Impressions from the Abstracts of the 7th Annual Meeting of the Oligonucleotide Therapeutics Society (Part 2)

The OTS Meeting has just finished and it looks like it was a success in that meaningful progress, especially on delivery, but also specific development candidates was presented. As the press releases by Tekmira and Alnylam today showed, the abstracts do not necessarily reflect the full progress of the respective studies as these have been submitted months ahead of the conference and also because some groups may want to hold back with the most exciting data points.

For this reason, I will first summarize what I consider to be the highlights of today’s press releases, and then complete my review of the abstracts following a blog post from last week.

Tekmira and SomaGenics report ~300-fold knockdown of HCV in chimeric mouse model

In last week’s post I had mentioned that Tekmira and SomaGenics reported in their abstract #30 the successful use of 'short shRNAs' together with SNALP LNP delivery technology in a model of HCV. This model comprised of an HCV-luciferase reporter gene driven by a liver-specific promoter which is delivered on a plasmid before the application of the LNP-formulated shRNAs. A 90% knockdown at 2.5mg/kg was noted with these somewhat unorthodox RNAi triggers. This type of system is somewhat similar to knocking down a 'normal' gene in the liver, and as such the abstract did not suggest that studies were also performed in the context of real viral replication.

Today’s press release, however, revealed that the companies went an important step further, namely that they applied LNP-shRNAs in a chimeric mouse model for HCV infection. As a reminder, animal models of HCV infection are hard to come by. The best system may be the chimpanzee model, but it is obvious that this system does not lend itself to large numbers, is very costly, and it has been challenging also for ethical reasons to conduct these types of studies. More recently, chimeric mice have been developed in which the mouse liver is repopulated by transplanted human cells under selection pressure. Unlike the hepatocytes of mice and almost all other animals, these chimeric livers can sustain a form of HCV replication.

Having demonstrated 2.0-log and 2.5-log HCV viral knockdowns following one and two administrations of LNP-shRNAs, respectively, is therefore intriguing news. Similar to Santaris’ anti-miR122 antagonist, such a treatment should have significant potential to increase cure rates in treatment failure patients and those patients with genotypes not served well by current therapies.

Alnylam reports more potent LNPs and liver cell-targeted GalNac-siRNA conjugates

Alnylam issued an OTS-related press release today on three different lines of RNAi delivery research, two concerning LNPs, one to siRNA conjugates. As ionizable SNALP LNPs already incorporate a hepatocyte-targeting mechanism (ApoE), the interesting aspect of the GalNac-siRNA conjugates (Abstract #32) is that they may better lend themselves to subcutaneous administration compared to the mostly intravenously delivered LNPs- although this may be less so if the LNP delivery efficiencies are in the single to low double-digit micrograms per kg. The 5 mg per kg ED50 of GalNac-siRNAs puts them right around where some of today’s RNaseH antisense technologies are just in terms of amount of oligonucleotides administered.

Alnylam claims that these numbers make GalNac-siRNA conjugates serious candidates for clinical development. Still, because of the superior, 1000-fold increased efficacies of LNPs and the less frequent dosing that can be achieved with them, this comment may be Alnylam trying to portray themselves as having multiple realistic delivery options, when in fact they are relying for essentially all of their relevant pipeline candidates on Tekmira’s LNPs- my prediction is also that the 5th and final 5x15TM candidate will be SNALP delivered. This cautionary note may also apply to the reported 0.002mg/kg ED50 MD1 lipidoid formulation, developed in collaboration with Professor Dan Anderson from the MIT, although I have yet to see the lipid structures and formulations to really conclude this.

The news on the ‘3^rd generation reLNPs’, probably belonging to the SNALP line of research, similarly relates to further increases in LNP potencies. Here, a ~10-fold increase was reported in therapeutic window over ‘2^nd generation LNPs’, including those containing the contentious MC3 lipid, with an ED50 of below 0.005mg/kg and being well tolerated at the much higher 10mg/kg dosage. Whether Tekmira has a claim also on reLNPs remains to be seen.

