Alnylam’s RSV Program Causes Excitement Among Lung Transplant Surgeons- An Example for a Shrewd RNAi Therapeutics Development Strategy?

“Yes, it’s interesting how what we think about most often begins to surround us” replied Chris, a friend from down the hall with whom I share an addiction of going to Stanford biomedical seminars, to an email of mine where I noted that RNAi Therapeutics is honestly popping up almost everywhere now. It happened this week Monday in a chemical engineering seminar on the delivery of crystal-like drug particles (think small 20-30nm, stable needle-like siRNA particles) or during a lung transplant talk on Friday.

Despite close to half a century of lung transplantation, with more than 2000 procedures performed annually world-wide, there has been very little progress in the 2-5 year morbidity and mortality, meaning that less than half of transplant recipients survive beyond 5 years. Community-acquired viral infections in the immuno-suppressed patients are responsible for roughly a third of such chronic rejection and declining lung function cases, with the respiratory syncytial virus (RSV) clearly topping the list.

Stanford is a fairly large lung transplant center, and the situation is not much different here. The seminar I attended concerned a review of the history of 25 lung transplant patients that had acquired either RSV or paraflu viral infections (23 of which with RSV) and were treated with the broad-spectrum antiviral ribavirin either alone or in addition to pavlizumab, a neutralizing antibody that is normally used for and really only effective in the prophylaxis of RSV.

Without going into the details, at the end of the presentation it was clear that, in the absence of any effective treatment, ribavirin and pavlizumab are given as a last resort and desperate effort (yes, despite of what you read in the press these days, these people really seem to care about improving the health of their patients) to make a dent against RSV, but that nobody was really convinced that this would more good than harm. Actually, inhaled ribavirin is even considered a safety hazard to attending nurses and docs.

Then suddenly, there was a commotion in the room as somebody mentioned the word “s-i-r-n-a”. Wasn’t there something in clinical trials right now that would attack the virus directly, a treatment that would even work in immuno-compromised patients? And yes, hadn’t it shown already some kind of antiviral activity in the clinic? Wow, maybe we should give it a try- anything that had been shown anywhere to inhibit RSV in man… There was a lot of excitement and confusion, for example about the mechanism of action, and "some commercial company” was mentioned. Probably worth revisiting.

This experience told me that, no, I am not living in an RNAi Therapeutics bubble, but that RNAi Therapeutics slowly, but surely is making its way into mainstream medicine. Given that there was confusion about what exactly RNAi was even among Stanford lung transplant surgeons, maybe some education would help. A better understanding should also help in recruiting the best centers for clinical trials and consequently facilitate drug development, and maybe if Alnylam reads this, they may want to approach them and spend a couple of minutes educating them what ALN-RSV01 is about. I'm confident they would find receptive ears.

It also changed my view about the prospect for ALN-RSV01 and the development path Alnylam has taken. It is clear that the experimental infection model studies were not, as sometimes criticized, an advertisement ploy irrelevant to the development path and future use of ALN-RSV01. With no alternatives, it appears that having shown some type of antiviral activity, ALN-RSV01, similar to ribavirin, could be widely applied for the treatment of RSV infection even if only approved for a small sub-population of RSV patients.

Testing ALN-RSV01 in the lung transplant setting therefore makes a lot of sense, as this may turn out to be the setting where ALN-RSV01 could be approved first. Lung transplant patients have the highest medical need for such a treatment, even more so than other immuno-suppressed transplant patients as the infection affects the graft itself and may lead to graft failure. Moreover, any type of therapy that depends on the immune system is unlikely to work in this setting due to the immuno-suppression therefore increasing the competitiveness of an RNAi Therapeutics. As the early detection of RSV should be critical for the success of ALN-RSV01, the fact that lung transplant patients are regularly monitored for and highly sensitized to the possibility of RSV infections is highly advantageous. And finally, as I learned this Friday, the viral shedding of RSV is prolonged in immuno-suppressed patients, meaning that instead of the typical 5 day RSV infection window, ALN-RSV01 gets more time to attack RSV. An interesting aside to the immuno-suppression theme here is that any efficacy of ALN-RSV01 would be much less likely due to non-specific immune responses elicited by the unmodified siRNA.

When the RNAi Therapeutics story has have been written and taught in business schools, one of the main lessons for which ALN-RSV01 could be a prime example should be that by applying innovation to areas of large medical unmet need, a sweet spot can easily develop into a large market opportunity. Due to its unique mechanism of action, RNAi Therapeutics is ideally positioned to repeatedly take advantage of that strategy.

