Big Pharma RNAi Therapeutics Backlash Shows Signs in Literature

There has been some unhappiness among certain Big Pharma companies about their adventures in RNAi Therapeutics. Roche, Pfizer, and Abbott Labs were among the publicized companies that discontinued RNAi Therapeutics platform development efforts not too long ago. Unsurprisingly, you could hear criticism from these companies about alleged immaturity of the technology and having been misled by the RNAi Therapeutics industry. Some of that backlash can now be felt in the peer-reviewed literature. Two recent papers by Abbott and Pfizer allow us to gain further insight into the causes of this unhappiness based on their actual practical experiences with the technology.

Pfizer: If it’s not ‘naked’ or involves invasive routes of administrations, we aren't interested

Pfizer, famous for its track-record of turning life-style drugs into blockbusters, apparently made the decision to pursue phosphorothioate LNA antisense over RNAi Therapeutics. As could be deduced from comments by then Pfizer oligonucleotide therapeutics chief Art Krieg that the need for intravenous administration makes a number of RNAi delivery technologies much less attractive, this was partly driven by concerns about patient convenience. Somewhat related and as could be seen from Pfizer’s conference abstracts, Pfizer also abhorred the complexity of some RNAi formulations. Consequently, instead of having therapeutic efficacy drive their research, Pfizer tried to make RNAi conform to their commercial principles (convenience and simplicity) meaning that they asked RNAi do what it is not suited to.

This type of scientifically conflicted approach was also exhibited in a paper by Pfizer published this month in Molecular Therapy (Moschos et al.: Uptake, Efficacy, and Systemic Distribution of Naked, Inhaled Short Interfering RNA (siRNA) and Locked Nucleic Acid (LNA) Antisense). The researchers set out to test the utility of unformulated siRNAs or LNA antisense for knocking down genes in the lung following intratracheal administration. To their disappointment, they were not able to observe target gene knockdown in the lung by either RNAi or LNA phosphorothioate antisense. This, of course, is in contrast to Alnylam’s studies that claimed that unformulated, unmodified siRNAs were an effective way to knock down genes in the respiratory epithelium and therefore fight respiratory syncytial viral (RSV) infection via RNAi gene knockdown.

Another surprising (this time for real) observation from these studies was that quite a bit of the unformulated oligonucleotides apparently made it into the systemic circulation following intratracheal administration, and as would be expected from the pharmacology of unformulated siRNAs and phophorothiote antisense, they were then either rapidly excreted into urine (siRNAs) or in the case of phosphorothioate antisense accumulated in various tissues, essentially identical to their biodistributions following intravenous or subcutaneous administrations.

For antisense therapeutics, inhalation may therefore be an interesting new route of administration for systemic therapy (e.g. knocking down ApoB in the liver). No more needles and injection site reactions.

In summary, Pfizer came out disappointed on RNAi Therapeutics as it approached it in one of the worst possible ways. Whether this ‘naked’ oligonucleotide thinking is really a choice or also reflects lack of know-how and technology access, only Pfizer really knows. As could be seen from the author affiliations in the paper, a number of scientists lost their jobs over this strange approach towards developing RNAi Therapeutics.

Abbott: Even the much-touted ‘SNALP’ cannot satisfy our cancer needs

The next technology to receive a scolding from Big Pharma, in this case the siRNA Therapeutics group from Abbott Labs, was…SNALP. The Li et al. paper published online in Gene Therapy at the end of September investigated the use of SNALP for knocking down genes in solid tumors. For this, they used their mouse cancer model where either a beta-gal or luciferase reporter gene in cancer cells was suppressed by the tetracycline repressor, tetR. Knockdown of tetR causes the derepression of the reporter genes which can be used as a marker of RNAi activity. They used SNALP because they had, in their own hands, previously identified it to be the most effective delivery system using this model system (Lin et al. 2011).

To summarize the results, Abbott found that SNALP-mediated gene knockdown was most effective in cells close to the vasculature. Cells in areas further removed did not exhibit knockdown sufficiently potent to cause the de-repression of the reporter gene. This in fact is in line with what is known from the pharmacology of similar liposomal particles and is relevant for the development of ALN-VSP02 and TKM-PLK1, two SNALP-delivered RNAi Therapeutics in clinical development for solid cancer: higher vascularized tumors will likely respond better, and the target gene should allow for strategies where a tumor can be killed from the vasculature outside in. It’s also not like all small molecules or antibodies would homogenously distribute within a tumor.

