Rational Design and Animal Studies Remain Gold Standard in Liposomal Delivery Development

In a recent paper (Chen et al 2012), the ‘Lipidoid Group’ from MIT admitted that their library screening method to find useful lipids for therapeutically relevant in vivo applications was flawed.  Essentially, the lipids in the much-heralded 2008 paper with over 200 citations (Akinc et al: 'A combinatorial library of lipid-like materials for delivery of RNAi therapeutics') were tested as crude lipoplexes in tissue culture, whereas successful in vivo delivery has very different and additional demands (stable circulation, extravasation, tissue penetration, biodegradability etc etc)- as is known from decades of liposomal delivery research.  To improve upon the predictive value of the lipid library screening approach, the group now reports, in a solid academic paper, a microfluidics-based approach for formulating lipids into more SNALP-like particles.

I’ve been following with a mix of amusement and annoyance that the Alnylam-funded MIT LNP effort has been getting so much attention to the point that many, if not most, observers believe that the next-generation LNPs are based on that research.  Needless to say, if one bothered to study the respective research coming from Vancouver versus those from MIT, it should be clear which effort alone is the therapeutically relevant one (the Vancouver one to remove any doubt).  The clinical SNALP pipeline says it all.

Who is to blame for this confusion and mis-attribution of credit?  Much of it comes down to scientific journalism looking for eye-grabbing headlines, best if high-profile institutions like the MIT are involved.  Of course, Alnylam does not always bother to point out the relative therapeutic relevance of company-funded research when it issues a press release to go along with a scientific publication.  At least in that regard does their inordinate investment in 10 MIT post-docs pay off.  A bit of a luxury in these times of lay-offs, if you ask me, and I don't expect it to be renewed as the original contract is running out any day now.  I respect Nature Biotechnology as a journal, but having such high visibility, it also has a responsibility to make sure that its prime criteria for selecting papers for publication is originality and research quality, and not renown as it sometimes seems to be the case.

This brings me to Prof. Rob Langer from the MIT and a talk by him at an OTS meeting a few years ago on his exciting ‘lipidoid’ research.  As most readers will be aware, Prof. Langer is famous, amongst other achievements, for fathering what must be dozens of biotech companies (and probably making a buck or two in the process) in various areas of nanotechnology.  Indeed, the apparent frequency of the spin-outs and the different technologies involved makes me wonder at times how a single person, no matter how genius, can be expert in all of them.  In any case, if VCs are keen to throw money after him that's up to them and their investors.  What bothered me about that talk though was that Prof. Langer played along the theme of ‘lipidoids’ having all these unique and interesting properties, including structural differentiation, without really showing proof for them and which, according to the latest paper, do not seem to exist: a SNALP formulation takes on the shape of a SNALP (in this case one with constitutive positive charge at physiological pH).

Looking at the positive side of things, the latest research shows that microfluidics promises to be a technology that allows you to test various SNALP formulations without it costing you an arm-and-a-leg as is current practice.  However, the research also shows that there is no substitute for testing them in vivo as a first indication of therapeutic utility (a lot of mice were used in that study to arrive at that conclusion).  The research parenthetically also strongly supported my impression that microfluidics is unlikely to yield the 20nm-sized SNALP LNPs as Alnylam said their microfluidics-based collaboration with Vancouver start-up Precision Nanosystem would yield (interesting to see Alnylam-supported LNP microfluidics in both Vancouver and Cambridge).  A recent publication involving Precision Nanosystem by the way suggests the same (Zhigaltsev et al 2012) .  In my opinion, 40nm is going to be a magic number for SNALP LNPs.

With the exception of perhaps 1^st-generation LNP delivery to phagocytes, and as had been known in the liposomal research community (including Tekmira, of course) for a long time, rational design approaches and animal experimentation are most likely to rule the delivery space for the foreseeable future.  