Quark Biotech Dazzles RNAi Therapeutics World with Expanding Pipeline, Adds to Evidence that Delivery Walls to Kidney Crumbling

Yesterday, the US-Israeli company Quark Biotech announced the filing of their now third RNAi Therapeutics IND. The latest IND candidate, DGFi, is the siRNA knockdown of p53 in the kidney for the treatment of kidney transplantation-associated ischemia-reperfusion injury. This program follows a similar systemic kidney-p53 oxidative stress RNAi program, Akli-5, for acute kidney injury, and a local RNAi program (RTP-801i) for age-related macular degeneration targeting the apoptosis-related gene RTP801/REDD1 that came out of Quark’s own gene discovery program.

This likely makes Quark Biotech, which specializes in the discovery of disease-associated genes that it then targets by in-licensed RNAi technology, the company with the most RNAi candidates in the clinic, unless, of course, Merck has early clinical programs that we haven’t heard about. This is quite remarkable given that very little is known about Quark’s RNAi science as judged by the literature and presence at leading RNAi conferences. My own patent search for Quark-related RNAi delivery technologies failed, although their IPO documents stated that they had been building an IP estate around RNAi Therapeutics, including proprietary delivery technologies [Note: the planned ~$80M IPO failed last year due to a difficult market; instead the company earlier this year raised around $27M from private Japanese investors].

So I can only speculate as to the systemic delivery technology employed. Given that unformulated and unmodified oligonucleotides have the propensity to end up being rapidly excreted by the kidney, it is well possible that some of these siRNAs get functionally taken up for gene silencing. In fact, the ground-breaking systemic siRNA delivery paper by Soutschek and colleagues from Alnylam employing cholesterol-conjugated siRNAs showed that the lipophilic siRNA conjugate was taken up reasonably well not only in the liver, but also jejunum, heart, adipose tissue, the lung, and kidney, albeit at quite high 50mg/kg dosages. Similarly, a recent publication by the Natarajan group from the City of Hope, CA, showed that subcutaneous administration of ~20mg/kg cholesterol-siRNA reduced gene expression in the kidney by about 50-80% with promising therapeutic effects in a mouse model for diabetic nephropathy. It is, however, possible that the apparently intravenous formulation is composed of a nanoparticle as suggested by the second name of the Akli-5 progam, I5NP (NP=nanoparticle?). In any case, the evidence is growing that the fact that siRNAs like to go to the kidney could be exploited for treating kidney-related disease by RNAi, slowly clearing yet another organ for RNAi.

Although I feel more comfortable judging an RNAi Therapeutics company with some scientific data at hand, Quark Biotech’s speed of entering the clinic while others are humbly optimizing their own candidates, particularly with regards to delivery, warrants some attention. In addition to the AMD program, the company has licensed a second RNAi program to the emerging RNAi superpower Pfizer, a program for COPD likely to be administered by inhalation. Overall, the Pfizer relationship has brought in over $25M of realized funding as of the filing of the IPO documents in March 2007. Another important relationship exists with Silence Therapeutics, although a report earlier this year suggested that there might be some frictions in that relationship. This would be consistent with Quark having subsequently licensed IP from Alnylam as well as Quark’s ambitions of developing proprietary RNAi trigger IP, whatever that is supposed to mean. I guess by providing a little more transparency, Quark Biotech might be able to attract more investor interest for a second IPO attempt. With so many clinical candidates and more coming up, such a cash infusion could be necessary soon.

In other news: The Pharmalot Blog posted yesterday that the approval rate of innovative medicines continues to be anemic. Only 5 new molecular entities were approved by the almighty FDA in the year through May. It’s time for RNAi to contribute to the development of more innovative drugs addressing unmet medical needs, and the regulatory agencies and society as a whole to understand that overdone conservatism and by killing the profitability of drug development aren’t helping in that regard.

Developing Multi-Functional RNAi Therapeutics

The great attraction of developing therapeutics based on RNAi is its inherent specificity and applicability to virtually any gene for the rational design of drugs. The specificity of gene silencing is not just a theory, but very powerfully attested by the many successes of applying RNAi even to genome-wide screens to uncover gene function (and also more targets for RNAi). Delivery of the RNAi inducer to the cells of interest would further add to such specificity and consequently safety.