Overall, the Abbott scientists did not seem all too pleased as the following concluding remarks of that paper show: ‘Although, in a general sense, the impact of tumor vasculature on nanoparticle-mediated delivery is not surprising, it is enlightening that the delivery efficiency of SNALP, the perceived current state of art in siRNA delivery, is still severely limited by tumor vascularity.’

Not surprising, but enlightening...Apparently, Abbott people have also lost their jobs over RNAi Therapeutics, so some of the bitterness is understandable. On the other hand, when I look at the Big Pharma RNAi literature and conference abstracts, perhaps with the exception of Merck, I see little problem solving or innovation. Instead, it is dominated by passive technology evaluations, often using home-made brews rather than the original stuff, or pretty mundane process development studies which, in the absence of viable delivery technologies, seems like putting the cart before the horse. It is even possible that having these internal RNAi groups has been harmful for allowing the technology to get a fair evaluation in Big Pharma as such groups may cause the companies to become too much inward-looking, protective of one’s own people, instead of tapping into what is already out there. Without owning a leading delivery technology themselves, the best strategy may be for Big Pharma to just license product candidates instead of trying to build their own RNAi Therapeutics platforms in-house.

Santaris Terminates PCSK9 Hypercholesterolemia Trial

Antisense company Santaris seems hardly able to catch its breath these days. After being sued by ISIS Pharmaceuticals for patent infringement, reporting first clinical proof-of-concept for a MicroRNA Therapeutic, it has now been revealed that the company prematurely terminated a phase I trial of its PCSK9 phosphorothioate LNA RNase H antisense inhibitor SPC5001 for the treatment of hypercholesterolemia. This study had been initiated in May of this year with an estimated enrollment of 40 healthy and familial hypercholesterolemia subjects.

Reasons for the trial termination were not revealed. However, since this was a phase I safety trial, it is likely that the trial termination was due to some safety issue. Supporting this is that, coinciding with the PCSK9 trial termination, enrollment has been halted in another phase I hypercholesterolemia trial sponsored by Santaris, this time targeting ApoB (SPC4955).

Consequently, after the premature trial termination last year of BMS/ISIS’ phosphorothioate 2’ MOE RNase H PCSK9 antisense inhibitor (BMS-844421), Alnylam’s SNALP-delivered ALN-PCS has suddenly taken the lead in the race to develop a PCSK9-targeting RNA Therapeutic, pitting it against the monoclonal antibodies by the likes of Regeneron and Amgen.

Without explanations for the trial terminations, it is difficult to pin down the exact cause(s) of the presumed toxicities. What the compounds by Santaris and BMS/ISIS Pharmaceuticals obviously share is that they both use phosphorothioate chemistries and work via an RNaseH mechanism. Phosphorothioates are widely used in antisense development because they bind to proteins in the plasma and various tissues, thereby allowing one to achieve tissue concentrations high enough such that mass action will allow for cell penetration and target gene knockdown. In my opinion, phosphorothioate oligonucleotides are promising for a number of local applications. The problem with systemic applications, however, is that the high concentrations reached in tissues such as liver, spleen, and kidney, ultimately limit their therapeutic window. Some industry experts have referred to this issue as the ‘hazardous waste’ problem of systemic phosphorothioate oligonucleotides.

ISIS supporters will point out that Santaris’ specific LNA chemistry is the most likely culprit, whereas ISIS’ chemistries are relatively safe as supported by ISIS’ comparative studies. Of course, these studies were published to make Santaris look bad, just as the recent lawsuit was aimed at blunting Santaris’ competitive threat. Conspiracy theorists may even suspect that the sudden turmoil around Santaris is no coincidence and was orchestrated to inflict maximum damage at a time that Santaris seemed to be on a roll with a number of trial initiations, positive HCV data, and depriving RNAi Therapeutics of a deal with Pfizer (Pfizer apparently being attracted to ‘naked’ oligonucleotide approaches over RNAi nanoparticle ones). However, if Santaris intended to go public to satisfy its increasing cash needs (maybe less so with the trial terminations), it can probably scratch that now.

RNA Therapeutics is intensely competitive as they compete for pretty much the same investment dollars. Investors, including Big Pharma, are easily influenced by public opinions and fashion trends. 4 years ago it was all about the fear of being left behind in RNAi Therapeutics, with little attention being paid to true enablement and expertise in a noisy field. This made antisense look like the ugly cousin. In the last 2 years, however, antisense has regained favor as the RNAi delivery ‘problem’ became widely publicized which made antisense look deceptively simple. Santaris especially made a living of that by advertising, e.g. at conferences and press releases, the delivery problem of RNAi Therapeutics and getting a few laughs by stating their motto of 'staying naked’ and ‘staying short’.