Abstract #35: Preclinical development of sd-rxRNAs for fibrosis and retinal disorders (RXi Pharmaceuticals)

RXi Pharmaceuticals reports interesting progress with their self-delivering siRNA for dermal scarring with more than 2 weeks of CTGF gene silencing following single intradermal administration. Also based on Excaliard’s antisense work, CTGF seems to be a quite promising gene target for an orphan indication of solid market potential. RXi Pharmaceuticals would probably aim to improve upon Excaliard’s candidate by increasing the extent and duration of CTGF knockdown, making it potentially a once or twice drug administration approach instead of one involving multiple administrations every couple of days.

RXi expects to file the corresponding IND later this year.

Abstract #43: Characteristic aspects of skeletal muscle as a target organ for siRNA (Dainippon Sumitomo and Koken, both Japan)

I found this abstract particularly interesting because it indicates that Japanese pharma company Dainippon Sumitomo has a quite broad interest in RNAi Therapeutics. As a reminder, Dainippon Sumitomo is one of Silence Therapeutics’ larger RNAi target and delivery collaborators and we should hear about that particular relationship soon.

Koken meanwhile is a company which has an interest in providing delivery solutions for RNAi Therapeutics using its collagen-derived atelocollagen formulation.

Abstract #59: Dicer-substrate siRNA exhibit improved guide strand selection and stronger RISC Loading Complex formation compared to canonical siRNA (Rossi lab, City of Hope)

This study concerns the molecular comparison between Dicer-substrate RNAi triggers, as also practiced by Dicerna, and more conventional (Tuschl-type) RNAi triggers. The abstract suggests that Dicer substrates were more potent and more specific than the competition. A big sweeping comparison between these structures remains to be performed and published to put this issue to rest.

Abstract #69: Glucan particles for selective delivery of siRNA to phagocytic cells in mice (University of Massachusetts)

Glucan-encapsulated siRNA particles (GeRPs) made headlines and raised a few eyebrows a few years ago when a study published in Nature claimed these to be a viable oral delivery technology for targeting gene knockdown in phagocytic cells throughout the body. RXi was the licensee to that technology.

The present update concerns combining GeRP-like particles with the Endoporter peptide-based delivery system. The micrometer-sized GeRP particles are meant to be taken up by phagocytes with the amphiphilic Endporter chemistry supposed to overcome the ‘last mile’, that is RNAi trigger escape into the cytoplasm which has been a challenge especially with phagocytic cells. In principle, these ‘first-localize, then escape’ two-component formulations are reasonable to develop. Whether this particular one can solve the problem, again, remains to be seen. It is probably noteworthy that the present abstract does not mention the oral delivery of these particles.

Abstract #72: RNA-based pancreatic cancer therapy by local delivery of ant K-RASMT siRNA (Silenseed, Israel)

The Israeli company Silenseed has been quietly pursuing the development of its siG12D LODER slow-release siRNA matrix for the local treatment of pancreatic cancer (gene target: mutated KRAS). This candidate is unique in the clinical RNAi Therapeutics staple in that it uses a biodegradable polymer matrix to locally deliver siRNA not at once, but over a sustained period of about 8 weeks. The matrix is placed into the pancreatic tumor mass by ultrasound-guided procedure.

While the concept is sound in principle, it is still not clear whether Silenseed has considered all of its technical requirements such as the intracellular delivery of the RNAi trigger following its local release and distribution. It would not appear an insurmountable challenge, but I have seen groups pursuing promising concepts, but really addressing half the technical requirements only.

Abstract #97: miRNA turnover in Dicer knockout cells (Monash University, Australia and IDT)

This is an interesting abstract as it addresses the inherent stability of small silencing RNAs in mammalian cells. This stability is responsible for allowing us to often observe 4 weeks and more of RNAi gene silencing in non-dividing tissues after single RNAi trigger administration in vivo. With the more potent LNPs, every two months (intravenous) dosing is already within reach.