However, as has long been known, certain nucleic acids elicit immune responses, and siRNAs are no exception to this. Although this does not apply to appropriately designed and vetted siRNAs, when it does, it may well interfere with the interpretation and predictability of the knockdown phenotype. However, instead of describing once again methods whereby such responses can be avoided and phenotypes more consistently obtained (siRNA length, structure, and modifications; bioinformatics etc), I would like to take the opportunity here to point out the potential for RNAi Therapeutics that include a immune-regulatory element. Moreover, as this is typically related to the uptake of the RNAi formulation in cells other than the primary target cells, I would also like to make us consider the potential for RNAi Therapeutics exploiting the entire biodistribution of a particular RNAi drug delivery system.

When we think about indications such as cancer and viral infections, the importance of the immune system in eliminating the disease cannot be underestimated. Cytokine therapies e.g. are well known to these areas of medicine and it is no coincidence that there have been long-standing efforts in harnessing the ability of nucleic acids to induce TLR and other immune signaling pathways to improve both cellular and humoral immune responses.

It is therefore conceivable that an immuno-stimulatory siRNA is not necessarily screened out during the siRNA selection process, but is deliberately packaged into a nanoparticle which in addition to the primary target cells (cancer cell, virus infected cell, etc.) would also be taken up by phagocytic cells where the cytokine stimulation would lead to enhanced antigen presentation or the augmentation of monoclonal antibody therapies. To further take full advantage of the biodistribution of the RNAi formulation, the RNAi drug could also contain two or more different siRNAs, each one designed to knock down a suitable gene in the various cell types that the nanoparticle is taken up in (e.g. in the case of a liver delivery system that enters both Kupffer cells and hepatocytes, an siRNA against a immuno-regulatory gene expressed in the Kupffer cells and maybe other phagocytes and one siRNA for a hepatocyte-specific gene).

In addition to immune-stimulation, certain siRNA formulations could be used for concomitant gene knockdown and immune suppression. As work by Protiva (now Tekmira) has shown, siRNA modifications may not only be used to avoid unwanted TLR signaling through siRNAs, but to inhibit these TLR responses in trans. A single modified siRNA could thus be employed in a two-pronged gene knockdown/TLR signaling inhibition strategy for treating autoimmune disorders.

Based on the acquisition of prior TLR company Coley by Pfizer, Alnylam’s vaccine spin-off intentions, and Tekmira’s IP and know-how on the immunological properties of nucleic acids, I would not be surprised if we should be hearing relatively soon more about such multi-functional RNAi Therapeutics.

Rather than considering innate immune responses and imperfect biodistribution as nuisances, it may well turn out that a number of RNAi Therapeutics may get the extra bit of efficacy out of simultaneously modulating immune responses and knocking down genes in multiple cell types. In my opinion, the medical and commercial opportunities for that are currently underappreciated.

Alnylam Starts Monetizing RSV Drug Candidate, but Keeps Options Open

As the biotech world is gathered at the BIO 2008 in sunny San Diego, Alnylam announced today the licensing of Asian rights to their lead, early phase II, RNAi Therapeutics program ALN-RSV01 for the treatment of RSV infection, to Kyowa Hakko, a Japanese company with an increased focus on biotech drug development. The deal involves an upfront $15M cash payment to Alnylam, with additional development and commercialization milestones of up to $78M and remarkable double-digit sales royalties.

Earlier this year, ALN-RSV01 has demonstrated proof-of-concept antiviral activity in an experimental infection model in healthy adult volunteers. This deal therefore comes at a reasonable value inflection point for the drug. Since the Asian rights for ALN-RSV01 were explicitly excluded from the platform licensing deal with fellow Japanese company Takeda, last month, today’s announcement may not come as a surprise to some observers. However, it shows that, supported by the strength of the RNAi platform, IP, and know-how, Alnylam management has executed on yet another strategic corporate goal. The exact timing may have to do with the convenience of signing contracts while assembled at the BIO, by the way taking place not too far away from where not only Kyowa Hakko’s parent company Kirin, but also Takeda have US operations, but possibly (pure speculation) also with the achievement of some clinical milestone (patients dosed in the current lung transplant trial etc.).