Needless to say, my favored technology is RNAi Therapeutics: ‘natural’ and ‘potent’ is my motto. The hypercholesterolemia arena will be a particularly good battleground for antisense and RNAi Therapeutics to test their metals: same targets, easy biomarkers, chronic applications. The next chapter will be top-line data from Alnylam’s ALN-PCS phase I trial which are expected by year-end.

Santaris Reports Clinical Efficacy of Anti-miR122 Treatment for HCV

Santaris reported yesterday intriguing antiviral HCV efficacy results from an ongoing phase IIa study of miravirsen, Santaris’ LNA-based antisense inhibitor of microRNA-122 (miR-122), miravirsen, an important host factor in HCV replication. Full interim results will be presented in a late-breaking oral session at the upcoming AASLD, The Liver Meeting.

The phase IIa study investigates 3, 5, and 7mg/kg of miravirsen, given weekly to treatment-naïve HCV patients subcutaneously for 29 days. According to the abstract, with the study now in the 3^rd and last dose cohort, patients in the second, 5mg/kg cohort showed very encouraging mean reductions in viral plasma RNA levels from baseline of up to 2.5logs when miravirsen was given as a single agent compared to placebo control. 5 of the 9 miravirsen subjects had reductions of more than 2 logs (>100-fold) with viral RNA in one patient becoming undetectable 10 weeks after the dose. Although the cohorts were relatively small, 9:3 drug:placebo, almost all efficacy measurements reached statistical significance.

What was interesting is that the decline in viral titers was quite prolonged, with the biggest viral reductions being observed after treatment had finished. While having prolonged drug activity per se is positive, the gradual decline is not optimal as it increases the chance of selecting for escape mutants, a major issue in HCV treatment in general and reason why the industry is busy developing new HCV treatment options to be added to the arsenal. In fact, a recent study out of Denmark showed that it is possible to select for HCVs that do not require miR-122 for replication, at least in tissue culture systems. On the other hand, given that miravirsen is targeted at a host factor and since the miR-122-viral interaction occurs at conserved sites, it may be relatively more difficult for the virus to develop such resistance as compared to for example direct antivirals. I would expect the company to report sequencing data at the conference next month.

Irrespective of the viral escape issue, slow clinical responses may also make it somewhat more difficult to integrate miravirsen into the newly emerging treatment paradigms, one aim of which is the reduction of treatment times. Here, RNAi Therapeutics would have an obvious advantage over miravirsen by acting much more rapidly than phosphorothioate antisense oligos which rely on tissue enrichment over time. This is also supported by the data that were recently reported by SomaGenics in collaboration with Tekmira and Roche.

On the safety front, the abstract noted the absence of drug-related serious adverse events. It did, however, note that among the supposed biomarker signals for anti-122 efficacy was an elevation of alkaline phosphatase (ALP) levels. ALP elevations are normally considered a marker of liver injury similar to ALT/AST, so I am not really sure how why this is not considered a safety signal. We will therefore have to for the conference presentation to learn more about the safety profile of miravirsen.

Overall, with the demonstration of antiviral activity and reductions in cholesterol levels which further support functional inhibition of miR-122, the abstract marks an important milestone in the development of microRNA Therapeutics: The first unambiguous demonstration of MicroRNA Therapeutic activity in Man.

Roche AASLD RNAi Therapeutics abstracts: HCV and LNP

Roche will present at the AASLD meeting on two RNAi Therapeutics studies. Both studies involve LNP01, which I assume involves ‘lipidoid’ LNP delivery chemistry. In one study, Roche and their academic collaborators targeted a host factor believed to be involved in the development of HCV drug resistance, especially to the interferons which are at the risk of becoming replaced and, unsurprisingly, Roche would like to revive that franchise. The abstract, together with the recent SomaGenics/Tekmira revelations, further demonstrates that Roche had been quite interested in RNAi Therapeutics for HCV. It also makes me think that maybe Novartis has not picked HCV as a target under Alnylam IP which would e.g. allow Tekmira to step into the void- well, if it saw any reason to do so, maybe out of strategic considerations.