In this particular system, the authors removed the critical microRNA enzyme Dicer by genetic ablation in mouse embryonic fibroblast. With this, the observed decline in miRNA abundance reflects the decay of the largely RISC-incorporated single-stranded miRNAs following Dicer knockout. An average miRNA half-life of ~5 days was observed this way. It is likely that knocking out the microRNA pathway upstream of Dicer at the Drosha step would have yielded even slightly prolonged half-lives and this would have better reflected the fate of double-stranded RNAi triggers. It will be critical to find out by how much these half-lives can be further increased using siRNA chemistry, as Merck has done, and how much of it is determined by the protein turnover of the RISC complex which ultimately may be a rate-limiting step difficult to meddle with.

The RNAi Trigger Marketplace in the Post-Tuschl World

This entry is the second of a 2-part series on the upcoming decision of who will control key intellectual property for therapeutic applications of RNAi. In the first part, I tried to provide an outline of the developments causing ownership of certain data in the Tuschl patent applications to become such an important issue. Here, I will try and delve more into the technical details of the scientific milestones that made RNAi a conceivable new class of human therapeutics, and based on that understanding make an educated guess about the outcome of the Tuschl Tussle and how this could shape the RNAi trigger IP marketplace in the future.

Back to Science.

Could RNAi be used as a therapeutic? That was the sort of topic of wild speculation in the lab where I worked as an undergrad in 2001 on a plant gene silencing project. Hey, Fire and Mello reported this cool stuff in worms 3 years ago and as we can see double-stranded RNAs can trigger the same process so beautifully also in plants thanks to some nice work by the Baulcombe group and another one in Australia. But humans? Well, unfortunately vertebrates seem to represent the exception when it comes to the existence of RNAi. That darn interferon response system...All this would change in a watershed moment when Tuschl and colleagues at the Max Planck reported in Nature the very existence of RNAi in human cells and taught a captivatingly simple technology to induce it there: siRNAs. The story therefore seemed quite simple until then. First it was Fire-Mello, then Tuschl's siRNAs.

Fire-Mello

Fire-Mello coins RNAi. The critical contribution by Fire and Mello in 1998 was their realization that it was in fact double-stranded RNA that was the effective inducer behind a variety of strange gene silencing phenomena in worm genetics and quite likely beyond (e.g. variegated Petunia flower color). While it was not necessarily obvious at the time that this would be applicable to humans as it was still very much doubted that RNAi existed in humans, the deeply influential nature of this eureka moment of the field of gene silencing and the non-exclusive licensing approach taken by the Carnegie Institution, the owner of Fire-Mello, established it as a widely respected patent. Add to this the endorsement by the scientific community as evidenced by the Nobel Prize in Physiology and Medicine this work entailed, there should be little doubt in the mind of patent examiners about the therapeutic relevance of that work. Prohhhhbably a fundamental patent.

Biochemical work in fly cell extracts by the inventors behind Tuschl I (Tuschl, Zamore, Bartel, Sharp) and involving the MIT, the Whitehead, UMass, and Max Planck on the same gene silencing phenomenon in flies aimed at the elucidation of the molecular fate of these long dsRNA RNAi triggers. Their main finding was that during RNAi, long dsRNA gave rise to 21-23 nucleotide small RNAs and that target RNA was cleaved at 21-23 nucleotide intervals also. This strongly indicated that it was the 21-23 nucleotide RNAs that were guiding the destruction of the target RNA. This, however, is different from demonstrating that the 21-23 nucleotide small RNAs are able to trigger RNAi themselves, something one would think would be important for claims to this effect to be considered enabled. To test this hypothesis, they therefore isolated and then reintroduced the 21-23 nucleotide mix of RNAs into fresh fly cell extract and asked whether those were able to induce RNAi gene silencing, too.

What may come as a surprise to a few: the silencing with these purified 21-23nt RNAs was actually quite mediocre, about 50% silencing compared to >>95% silencing with the long dsRNA (Figure 12 of US Tuschl I application). Similarly, when the dsRNA length dependency of RNAi was tested, the shorter the dsRNA, the worse the silencing. Together, these types of findings described in Tuschl I seriously calls into question claims that Tuschl I technically enabled human RNAi. Some may even cite such data as proof to the opposite, namely that this work made it even less likely that short RNAs would be useful RNAi triggers.