While Alnylam Focuses Suit on Whitehead and UMass, a New Tuschl Loophole Approach Gains Traction

Not a day goes by in RNAi Therapeutics land without hearing the sounds from the RNAi trigger IP battle grounds. Today is no exception…

Wading through the court documents from the Alnylam-Max Planck suit on how the Whitehead prosecuted the Tuschl I (T-I) patent application (aka the 'Tuschl Tussle'), it struck me that while it was Whitehead, their hired patent counsel, and UMass that apparently conceived of the strategy of how to incorporate data from the T-II patent into T-I against the interests of Alnylam and Max Planck, the MIT seemed to merely go along unwittingly. As a result, MIT not only became a victim in that they were deprived of the benefits from the therapeutic agreement they were part of with Max Planck and the Whitehead, which to my knowledge anticipated equal sharing of the profits from therapeutic licenses derived from the combined T-I and T-II estate, but also because they ended up as defendants in the suit.

Perhaps realizing this, Alnylam announced today that it will leave the MIT off the hook in return that they will be bound by any ruling in favor of Alnylam/Max Planck. It could also help drive a wedge between the former co-defendants as the MIT will now feel less fearful about telling their side of the story which could turn out to be quite revelatory. The MIT should have every reason to be unhappy with the Whitehead and the way T-I was handled. The argument that Whitehead, and implicitly, the MIT, aided UMass for political reasons, thereby leaving therapeutic licensing money on the table, never really flew with me since a) institutions just don’t give away money like this, and b) I could never imagine a research institution of the stature of Whitehead as being nationalistic and therefore anti-Max Planck.

While the Tuschl Tussle is going on, there is another Tuschl loophole movement in the field that appears to be gaining some traction. It initially started with mdRNA’s claims that the mere inclusion of a single ‘funny-looking’ nucleotide into an siRNA, unlocked nucleic acids (UNAs), would help it get around the Tuschls. It long cited evidence by outside patent counsel that supposedly supported their belief that they had freedom-to-operate in the RNAi trigger space. The news that Quark Pharmaceuticals had just initiated dosing for their 5^th (!) clinical RNAi program for a neuroprotective agent of the eye, it reminded me of their similar claims about ‘proprietary siRNAs’. To find out about the nature of these claims, I looked up what potentially applicable patent applications they had filed, and came up with the following main claim:

"1. A compound having structure (IX) set forth below:

(IX) 5' (N)x - Z 3' (antisense strand)

3' Z'-(N')y- z" 5' (sense strand) wherein each of N and N' is a ribonucleotide which may be unmodified or modified, or an unconventional moiety; wherein each of (N)x and (N')y is an oligonucleotide in which each consecutive N or N' is joined to the next N or N' by a covalent bond; wherein Z and Z' may be present or absent, but if present is independently 1-5 consecutive nucleotides covalently attached at the 3' terminus of the strand in which it is present; wherein z" may be present or absent, but if present is a capping moiety covalently attached at the 5' terminus of (N')y; wherein x =18 to 27; wherein y =18 to 27; wherein (N)x comprises modified and unmodified ribonucleotides, each modified ribonucleotide having a 2'-O-methyl on its sugar, wherein N at the 3' terminus of (N)x is a modified ribonucleotide, (N)x comprises at least five alternating modified ribonucleotides beginning at the 3' end and at least nine modified ribonucleotides in total and each remaining N is an unmodified ribonucleotide; wherein in (N')y at least one unconventional moiety is present, which unconventional moiety may be an abasic ribose moiety, an abasic deoxyribose moiety, a modified or unmodified deoxyribonucleotide, a mirror nucleotide, and a nucleotide joined to an adjacent nucleotide by a 2 '-5' internucleotide phosphate bond; and wherein the sequence of (N)x is substantially complementary to the sequence of (N')y; and the sequence of (N')y is substantially identical to the sequence of an mRNA encoded by a target gene."