The other abstract concerned potential innate immune stimulation elicited by LNPs. Consistent with what had already been known or suspected, TLR7/8 are the major innate immune receptors and cause of LNP hepatotoxicity, and this can be alleviated by simple 2’-O-methylation as demonstrated before by Tekmira before. What was particularly nice though in this particular study was that with the use of TLR3 knockout mice, TLR3 can now essentially be excluded as a significant tox factor for LNP delivery.

Read also: Miravirsen shows efficacy in HCV chimpanzee models.

Silence Therapeutics Signs Lung Delivery Deal with Mystery Partner

As Tekmira is struggling to regain possession over its delivery technology from mighty Alnylam, it is foremost Silence Therapeutics' business development that is benefitting from having one of the most advanced, clinically tested, and commercially uncontested systemic delivery technologies in RNAi Therapeutics. Less than a month after closing a deal with Dutch company InteRNA under which Silence’s AtuPLEX lipoplex delivery technology will be evaluated for the delivery of microRNA cancer therapeutics, the company has just announced another delivery collaboration, this time for the related DACC delivery system. Like AtuPLEX, DACC targets vascular endothelial cells, but unlike AtuPLEX it does so with a high preference for the vascular endothelia of the lung.

Curiously, the identity of the partner was not disclosed, although it was mentioned that it was a ‘Top 10 Pharma Company’. 'Top 10 Pharma Company'....didn't Takeda just become Number 10 with the acquisition of Nycomed? Moreover, the Nycomed acquisition included a fresh COPD drug, a pulmonary disease with an inflammatory component for which the DACC delivery system could very well be useful.

You can imagine that Takeda is a bit hesitant to talk about RNAi Therapeutics, maybe having overpaid a bit for what Alnylam really sold them, but with their significant investment in RNAi already it has every incentive to get things moving while Tekmira and Alnylam are fighting it out. Maybe following this rationale, Takeda and South Korean company Samyang announced earlier this year a somewhat surprising delivery collaboration.

The press release also noted that it is the partner that will provide the RNAi triggers. This pretty much excludes Silence’s existing Big Pharma partners AstraZeneca and Dainippon Sumitomo, the latter of which is not a Top 10 Pharma anyway. On the other hand, Takeda licensed RNAi triggers from Alnylam only for the metabolic and oncology fields, something that would probably add to the apparent sensitivities. I highly doubt that Takeda will hand over another $50M to Alnylam for pulmonary.

Of course, it is always possible that RNAi Therapeutics has gone so much out of fashion that Big Pharma companies are embarrassed to be publicly associated with it. Not only that, their investors shun the word ‘Research’ like the plague.

More seriously, the data that Silence reported for the DACC system has been quite impressive. Working on a model for acute lung injury, it has demonstrated potent and persistent gene knockdown in the pulmonary vascular endothelium following a single intravenous administration. While less is known about the clinical safety profile of DACC, it can be considered somewhat de-risked by the fact that its chemistry seems to be very similar to the clinically proven AtuPLEX system (see the highly encouraging safety profile of their phase I Atu027 clinical candidate so far).

RXi Pharmaceuticals to Write Off and Spin Out RNAi Assets

If you just blinked, you may have missed it: RXi Pharmaceuticals, now re-named Galena Biopharma, is to be a cancer vaccine company. As you may remember, when RNAi was still a hot technology in 2007, the parent company of RXi, small molecule biopharma CytRx, under the helm of corporate maverick Steven Kriegsman, acquired and then spun out RNAi assets in the form of RXi to be a pure-play RNAi Therapeutics company and cash cow for CytRx.

The financial details provided today on the transaction reveal that RXi has pretty much written off the value of its RNAi assets, chiefly among them the self-delivering RNAi trigger platform and the anti-scarring candidate RXI-109 which is about to enter clinical development, with the incoming investors taking over the lion's share of the company (83% of the shares) for their $9.5M in cash. Before RXi decided to be a cancer vaccine company, that is before the acquisition of Apthera in April, the then pure-play RNAi Therapeutics company had about 20 million shares outstanding, $10M in cash and was trading between $1.0-1.5, giving it an enterprise value of $10-20M. 5 months later, the net value of the same assets has become perhaps $2M.

On the other hand, the revaluation of the seemingly dead breast cancer vaccine candidate NeuVax was remarkably positive. From a value of essentially zero, it is now largely responsible for sustaining a market cap of ~$40M. Subtract about $20 in contributed cash, this still leaves a remarkable gain of ~100% for the asset (ignoring the value of the warrants and milestones). Having said that, it was definitely not the shareholders that profited from all that. Indeed, all these transactions make it difficult to follow where the money ended up.

