good fortune.

Anonymous said...

If I understand this correctly Isis perceives RNA duplexes as antisense pro-drugs converted by RISC into the "active" form.
For this to be correct, single stranded antisense RNA alone ("real drug") would need to be equally or more efficient, what does not seem to be correct.

On the other hand,in case of Dicer-substrates designed to circumvent Alnylam's patents, perceived "drug" is siRNA duplex, clearly equally (or more?) potent to the "pro-drug" - Dicer substrate.


Dirk Haussecker said...

Bion4maticist- on days like these when Wall Street analysts such as Jon Alsenas from Leerink Swann come out with ludicrous research reports on Alnylam, and essentially all nucleic acid therapeutics, I somewhat understand your comments.

My blog entry was meant to highlight ISIS' assertions on FUNDAMENTAL RNAi IP based on research dating back to before the discovery of RNAi.
Saying that RNAi is antisense, and that they have found before the time of Fire-Mello that single-stranded antisense induces RNAi, equates to claiming the discovery of RNAi- there is no way around it. I, however, believe that it was the explicit introduction of double-strand RNAs and the observation of gene knockdown that marked the discovery of RNAi and heralded its use as a widely applicable technology.

Anonymous said...

Dear Bion4maticist:

The style of some of your comments (most notably the last one) is quite unnecessary and, indeed, makes me wonder whether it is debate or confrontation that you wish to elicit.

I have listened to and read some of the recent Isis presentations (and do not bother to challenge me as to which one, when, by whom or page reference – I am not keeping notes and have no intention of writing thesis on what and when Isis said). On this basis, it is my impression that Isis is in fact looking to frame RNAi as a subset of antisense. While this may not be technically tantamount to claiming that they "discovered" RNAi, from an IP perspective it really makes no difference. Isis' purpose here is not to receive applause for RNAi discovery from the scientific community but to expand their IP claims into this promising new technology. From Isis' perspective it is understandable but their arguments strike me as self-serving.

The point with the engine analogy was that it is an entirely different mechanism of action that results in the same or similar end effect. Take cancer as another example and one that is closer to the field. Would someone with a technology (whatever technology that might be and regardless of whether it works) aimed at destroying tumor cells be entitled to make claim to a different technology with a different mechanism of action that also results in the destruction of tumor cells? I think not.

I do appreciate your comments and it is helpful to see different perspectives (in particular from people that appear to be knowledgeable). But please stop being petty in trying to "catch" Dirk on any particular choice of words. Especially as that choice of words is irrelevant to the gist of the post in that Isis appears to be trying to expand its IP claims into RNAi on (what I consider to be) self-serving arguments.


Anonymous said...

i could be wrong but as a benitec investor it seems to me the uspto has raised much antisense prior art in their non final rejections of benitec's graham patent for ddRNAi. i'd be cautious with the sounds coming from ISIS


bion4maticist said...

Moler: Yes, I suppose the anti-guide strand could be considered to be some kind of adjuvant. But your conclusion that ssRNA would therefore have to be equally effective is false. Adjuvants make a drug more effective while being themselves completely uninvolved in the mechanism of action. And that's obviously the case here: RISC loads dsRNA and discards the anti-guide strand. The guide strand then directs the destruction of complementary RNA.

Dr. Haussecker: Claiming to have observed a phenomenon without having described it is not the same as a claim to have discovered the phenomenon. I still feel that your accusation is unsupported, tendentious, and contentious. You are putting obviously false words in their mouths, and then pillorying them for what they have never said.

Martin: I apologize to you (and to Dr. Haussecker) if my words appeared unnecessarily confrontational. (See paragraph above: I felt provoked by what I perceived to be calumny.)

I agree with you that Isis is trying to describe RNAi as operating via an antisense mechanism (or perhaps reminding them that it does so). And that it's self-serving. I also think that their position is actually quite objectively true according to a commonly accepted definition of that mechanism. Please go read the original paper by Dr. Fire and look at how he uses the word "antisense" to describe his work in worms, the work that first described a mechanism that would come to be called RNAi and which would win a Nobel Prize.

We disagree on whether it's fair to characterize as "an entirely different mechanism of action" these two systems which use Watson-Crick pairing with a guide oligo to capture an mRNA so as to cleave it with an endonuclease. I can see how you could see it that way, since the proteins involved are different. But I can easily see a perspective that says that these are highly analogous systems.

I do understand that it is frightening to Alnylam partisans to see Isis try to encroach on what had been considered to be Alnylam-exclusive turf. Wouldn't you expect Alnylam to be just as aggressive in pursuing and extending its IP claims?

If you want something to be outraged about, how about that "satellite company" talk. They really did say that -- that Alnylam was one of Isis' satellite companies -- and I think that was outrageous and provocative.

Anonymous said...

Isis is not trying to expand their IP claims. They have ALWAYS claimed to have fundamental IP in the RNAi area. It is called the Crooke patent estate and is based on their early work with ssRNA. They were also the first to clone and sequence the enzyme that is now called Drosa. The Crooke patents, along with the plethora of chemical modification patents, are why Alnylam had to do a deal with Isis early on. If you read many of the Alnylam press release exposing their dominant patent position they often include by name the "Crooke" patents.

Dirk Haussecker said...

It is true that Alnylam frequently refers to the fundamental importance of ISIS patents for RNAi Therapeutics. This, however, relates to a) dsRNase mechanism of action, and b) chemical modification of oligonucleotides, and not every claim that ISIS has made such as that RNAi was merely a form of antisense.

Of course, since they have exclusive access to that IP (a+b) for RNAi, it is also entirely self-serving to add these patents to the fundamental RNAi Therapeutics patent estate. While I agree that the chemical modification patents are important, I feel less strongly about the dsRNase mechanism of action for which no real therapeutically relevant in vivo data was provided. Just in case you wondered whether I was an Alnylam spokesperson.

Bion4maticist- if I would just copy-and-paste press releases then there would be no point to write this blog. It's more important to me what somebody means when he says something, and in the case of ISIS it is clearly their intent to claim discovery rights to RNAi, not, as Martin rightly notes, for scientific accolades, but IP rights. And, yes, I meant to be provocative when I translated that claim into its scientific meaning.

Dirk Haussecker said...

I have now added to the beginning of the blog the abstract of the Fire-Mello paper as a refresher.

Anonymous said...

is this award as broadly impacting on siRNA IP as the press statement is claiming?


Dirk Haussecker said...


Good question. I've commented on the patent in question (dsRNase MoA) in a previous entry of this discussion thread. It's certainly possible to relate these findings to RNAi, but I do not think it's anywhere as strong as the other fundamental patents held by and licensed to ALNY. You also have to see that if ALNY and ISIS get along well, this makes it very tough for the competition patent-wise. JMHO:

"It is true that Alnylam frequently refers to the fundamental importance of ISIS patents for RNAi Therapeutics. This, however, relates to a) dsRNase mechanism of action, and b) chemical modification of oligonucleotides, and not every claim that ISIS has made such as that RNAi was merely a form of antisense.

Of course, since they have exclusive access to that IP (a+b) for RNAi, it is also entirely self-serving to add these patents to the fundamental RNAi Therapeutics patent estate. While I agree that the chemical modification patents are important, I feel less strongly about the dsRNase mechanism of action for which no real therapeutically relevant in vivo data was provided. Just in case you wondered whether I was an Alnylam spokesperson."

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