As the red highlight shows, the ‘proprietary’ claim of the Quark siRNAs rests on the sense/passenger strand similarly containing at least one ‘funny-looking’ nucleotide. I was disappointed, however, that the specifications did not explain why this should have any unique advantages. Since in addition to novelty (that's where they will likely try to focus their arguments on), a patent has to also fulfill the demands of non-obviousness and utility, I have my serious doubts that this and the usiRNA patent applications will stand up to closer scrutiny, and if they did get by any patent offices, Alnylam would ultimately challenge them. It suggests, however, that while patent protection for these RNAi triggers is unlikely, a number of players increasingly view this strategy as a way to at least gain independence from the Tuschls. The exact reasoning behind this is mysterious to me, since these modifications should already be explicitly covered by the Tuschls when they refer to 'nucleoside analogues' which usiRNAs are. And even if the Tuschls did not explicitly mention them, it would appear obvious that an siRNA is an siRNA whether it contains a limited number of ‘funny’ nucleotides or not.

As I said, I have yet to hear a convincing argument by the companies what their belief is based on. Just stating that they believe so is not enough to convince investors and potential partners. I am always willing to listen to such arguments.

Lipidoids Expand Chemical Space for Cationic Liposome Delivery of RNAi Therapeutics

[Important update, see end of entry]

Following a long series of presentations and publications involving Protiva, Tekmira, Alnylam, and Merck/Sirna, PEG-stabilized cationic liposomes known as SNALPs have to be considered one if not THE most advanced systemic RNAi delivery technology to date. While efficacy was very potent at single-digit mg/kg doses, the major drawback of the first studies on SNALP-siRNA delivery were slight elevations in liver enzymes, an indicator of toxicity (Zimmermann et al. study). It was therefore important to expand on those studies and search for formulations with even better knockdown efficacies and inherently less toxicity, thus pushing the therapeutic index well into predictably clinically safe ranges.

The recent announcements 0.1mg/kg IC50 knockdown efficiencies for SNALP-like formulations by both Protiva and Tekmira (due to their upcoming merger from now on referred to as Tekmira for simplicity) support the notion that the exploration of new chemistries should facilitate the development of SNALP RNAi for clinical use, and although only the surface has been scratched, a first IND with realistic chances at therapeutic success may not be very far away.

To exploit the chemical space available for SNALPs though requires the ability to generate new and diverse lipid chemistries as well as the ability to manufacture and formulate these chemistries to scale. The latter has been achieved by Tekmira’s spontaneous vesicle formation by ethanol dilution method allowing for the speedy manufacture of SNALP-siRNA formulations that can support late pre-clinical and clinical studies, while the long-awaited paper by Akinc and colleagues from the MIT (Langer/Anderson lab) and Alnylam Pharmaceuticals on so called lipidoids and that has now been published in Nature Biotechnology [Akinc et al. (2008): A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.], will now faciliate the efficient exploration of novel, SNALP-compatible lipid chemistries.

Rather than laboriously synthesizing and testing one lipid after the other on a hypothesis-driven basis, Akinc and colleagues developed a synthesis method that allowed them to generate libraries of cationic lipids with quite unusual and diverse characteristics to systematically evaluate them for siRNA delivery. A first library gave an indication of which chemistries worked better than others, and a second library was generated based on the characteristics of the best performing ones in the first set.

Initial tests were based on silencing reporter genes in tissue culture. It should be noted that for these high-throughput experiments, probably for speed and ease, simple siRNA-lipoplexes were used (siRNA-lipid mix), instead liposomally formulated siRNA as for later tests in vivo (siRNA captured inside liposomes), and this may be one limitation as it could have caused them to miss even more promising in vivo silencing chemistries. In any case, the best candidates were then taken forward into rodent and non-human primate studies, this time formulating the lipids together with cholesterol and PEG-lipid into cationic liposomes, essentially based on the same principles as SNALPs (stabilized liposomes containing diffusible PEG-lipids, the latter interestingly manufactured by Alnylam itself).

Overall, IC50s in the low mg/kg range were routinely observed for a number of liver targets. This was achieved without significant toxicities based on careful safety analysis, and only in some cases mild elevations, less than 2-fold, of liver enzymes were observed. Similarly, the absence of negative interference with endogenous microRNA pathways was reported last year. This is a good start and may not have employed the most efficacious siRNAs, but by optimizing the formulations further, e.g. by engineering additional fusogenic lipids and other properties into these particles, sub-mg/kg doses that would be desirable in the clinic should be achievable and still be compatible with the more scalable manufacturing technologies practiced by Tekmira (although 50nm particles and high encapsulation efficiencies were achieved, the extrusion-based method as employed in the present study may limit scale).