For RNAi Therapeutics, there is still a hopeful message in all of this. The $9.5M in new investments is considerable. The confidence seems justified by the fact that RXi has found itself a nice niche by focusing on interesting self-delivering RNAi triggers, particularly for local and localized gene knockdown. The anti-scarring program also has potential to be an opportunity for the RNAi Therapeutics field to show relatively rapidly its clinical potential, especially since it can be measured against a promising late-stage antisense compound by ISIS satellite company Excaliard, EXC 001. So on the face of it the new investors seem to have gotten a good deal, although it is unclear how toxic the $45M in potential milestones to Galena are.

Today’s news shows how arbitrary the valuations of pre-clinical biotech assets can be and how fashion trends and promotion are often their main drivers. I’m so ready for the new season.

ISIS Files Aggressive Lawsuit against Santaris

It is not a big secret that antisense therapeutics companies ISIS and Santaris are fierce competitors. Today, ISIS filed an unusual, because rather aggressive lawsuit against Santaris Pharma that alleges the Danish company to be selling to the industry technology that is covered by at least two of ISIS’ literally thousands of patents. Because ISIS considers itself the gate-keeper of oligonucleotide therapeutics, and because some of the business development in RNAi Therapeutics has probably occurred under the mantle of the Research Exemption, a ruling in favor of ISIS Pharmaceuticals could have wide ramifications, actually well beyond oligonucleotide therapeutics.

Under the Research Exemption doctrine, the result of Merck vs Integra, it is generally assumed that patented technologies can be used for research purposes quite broadly as long as product, in this case drugs, are not marketed. Without this safe harbor, much of the preclinical pharmaceutical research and academic research would be a legal nightmare.

Nevertheless, ISIS believes this standard does not apply here, because Santaris in a way is selling ISIS technology as part of its platform partnerships. These include relationships with Pfizer, Enzon, GSK, and Shire. Moreover, it probably irks ISIS that Santaris has been rather successful in its business development efforts, and even had the guts to hire a former top ISIS executive, Art Levin, to set up a business development branch in ISIS’ backyard San Diego.

My impression is that Santaris’ LNA-based antisense compounds are more potent than ISIS’ generation 2.0 2’-MOE phosphorothioate gapmers, and this is why Santaris is likely to be the more attractive company to partner from a technical point-of-view. ISIS apparently has realized this as well as it is following Santaris' example in developing conformationally constrained (‘locked’) nucleic acid chemistries (expect Santaris to counter-sue ISIS on that). Anti-miR122 for the treatment of HCV infection is one example where Santaris’ data have been more promising than Regulus’ using ISIS chemistries. Ironically, GSK dumped Santaris’ stronger science on HCV in favor of Regulus for what had to be concerns about IP (I believe though that it was miR-122-specific IP, not antisense platform-related IP that was responsible for GSK’s move).

I don’t want to speculate whether Santaris in fact makes use of technology covered by ISIS patents or not. It is, however, a case worth watching for the entire pharmaceutical industry. Closer to home, it illustrates how aggressive, and so far successful ISIS is in using its sheer number of patents in ‘extorting’ concessions from other oligonucleotide therapeutics companies. Wherever you look, ISIS is getting a piece of the pie. I have been studying ISIS’ patents with regard to RNAi Therapeutics, and cannot really find anything of value there, except maybe if you are interested in using the 2’-fluoro modification which is useful, but dispensable. Still, ISIS was able to extract surprising concessions from Alnylam when Alnylam IPO’d a few years ago and has even called Alnylam a ‘satellite company’ without much public protest by Alnylam. Same story with the multi-million $ that Alnylam gave ISIS for the ill-fated single-strand RNAi collaboration. It goes to show that when it comes to public perceptions and business development, quantity and brand recognition often still trump quality and due diligence.

Post-scriptum (9 October, 2011): On October 7, Exiqon and Santaris on October 7, 2011, settled their legal differences that resulted from Santaris suing Exiqon for selling LNA-based reagents that were used for the development of drugs incorporating LNAs,,,and thus would not fall under the Research Exemption- an ironic twist of fate. In the settlement, Exiqon paid Santaris a minimal amount. It is difficult to conclude from this anything about the outcome of the ISIS-Santaris litigation.

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.