What is the explanation for this somewhat surprising finding? In retrospect, it is most likely the fact that when the 21-23nt small RNAs were introduced they were single-stranded and not double-stranded and demonstrates that at that time, the authors did not know about the requirement for double-strandedness also of the small RNA intermediates for triggering RNAi . Consequently, the patent contemplates both single-stranded and double-stranded RNAs as candidate RNAi triggers. Thus, while an important piece of the puzzle of RNAi molecular biology history with ~1600 citations to the underlying Year 2000 paper, it by no means was the catalyst leading to the adoption of RNAi in humans. I would not even be surprised if the authors did test the hypothesis of whether such isolated 21-23nt small RNAs were able to silence genes in humans cells (not very difficult to do) and came up short. It is also worth noting that their discovery of small RNAs during RNAi was not entirely new to gene silencing scientists then, a year after Hamilton and Baulcombe reported such an observation in Science, something that also has not escaped the patent examiner.

Tuschl II

Tuschl II coins ‘siRNA’. The critical insight that, first of all, proved the existence of RNAi in Man and even more importantly in terms of enablement, taught a straightforward method for triggering this process in humans, came from very elegant work led by Tom Tuschl at the Max Planck in Goettingen and forms the basis for Tuschl II.

The spark of ingenuity by the people at Max Planck, not involving those at the MIT, Whitehead, or UMass, was that the small RNAs had to be in double-stranded form to serve as useful triggers of RNAi. To prove this, they generated short dsRNAs, which they coined siRNAs, through chemical synthesis, also a first, and found them to be potent triggers of gene silencing not only in fly lysates, but subsequently also in human cells. The fact that the fly lysate work was reported separately by the Max Planck group and temporally between the Tuschl I 21-23 nucleotide RNA paper and the human RNAi findings, further illustrates the temporal, geographic, and intellectual separation of Tuschl's work in Massachussetts and then as a group leader in Germany.

Importantly, these siRNAs allowed for gene silencing that was specific and independent of the interferon response, again something speculated about, but not clearly proven in Tuschl I. The Tuschl siRNA-template is now used by thousands of laboratories around the world, with an amazing 6000 citations to the underlying paper further illustrating its importance.

In the Max Planck vs Whitehead case, the Whitehead argues that 3’ overhang siRNAs that are at the core of the Tuschl II patent application were already part of Tuschl I. On the surface this is true. This is because for some strange reason and that is the biggest mystery to me in all of this and that I hope the next months will shed some light on, the human siRNA data miraculously appear at the end of the Tuschl I application, as does the term ‘siRNA’ emerge without prior definition. If this data were to remain part of Tuschl I, there is the real possibility that Tuschl II could be declared invalid on a technical basis because of Tuschl I’s priority status (in a temporal sense) and double-patenting laws. Not good for Alnylam!

The two related questions of which the answer will rock the RNAi Therapeutics universe are therefore: a) Has the human siRNA data that in light of the weak activity of the ’21-23 nucleotide RNA’ in fly lysates and very uncertain translation of those results into humans now form the inventive basis for the broad human RNAi claims in Tuschl I, been rightfully included? b) In doing so, has the Whitehead, responsible for prosecuting Tuschl I also on behalf of Max Planck, fulfilled its fiduciary duty towards all its partners?

First of all, as I explained in my previous post, the data critical for the siRNA claims of Tuschl I had been generated by the inventors behind Tuschl II (most importantly, in addition to Tuschl, Elbashir and Lendeckel; both of them also at the Max Planck then), but who are not named as inventors on Tuschl I. On this technical ground already, Tuschl I in its present form is invalid. Beyond that, there appears to be early communication in which Max Planck confirmed with the Whitehead that the human siRNA data were the domain of Tuschl II. So even if Max Planck and the inventors of Tuschl I had been wrongly convinced by the Whitehead and their hired patent attorneys that this should not pose a problem for the approval of both patents, any patent attorney worth his salt should have known this to be a fundamental omission. It would therefore seem to be wise to remedy this deficiency either by including the inventors on the Tuschl I patent or by leaving out the data as stipulated by Max Planck, before the specter of 'malpractice' was raised. And obviously, Whitehead now is clearly not acting on Max Planck’s behalf and this should be sufficient cause to give back Max Planck de facto veto power in Tuschl I by confirming that the Whitehead does not have Max Planck's power of attorney any more.