Beyond the liver, the lipidoid formulation showed some promise for the delivery of siRNAs to the lung as demonstrated in an RSV model. Interestingly, inhibition of RSV replication was enhanced by lipidoid-siRNAs (almost 3-log knockdown at 2mg/kg) over unformulated, naked siRNAs (1-log knockdown), in contrast to previous studies on which Alnylam’s ALN-RSV01 is based that showed somewhat less viral knockdown with naked siRNAs and that suggested no enhancement of siRNA delivery to the lung by formulation with other delivery chemistries. In addition, given the propensity of such nanoparticles to be taken up by phagocytic cells of the immune system and the largely unmodified siRNAs used for targeting RSV, follow-up studies need to look at any innate immune responses elicited by such lipidoid-siRNA combinations.

In summary, this study opens up a wide chemical space for the systematic evaluation of cationic liposome-mediated delivery of drugs, particularly siRNAs, but also microRNA antagonists (demonstrated in this study) and beyond. Following some of the recent breakthroughs and due to the triangular relationship between Alnylam, Tekmira, and the MIT, complementary in terms of both know-how and IP, progress of SNALP-siRNAs into the clinic may hopefully occur within the next few months and should be followed by next-generation chemistries.

[Update May 1, 2008: According to a report by RNAiNews , an Alnylam spokesperson indicated that the company had not given guidance on the specific liposome formulations to be used for their hypercholesterolemia and liver cancer clinical programs. This is in contrast to an earlier report by RNAiNews from last year’s Beyond Genome conference in San Francisco which indicated that Alnylam had chosen “choose lipidoids over SNALPs” for these indications (also discussed in a blog entry here). It therefore seems that Protiva/Tekmira's 0.1mg/kg IC50 liposomal nanoparticle formulations may be the current frontrunners in entering the clinic (see also a recent PR).

The sometimes imprecise use of the terms SNALPs and lipidoids may be partly to blame for the confusion. SNALP refers to a liposomal formulation technique, while lipidoids are a new class of lipids generated by combinatorial chemistry which can now be evaluated for liposomal drug delivery. As such, lipidoids could be used within the context of SNALPs, and both of these approaches are therefore complementary to each other.]

Nature Publishes Reassuring Study by Alnylam on the in vivo Delivery of siRNAs and their Effect on the Endogenous microRNA Pathway

Given that therapeutic RNAi takes advantage of an endogenous biological pathway, the introduction of very high levels of small RNAs certainly has the potential to interfere with related small RNA pathways, such as microRNA function.

Indeed, competition with the microRNA pathway in vivo which in some cases caused the death of mice, were first reported in a study by Grimm and colleagues in the journal Nature last year. Ironically, rather than a demonstrating a failure of the viral delivery system used in that study, it was the extreme efficiency with which small RNAs could be expressed with the double-stranded AAV vectors that allowed the competition to be observed. In what is an often overlooked aspect of these studies, compared to non-viral delivery methods, silencing efficiencies of over 95% can be easily achieved with these vectors at doses that have no adverse effects on either the microRNA pathway or the viability of mice.

In an ideal world, the Grimm et al. studies would have been embraced as an opportunity to study the dose-limiting steps of therapeutic RNAi to inform future RNAi therapeutics strategies. Instead, as the Press lives from feeding the public the simple messages, rather than reporting complicated truths, it decided to label the studies as yet another example of the dangers of gene therapy, and - somewhat understandably- caused some companies involved in developing RNAi Therapeutics to distance themselves from DNA-directed RNAi for political reasons.

