Monday, September 19, 2011

Bass versus Tuschl: Patentability of 3’ Overhangs Could Depend on Meaning of ‘Person of Ordinary Skill in the Art’

2000-2001 was one of the most exciting, fast-moving periods in RNAi history. All the evidence from the various fields converged to unravel the central mechanisms of RNAi resulting, amongst others, in the discovery of how RNAi can be triggered in human cells. Plant scientists discovered that small RNAs were involved, a group in Cold Spring Harbor found that the 3’ overhang-generating enzyme Dicer played a role, fly biochemists, led by Tom Tuschl, found that synthetic siRNAs could induce RNAi first in fly extracts, and then also human cells. Eureka!

The discovery of RNAi in human cells would not have been possible without the contributions by so many other bright minds. In addition to the research publications, the scientific discourse that happens at conferences and the hypotheses put forward in review articles are a vital part of the scientific process. Everybody stands on the shoulders of others, but it is also acknowledged that the peer recognition belongs to the person/group that publishes first. It is this peer recognition more so than monetary compensation that is the satisfaction of many a scientist. If I’m honest, yours truly probably wasn’t all that different.

Tom Tuschl clearly deserves the scientific recognition. Being the first to demonstrate in a robust manner RNAi in a range of human cells in such a competitive field is no minor feat, nor did it come out of nowhere. Tuschl, partly in collaboration with Zamore, Bartel, and Sharp, did a considerable part of the groundwork leading up to the breakthrough. One should not forget that Brenda Bass, who (or is it really the University of Utah) contests the intellectual ownership over the 3’ overhang feature of the ‘Tuschl siRNA’, was in fact commenting on a paper involving Tuschl during his time as a post-doc in Cambridge, Mass, when she speculated about the possibility of 3’ overhangs in the Cell Review. The other discussions that she claims to have had with the Tuschl siRNA inventors and Zamore were also likely strongly influenced by that research…although I cannot say that for sure. In that sense Tuschl stood on the shoulders of many RNAi giants, Bass on top of Tuschl, and then (maybe) Tuschl again on top of Bass. You get the picture.

Whether having earned the uncontested peer recognition will be followed by the issuance of strong patents, however, is an entirely different question, as I believe that there is merit to Brenda Bass’ contention that Tuschl was at least in part inspired by her work and ideas. On the other hand, I should repeat that because Bass chose to publicly disclose her ideas without filing for patent protection, her ideas became a free-for-all and part of the prior art so that trying to get her name on the patent would seem like inequitable conduct in broad daylight. Therefore, the consequences of the decision whether Bass was instrumental for Tuschl to choose the 3' overhangs in his siRNA design would seem one of patentability of 3’ overhangs, not ownership.

Why do I believe the University of Utah has a point with regard to Tuschl having been inspired by its very own Bass? Some evidence can be found in the commentary of the early papers themselves which I re-read with the question in mind of what gave Tuschl the actual impetus to choose the 3’ overhang feature.


Chapter 1: Long double-strand RNA is processed into small RNAs which direct RNAi in fly lysates (Zamore, Tuschl et al. Cell March 31, 2000)

In this paper, which is a central component of the Tuschl I patent application, Zamore and Tuschl studied the processing of long double-stranded RNAs in fly lysates and found that 21-23 nucleotide small RNAs were generated. They subsequently mapped the cleavage sites in the target mRNA and found that it was cleaved at 21-23 nucleotide intervals. Consequently, the authors suggest that the 21-23 nucleotide RNAs direct RNAi target cleavage- a hypothesis that would survive scientific scrutiny.

Of note, their proposed model of RNAi in Figure 7 shows siRNA without any evidence of 3’ overhangs.


Chapter 2: Bass proposes that small RNAs observed in Zamore et al. paper form 3’ overhangs (Cell April 28, 2000)

In this review in Cell, which shortly followed the Zamore et al. paper and which is a key exhibit in the Utah case, Bass proposes, for the first time, that since only RNase IIIs were known to be able to cleave double-stranded RNAs at specific sites, the small RNAs observed in the Zamore et al. studies ought to form 3’ overhangs as the typical feature of RNase III cleavage.

Importantly, she further speculates that the predicted instability of these 3’ single-stranded overhangs explains why Zamore et al. observed a mixture of small RNAs between 21 and 23 nucleotides, instead of small RNAs of a single size (i.e. 23 nucleotides) as one might have expected from the involvement of a single enzyme, since the single-stranded RNA 3’ overhangs of the initially generated siRNAs would be susceptible to degradation in the fly lysate. As we will see, this idea may have been picked up by Tuschl in his subsequent studies, providing additional support that he and his group indeed read this review very closely. Is Bass’ speculation in fact the reason why half the RNAi Therapeutics world and research/reagent providers still cling to the (actually largely useless, and sometimes indeed harmful) 3’ dTdT feature?


