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. fire@mail1.ciwemb.edu

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.