As the trusted leader of RNAi Therapeutics, Alnylam was therefore given the platform to reassure the RNAi community this week in Nature that, unlike AAV-RNAi, liposomally delivered siRNAs had no obvious adverse effects on the endogenous microRNA pathway (John et al., 2007). The study further highlighted that liposomal siRNA delivery has advanced to a point where around 80% gene silencing in hepatocytes can routinely be achieved following systemic administration of therapeutically viable doses of siRNAs, including their repeat administration.

Although I welcome Nature’s decision to document progress in the important area of RNAi therapeutics, and understand Alnylam’s desire to publish in the highest profile journals, I would like to take this opportunity to address a few misconceptions about the studies. One important misconception is that delivering RNAi with AAV per se is more toxic. To make this point, a direct comparison of the intrahepatic levels of small RNA levels following both routes of administration would have been necessary. Given the >99% transduction efficiency of double-stranded AAV in mice and the consequently extremely high gene silencing efficiencies, it is quite likely that double-strand AAV vectors are currently the most potent delivery system to the liver in terms of small RNA delivery and gene knockdown.

I would therefore not be surprised at all to see similar competition with microRNA function following administration of very high siRNA dosages. This is supported by numerous studies that have shown competition for gene silencing when very high levels of two or more siRNAs were introduced simultaneously into tissue culture cells. However, given the ability of hundreds of microRNAs to function in a given cell at any time, such observations represent only extreme cases and suggest a wide therapeutic index. Unfortunately, the relatively small range of doses used in the John et al. studies (2mg/kg to 5mg/kg) did allow for a careful evaluation of related competition in vivo and concomitant dose-limiting toxicities.

I guess the purpose of this Blog really is my plea to the field of RNAi Therapeutics to keep learning from each other, instead of letting the Press and uninformed “analysts” play on the fears of investors, through their indiscriminate use of buzzwords, thereby polarising and separating what really belongs together. In this spirit, I would like to stress that this study is yet another proof-point of the viability of RNAi for therapy leading up to the possibly first proof-of-concept gene silencing results in Man to be revealed in the coming months- once again by Alnylam.


PS: Although a combination of liposomal delivery methods were used in these studies, the details were not disclosed. Apparently, another study on lipidoid-delivered siRNAs, a technology developed by the Langer and Anderson groups at the MIT, has been submitted to Nature Biotech and is about to be published. Lipidoids differ slightly from the Tekmira-owned SNALP technology, and looks likely to be the technology used for Alnylam’s first clinical systemic RNAi program (liver cancer or hypercholesterolemia), for which an IND is expected by the end of 2007. More than knockdown efficiency, we should be looking for the toxicity profile as I regard this to be the big unknown that will determine the success of this program, particularly if it turns out to be for hypercholesterolemia.

Alnylam Chooses Lipidoids over Cationic Liposomes for their First Systemic RNAi Clinical Studies

[Important update at end of this entry]

In an interesting twist, Alnylam announced at the Beyond Genome conference held last week in San Francisco the use of MIT’s lipidoid technology for their first systemic RNAi programs. These formulations will be used for knocking down PCSK9 for the treatment of hypercholesterolemia and the dual siRNA cocktail ALN-VSP01 for liver cancer. This was somewhat surprising, following a proof-of-concept Nature study last year that demonstrated efficient systemic RNAi delivery in primates using cationic liposomes. These were developed by Protiva/Tekmira, and Alnylam consequently established a broad alliance with Inex Pharmaceuticals, now Tekmira, that comprised an exclusive license to Alnylam to Tekmira’s liposomal delivery IP estate. It was therefore largely expected that Alnylam would use Tekmira’s cationic liposome SNALP technology in their systemic RNAi programs which are scheduled to enter the clinic by the end of this year.

As I pointed out in some of my earlier blogs, I was concerned that Alnylam’s plans to move into systemic clinical trials so quickly were too aggressive. This concern was largely based on the considerable, albeit transient elevation in liver enzymes, a measure of liver toxicity, at the 2.5mg/kg dose range reported in last year’s Nature study. Moreover, combined with the non-linear dose response for cationic liposomal siRNA delivery that Alnylam reported at the Keystone RNAi meeting earlier this year, this made choosing the right dose range for human studies less than certain.