Chapter 3: First demonstration, by the Tuschl lab, that synthetic siRNAs, especially those with 3’ overhangs, can trigger RNAi (fly lysates; Elbashir et al. Genes and Development January 15, 2001)

Before the Tuschl group famously published in Nature on the finding that synthetic siRNAs with 3’ overhangs can trigger efficient RNAi in human cells, they published initial results with such siRNAs in fly lysates in Genes & Development. This paper is critical as it is the first use by the Tuschl group of siRNAs with 3’ overhangs. How would they explain the adoption of this feature for their RNAi trigger design?

In addition to mapping the RNAi cleavage sites in the target RNA, Elbashir and colleagues cloned the ~21-23nt small RNAs themselves. These were then analyzed for the modifications at the 5’ and 3’ ends, upon which they found that the small RNAs contained 5’ phosphates and 3’ hydroxyl groups. They then speculated that these termini indicate that they had been processed by RNase III as such ends are characteristic of cleavage by this enzyme. They then go on to show that synthetic siRNAs with 3’ overhangs were more efficient triggers of RNAi in fly lysates than the corresponding (blunt) RNAi triggers without them. The explanation for the choice of the 3’ overhangs was the RNase III speculation.

Importantly, Tuschl and colleagues acknowledge in their model at the end of the paper that Bass, in her 2000 Cell review, already proposed the involvement of an RNase III. I guess this is pretty good evidence that there was at least some inspiration, although they also suggested earlier in their description of the experimental results that this conclusion could also been drawn independently from the small RNA end modifications that they determined. Here, I would disagree with Tuschl and colleagues as there are other RNases that leave 5' phosphates and 3' hydroxyls, and it is really the understanding of a dsRNA expert like Bass that long double-stranded RNAs get processed by RNase III. That an RNase III was involved in RNAi was shown around the same time by the Hannon group in Cold Spring Harbor. But even if we assume that the conclusion of the RNase III involvement could have been drawn independently, the Bass review was part of the prior art at this point.

I was surprised that it was the RNase III speculation and not direct experimental evidence that led Tuschl and colleagues choose the 3' overhangs in this research. Without re-visiting this issue, I had been under the impression that it was the small RNA cloning results that allowed Elbashir et al. to reconstruct the 3' overhang siRNAs.


Final Chapter 4: Synthetic 3’ overhang siRNAs trigger RNAi in human cells (Elbashir et al. Nature May 24, 2001)

This is the famous paper where Elbashir and colleagues from the Tuschl lab showed for the first time RNAi gene silencing in human cells, using synthetic 3’ overhang siRNAs. Unlike the preceding Genes&Development paper, this one discloses the exact nature of the overhangs. With the exception of one siRNA, all of them contained a dTdT in the overhang (deoxy-ribose, not ribose). This immediate adoption of deoxy would seem strange initially, because the siRNAs that are generated from long dsRNAs would have ribonucleotides in their overhangs and why risk a loss of silencing activity by substituting them with deoxy? The cited reason was that it would reduce the cost of synthesis, and probably also stabilize them from degradation. The synthesis cost argument clearly is not a valid reason for using deoxy for a basic science study such as this one, so it makes sense that it was actually instability that was on their mind, an instability first suggested by Bass in her review.

In light of the evidence in the above publications alone, I believe that it will be very difficult to make the argument that Bass did not influence the 3’ overhang feature in Tuschl’s siRNA design. Nor do I see much evidence that Tuschl made an effort to hide this and quite fairly referenced the Bass review in the Genes & Development paper. I have seen much worse examples of groups deciding not to cite the work of others because it may take away from the novelty of their research. And furthermore, if the Bass review was prior art, why didn’t others jump on the idea and published before Tuschl? Was it because he had an unusual understanding of what was going on in the field and the Bass review, presentations, and discussions only gave an extraordinary person like Tuschl the right insights, or was it ‘just’ because he was already set up to do the experiments and that there would have been many others that were inspired by Bass and would have come up with the 3’ overhang siRNAs if Tuschl had gotten into a bad accident.

The decision whether a person in the art of ordinary skill would have been able to exploit Bass’ speculations to come up with the 3’ overhang siRNAs, similar to how it apparently influenced Tuschl and colleagues, is one of the critical questions challenging the patentability of the 3’ overhang feature, possibly the only RNAi trigger feature of relatively broad scope and value that Alnylam can still hope to control.

1 comment:

John said...

In the video of a conversation between Tuschl and Klaus Weber, Klaus states that their demonstration of siRNA mediated silencing in human cells was likely only a few months ahead of other labs. Things were moving fast back then. I am still amazed that Bass did't chose to test her own hypothesis. Perhaps she tried...

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