Because some of these issues are civil ones that are not the domain of the USPTO, it is important to sort them out before it goes back to the patent office and can cause lasting damage to the patents. For the stated reasons, I am quite confident that Max Planck and Alnylam will prevail and regain control of the human RNAi data and some sort of declaratory judgement that the way that data had been used in Tuschl I cannot be construed to contest the validity of Tuschl II in the future.

Before I consider the ramnifications of the two main outcomes of the Tuschl Tussle for the RNAi Therapeutics RNAi trigger IP space, it should be noted that Tuschl II already disclosed the observation that blunt-ended siRNAs can silence, too, just not as efficiently as 3' overhung siRNAs on average. 3' overhangs were therefore taught to be a preferred characteristic of siRNAs when used for mammalian RNAi applications. Hence, with many more reports confirming that RNAi in fact is so robust that all sorts of exogenously introduced small dsRNAs can efficiently induce RNAi in humans, it will become more and more difficult to convince the patent offices of a proprietary nature of not only overhung siRNAs, but also those without overhangs. One exception may be Silence Therapeutics’ blunt ‘Atu-siRNAs’ which because it was a relatively early disclosure Silence/Atugen was able to convince the US and European patent offices of their arguably surprising stability, an important feature for most RNAi Therapeutics approaches.

Outcome 1: Tuschl II becomes dominant, Tuschl I essentially irrelevant

The most likely outcome. This will confirm Alnylam to be the most desirable partner based on RNAi trigger IP alone and leave Merck and RXi Pharmaceuticals empty-handed. Clear freedom-to-operate and exclusivity for the most efficient RNAi trigger that is also highly competitive with regards to other challenges such as innate immune activation. However, since Tuschl II does not claim blunt siRNAs, something I believe should have been done at least initially and maybe wasn’t because of an integrated Tuschl I-II strategy that now clearly has fallen apart, there remains scope for plenty of blunt-ended workarounds in the important 19-24 base-pair range. These workarounds, however, are not very attractive for licensing purposes if they cannot be protected by patents. Again, the exception here is Silence Therapeutics which, assuming that Kreutzer-Limmer's staying power is questionable, would be a beneficiary of such an outcome since it would now free Atu-siRNAs from the Tuschl I threat. There are, however, some significant limitations with Atu-siRNAs, since the scope of the patents is quite narrow in terms of allowed siRNA patterns and chemical modifications. Thus, while Tuschl II will offer a platform that should be applicable to RNAi Therapeutics for many years to come, Atu-siRNAs may not be able to adapt to the evolution in cutting-edge siRNA modification technology and hence its value should decline over time relatively quickly.

The IP position of Dicerna is probably least affected by the Tuschl outcome among the synthetic siRNA Therapeutics companies. There may be some uncertainties with whether and how the Tuschl patents may be applicable to Dicer substrates, but unless there will be a messy outcome in which both Tuschl’s go up in fire (highly unlikely), neither outcome 1 or 2 should change this much.

Under outcome 1, the market would have to balance the luxuries that Tuschl II offers, namely patent protection and overhangs, with the lower price, but added liabilities of the Silence Therapeutics and Dicerna platforms or even non-patented siRNA workaround designs. Last but not least, due to its use of overhangs, mdRNA’s overhung ‘usiRNAs’ would be a loser under this scenario.