Interestingly, at the same Keystone meeting, Dan Anderson together with Rob Langer, a world authority on drug delivery and scientific advisor for Alnylam, presented impressive systemic delivery data using so called “lipidoids”. “Lipidoids” were discovered as part of a library approach as an apparently new class of lipid-like molecules that were very effective in delivering siRNAs systemically to the liver and were structurally sufficiently distinct to conventional lipids and cationic polymers to give it a new name. Only a few months after that, Alnylam and the same groups at MIT announced a broad systemic RNAi delivery initiative in which Alnylam would fund 10 post-doctoral researchers for 5 years in return for exclusive rights to the lipidoid technology and an exclusive option for any new RNAi delivery technologies resulting from the sponsored fellowships.

Importantly, the tiny lipidoid formulations were reported to have a favourable safety profile and showed dose-dependent gene suppression without compromising RNAi knockdown efficacy.

In hindsight, it is very reassuring to see that Alnylam did not go out on a limb by promising an aggressive systemic RNAi timeline and gamble an early program on a potentially unsafe delivery technology. It speaks to the quality of the management and Alnylam’s reputation as the leader in RNAi Therapeutics that it always had valid options outside cationic liposomes.

Where does this leave the Tekmira-Alnylam alliance? Although “lipidoids” appear to be somewhat distinct to cationic liposomes, it is certainly a good insurance to be covered by Tekmira’s important IP estate in the field of liposomal drug delivery. Moreover, as part of the alliance Alnylam invested in Tekmira’s manufacturing capabilities, and it appears this investment will pay off as Tekmira is manufacturing the lipidoid-siRNA formulations for the PCSK9-hypercholsterolemia and liver cancer trials. According to David Bumcrot of Alnylam, IND-enabling studies for these programs are well underway.

PS: In another interesting twist, David Bumcrot noted that part of Alnylam’s decision to target PCSK9 in their hypercholesterolemia program is due to a fatty liver problem seen in targeting the former front-runner ApoB100. This appears to be a target-specific phenotype as this phenomenon has been seen with a number of siRNAs targeting ApoB100. Interestingly, ISIS Pharmaceuticals which has an ApoB100 antisense compound in late phase II trials, but has not reported on that problem has followed Alnylam’s lead in targeting PCSK9 in a new hypercholesterolemia program (see May 9, 2007 post). Once again, Alnylam has demonstrated a characteristically circumspect development approach that includes bringing together the best scientists in a given disease area to carefully characterise RNAi knockdown phenotypes.

[Update May 1, 2008: According to a report by RNAiNews , an Alnylam spokesperson indicated that the company had not given guidance on the specific liposome formulations to be used for their hypercholesterolemia and liver cancer clinical programs. This is in contrast to an earlier report by RNAiNews from last year’s Beyond Genome conference in San Francisco which indicated that Alnylam had chosen “choose lipidoids over SNALPs” for these indications (also discussed in a blog entry here). It therefore seems that Protiva/Tekmira's 0.1mg/kg IC50 liposomal nanoparticle formulations may be the current frontrunners in entering the clinic (see also a recent PR).

The sometimes imprecise use of the terms SNALPs and lipidoids may be partly to blame for the confusion. SNALP refers to a liposomal formulation technique, while lipidoids are a new class of lipids generated by combinatorial chemistry which can now be evaluated for liposomal drug delivery. As such, lipidoids could be used within the context of SNALPs, and both of these approaches are therefore complementary to each other.]

RNAi Therapeutics IP: On the Importance of Being Tuschl

A strong patent position is essential for companies to attract investments for the development of RNAi Therapeutics. Although the number of patents and patent applications surrounding RNAi Therapeutics continues to grow, arguably the two or three key early patents are 1) Fire and Mello describing the discovery and use of long double-stranded RNAs for inducing RNAi-mediated gene knockdown in worms and by extension other organisms, and 2) the Tuschl I and II patent series describing the identification of small 21-23nt small RNAs as the mediators of RNAi (Tuschl I) and the use and properties of synthetic siRNA duplexes for RNAi in human cells (Tuschl II).

While Fire and Mello has been granted in the US and can be licensed by almost anyone that wants it, there is much more controversy surrounding Tuschl I and II. It is undisputed that scientifically Tuschl’s studies describing the use of synthetic siRNAs for RNAi in mammalian cells is what opened up the prospect for RNAi to become the next platform for drug development. Tuschl II which is based on the work by Tuschl, Elbashir, and Lendeckel and owned by the Max-Planck Institute in Germany has been exclusively licensed to Alnylam Pharmaceuticals and has already been granted in the US and some other territories (Tuschl is a scientific co-founder of Alnylam). The patent application was provisionally filed in the US on March 30, 2001, and in Europe on December 1, 2000, and describes in great detail the anatomy of effective synthetic siRNAs.

Meanwhile, much of the work in Tuschl I is focussed on the identification of small RNAs as the mediators of RNAi based on the fact that isolated small RNAs derived from processed double-stranded RNAs in Drosophila cell extracts trigger specific gene knockdown and cleavage of a target message at 21-23nt intervals. Interestingly, most of that work does not mention the fact that these 21-23nt small RNAs should be double-stranded. This conclusion could not be derived from the observation of 21-23nt small RNAs on denaturing polyacrylamide gels, but was deduced through the cloning of these small RNAs which is described in Tuschl II, not I. It is therefore very surprising that, out of the blue, Tuschl I demonstrates the use of synthetic siRNAs with preferably 2-nucleotide overhangs for RNAi in mammalian cells. Subsequent claims then focus on the use of such siRNAs for human therapeutic development. This is rather surprising given that the basis for choosing 2-nucleotide synthetic siRNAs is lacking. It therefore appears as if this example had been appended later so as to make the patent more relevant for human therapeutic use. Otherwise, only fruit fly work, albeit important, would have been described. Given the near-identity of this last claim of Tuschl I with work described in Tuschl II, it is difficult to imagine how Tuschl I could be granted in full in the presence of Tuschl II.

Similarly intriguing is the fact that Tuschl I, which by the way has not issued yet, is co-owned by the Whitehead Institute for Biomedical Research in Cambridge, MA, the MIT, the University of Massachussetts, Worcester, and the Max-Planck Institute. At the same time, from the publication record it is clear that synthetic siRNAs were pioneered by Tuschl, Elbashir, and Lendeckel while at the Max-Planck Institute. Interestingly, the UMass has chosen to co-exlusively license their rights to the patent to Sirna Therapeutics (now a Merck subsidiary), CytRX (now RXi), in addition to Alnylam. Clearly, Merck and RXi would benefit most if Tuschl I would be granted eventually and somewhat limit the dominance that Alnylam currently enjoys in the RNAi patent space. Nevertheless, the fact that Tuschl I, filed on the very same December 1, 2000 date as Tuschl II in Europe, has not issued yet and the sudden appearance of synthetic siRNAs and their use in human cells at the very end of that patent application, raises questions about conflicting interests between the involved parties.

Based on publicly available information, the claims of the Tuschl patent series could therefore be divided as follows: Tuschl I getting credit for identifying 21-23nt small RNAs for mediating RNAi in fruit flies and by extension in other organisms, and Tuschl II for characterising effective siRNAs to be double-stranded with preferably 2-nucleotide 3’ overhangs and the ability of synthetic versions thereof to mediate RNAi in mammalian cells. In such a scenario Tuschl II would carry considerably more weight for the development of RNAi Therapeutics which would in turn reinforce Alnylam’s already leading IP position.

I am aware that parts of my interpretation are based on conjecture and criticisms are welcome (email: dirk_haussecker@yahoo.com). For those interested in the patents themselves, please visit

Tuschl I: http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PG01&s1=tuschl.IN.&s2=zamore.IN.&OS=IN/tuschl+AND+IN/zamore&RS=IN/tuschl+AND+IN/zamore

Tuschl II: http://www.google.com/patents?id=BlV6AAAAEBAJ&dq=rna+sequence+specific+mediators+of+rna+interference