Outcome 2: Tuschl I becomes (almost) gate-keeping, supersedes Tuschl II

If Tuschl I were allowed in the US in the form now proposed by Whitehead, then Tuschl II may go up in flames with Tuschl I covering blunt and overhang siRNAs comprising RNA strands of 21-23 nucleotides. Under this, albeit very unlikely scenario, Alnylam would have to share gate-keeper privileges for the most direct route to RNAi Therapeutics with Merck and RXi. Further risking to put pressure on price would be RXi selling such rights for a pittance as well as uncertainty about UMass’s ability and willingness to further grant rights to Tuschl I. Not all would be lost for the rest of the field even under this scenario. Silence Therapeutics, for example, would still be able to operate in the 15-20 base-pair range, with maybe 19 and 20 base-pair offering quite good opportunities of discovering efficacious and non-immunostimulatory siRNAs with acceptable efficiency. Similarly, 19 to 20 base-pair siRNAs may also become the preferred space for other non-patented siRNA designs, though all of this is dependent on what happens to Kreutzer-Limmer. Again, Dicerna would be little affected by all of this, and mdRNA may be well advised to try its luck with ‘usiRNAs’ outside the 21-23 nucleotides range, although I still feel chances are slim that one or two supposedly ‘non-nucleotide’ nucleotides will allow them to call what look and behave like siRNAs by another name.

Where does Big Pharma stand in all of this? Those interested in taking broad platform licenses to RNAi trigger IP can probably be classified into into two categories: 1) those like Pfizer and GSK that have diligently done their homework and will already have made up their minds about what type of RNAi triggers are required, including whether they consider overhangs to be an essential feature or not. Such companies can simply await the outcome of the trial and then choose the most economical option everything else (e.g. access to delivery and other know-how) being equal; 2) those companies that have shied away from heavy investments thus far and would prefer to get started with a pure-play RNAi Therapeutics partner providing patent-protected siRNAs and other basic RNAi capabilities. These companies may be most swayed by the outcome of the Tuschl Tussle, since they may be more relaxed in terms of what they consider acceptable siRNA designs.

Tekmira is a pure-play RNAi Therapeutics company that should be uniquely affected by the outcome, because it does not tout having invented unique siRNA triggers, although it certainly could make up such claims to the same degree that others do in the space, and because of its complex relationship with Alnylam. For one, it may determine whether potential partners consider it to be necessary to access SNALP delivery via Alnylam or whether they can go directly to Tekmira and get the same for probably considerably less. Moreover, in the unlikely case of a Max Planck/Alnylam loss, it may become even more difficult to insist on controlling SNALP delivery for RNAi Therapeutics all the while it is obvious that Alnylam is intent on minimizing the importance of Tekmira for their delivery efforts (the whole issue of what is called a SNALP which clearly differs between Alnylam and Tekmira). If SNALP is old and first-generation where is the harm in letting Tekmira fully exploit this technology by partnering it out ex-ALNY instead of letting it wither on the vine? I guess something ought to be worked out here to the satisfaction of both companies.

After 8 years of spending enormous efforts on confusing the investor world with what is valuable RNAi trigger IP, it looks like everything will come down to basic science. The collective scientific community based on the number of citations a paper gets and how it has recognized critical inventions for example in the form of scientific awards, would have been a much more straight-forward, fairer, and infinitely cheaper way of determining ‘good’ RNAi IP. Who else was better suited to spot critical contributions in technically demanding areas such as RNAi than scientists themselves? One would hope that the judge will concur, also in the interest of what RNAi Therapeutics could do for society.

Disclaimer: The above are my own interpretations of the case, based on publicly available documents from the USPTO and court sites, interviews, and press releases. Accuracy cannot be guaranteed as I may have overlooked critical elements of the case, and am neither trained in intellectual property nor contract law. Information provided herein cannot be relied upon for making investment decisions. Investments in RNAi Therapeutics are very risky and not suited for most. Consult with your own professional advisor before doing so.

RNAi Therapeutics Investment Tracker Update

Following today’s announcement by Protiva and Tekmira, I was finally able to face the reality of the plunging stock market, and updated the investment tracker. Due to the spin-out of RXi out of CytRx, I have liquidated as of today the CytRx part of the deal and reinvested the proceeds into RXi so that there are now 40.85 shares in the portfolio purchased at an adjusted share price of $12.24. Other positions were left unchanged, although progress was noted for Nastech and Rosetta Genomics.

Nastech has successfully appointed a scientific advisory board for mdRNA, collecting Nobel laureates as if they were stamps and suited for their RNAi delivery efforts. It is now critical to build on this with a successful fund-raising round and hopefully a financial separation between the nasal delivery and RNAi Therapeutics businesses. I do understand that there are overlaps, but experience has shown that exposing RNAi Therapeutics to the risk of an unrelated technology has hurt RNAi Therapeutics development efforts rather than helped it. Also, the meroduplex poster at the Keystone meeting did not make much sense to me as the whole idea is based on the premise of circumventing Alnylam’s IP no matter whether it makes sense scientifically or not. I’d rather see a company focus on delivery and license the target rights accordingly, than a company trying to re-invent the wheel and consequently making drug development for themselves even more difficult than it already is.

Rosetta Genomics has impressed me at the Keystone conference by their presence and breadth of science. The RNAi/microRNA world is watching them as they prepare the launch of their first microRNA diagnostics this year.

Day 1 of Keystone RNAi Conference: New Small RNAs and Oral RNAi Delivery

More recently discovered types of small RNAs were the focus of the keynote session on the first night of the influential annual gathering of the RNAi community, the Keystone conference on RNAi, this year held in Whistler resort near Vancouver, Canada.

Following introductions by the scientific organizers Judy Lieberman (Harvard) and Phil Sharp (MIT), Craig Mello, Nobel laureate and faculty at the University of Massachusetts, gave an account of a variety of relatively recently discovered small RNA populations that are enriched in the worm germline and are characterized by specific structural features, such as 5’ triphosphorylations or 3’ methylations, and the Argonaute proteins (worms have over 20 of those, whereas humans only have 4) with which they are associated.

For many of these small RNAs, the biological function is still somewhat unclear and the field, as was also echoed by the second keynote address by Greg Hannon (Cold Spring Harbor) is busy using high-throughput sequencing techniques and bioinformatics to catalogue them. As an aside, with the speed sequencing technology is progressing, it could very well be that the future of microRNA diagnostics will entail the quantitative high-throughput sequencing of microRNAs rather than interrogating a limited number of defined microRNAs by microarray or PCR.

A particularly interesting small RNA population is one that is marked by 5’ triphosphorylation, unlike siRNAs that are 5’ monophosphorylated. It appears that in their biogenesis, a primary small RNA (siRNA or microRNA) cuts a target mRNA which then becomes the template for RNA-dependent RNA polymerases (RdRPs) that generate the 5’ triphosphorylated RNAs (aka secondary siRNAs). Surprisingly, the secondary small RNAs are the ones that carry out the majority of the gene silencing, while the primary small RNAs are present only in very small amounts and appear to function only in triggering the amplification of gene silencing.

It is, of course, now interesting to speculate whether synthetic small RNAs with various 5’ modifications could likewise function in human cells, possibly with different biological activities from siRNAs and microRNAs. However, in the case of the RdRP-dependent 5’ triphosphorylated small RNAs, one has to keep in mind that a dedicated RdRP appears to be absent in the human genome and that 5’ triphosphorylated RNAs in the cytoplasm may trigger unwanted cytokine responses. But maybe there will be some other type of modification around which one could build a therapeutic platform.

At the start of his presentation, Mello noted that one way to induce gene silencing in worms is by feeding them with bacteria expressing double-stranded RNAs (somewhat reminiscent of Cequent Pharmaceutical’s transkingdom RNAi approach). He finally came full circle at the end of his talk, when he showed some intriguing slides from colleagues at the University of Massachusetts, Michael Czech and Gary Ostroff, on the oral delivery of therapeutic RNAi. Starting with yeast ghosts consisting of essentially a shell of beta-glucans that they filled layer by layer with RNAs, including siRNAs, to create nanoparticles that would be taken up by the Peyer’s patches in the gut. Unfortunately this part of the presentation was quite brief, but it appears that they have succeeded in ameliorating inflammation in a mouse model by targeting TNF-alpha. It will now be important to demonstrate the generality of this phenomenon by targeting a number of other genes unrelated to immune responses. As this approach appears to target immune cells, it may at least initially be applicable for orally delivering RNAi for a number of immune related disorders.

This development could be particularly interesting for RXi given the close relationship between Mello and Czech and this new public company. Also taking into account that UMass RNAi efforts will be a major beneficiary of the Massachusetts life science initiative, it seems that RXi is finally showing some signs of scientific life.