Importantly, while Alnylam is thus starting to monetize ALN-RSV01 thereby lowering the risk that its broad RNAi Therapeutics platform may be unduly predicated on this first-generation RNAi Therapeutics candidate, this arrangement leaves Alnylam almost all options open with regards to ALN-RSV01. It leaves them with the clinical development responsibility which is a good thing for a company that aims to become a vertically integrated drug company and, despite its young age, may be the best to shepherd such an RNAi drug candidate through clinical development due to its intimate familiarity with the technology. On the other hand, should one of Alnylam’s upcoming programs for hypercholesterolemia, liver cancer or Huntington’s Disease, show even more promise than ALN-RSV01 early on in the clinic, Alnylam may decide to lower their exposure to ALN-RSV01 through further partnering, potentially on even more lucrative terms following results from ongoing phase II studies. If not, Alnylam may decide to invest more and thus retain most of the rights to ALN-RSV01 for itself.

The terms of the agreement are very favorable indeed and illustrate the virtue of developing innovative therapeutics based on novel mechanisms of actions for diseases of high unmet medical needs- one of the attractions of RNAi Therapeutics. By this, even programs that may ultimately fail in the clinic could actually pay for themselves. The deals just keep coming, and it is only a question of time until even Wall Street realizes that as Alnylam starts paying taxes on the resulting profits, that this is actually part of a sustainable business strategy.

Capped Small RNAs Expand Universe of Small RNAs

Findings from research by myself and colleagues here in Stanford on the discovery of a new species of distinct small RNAs, capped small RNAs, have just been published online at Nature Structural & Molecular Biology (Haussecker et al.: Capped small RNAs and MOV10 in human hepatitis delta virus replication).

It is becoming clear from large-scale sequencing efforts and hypothesis-driven research such as ours, that siRNAs and microRNAs are just two classes in an ecosystem of other small and non-coding RNAs. This particular class of small RNAs carries mRNA-like 5’ cap structures and was discovered during our studies on human Hepatitis Delta Virus (HDV) replication.

HDV is the smallest virus known to the animal kingdom and is even more so remarkable in that it does not encode for its own polymerase for viral replication, as all other viruses do, and instead relies on host RNA Polymerase II (Pol II) for its replication. HDV accomplishes this with carrying the genetic information for just one non-catalytic protein, the hepatitis delta antigen (HDAg), while its RNA genome calls all the other shots. It is also the only known example of RNA-directed transcription by Pol II in vertebrates, as Pol II is largely thought to use DNA, not RNA, as a transcription template.

Intrigued by this and by the possibility that within the complexity of our transcriptome there may be hidden RNA-directed transcription, vestiges of our RNA World heritage, and HDV may be the key to its understanding, we set out to investigate whether small RNAs would also play a role in HDV replication. Sure enough, a few Northern blots later, it became clear that RNA secondary structures previously associated with the initiation of HDV transcription harbored small RNAs. Long story short, the occurrence of capped small RNAs from hairpin secondary structures suggests that maybe analogous RNAs in our genome could be involved in analogous processes.

As we were studying the capped small RNAs, we also sought to determine the host factors that HDAg interacted with. Knowing this may give us further insights into HDV-related RNA-directed transcription. Intriguingly, the mass-spectrometry screen yielded MOV10 which is a gene that in plants had been implicated in RNA amplification during RNAi. Knocking down MOV10 with siRNAs inhibited HDV RNA accumulation, consistent with a function in RNA-directed transcription in vertebrate cells as well. Given that triphosphorylated small RNAs, which like capped small RNAs, should be derived from short transcription initiation events, had been found during RNAi amplification in worms, we speculate that HDV replication may indeed tap into an evolutionarily conserved process which makes it also more likely that non-viral RNA-directed transcription indeed occurs in our cells.

While RNAi-related factors such as Dicer and Drosha do not appear to play a role in HDV replication, we found in the course of our studies that knockdown of Argonaute 4 (AGO4) significantly affected HDV replication. AGO4 is related to AGO2 which is the Slicer in RNAi that cleaves target mRNA, and yet very little is known about AGO4. Determining the roles and preferences of the various AGO proteins in humans will be an important area of RNAi Therapeutics research as it promises to yield improvements in the design of RNAi triggers both in terms of safety and knockdown potency.

It remains to be seen whether capped small RNAs are specific for RNA-directed transcription, or whether they are a reflection of a more widely used gene regulatory mechanism. In fact, high-throughput sequencing efforts by others indicate a population of small RNAs associated with gene promoter regions. It is possible, although not yet demonstrated, that these are capped, and since the HDV small RNAs are abundant enough to be detected by Northern blot, HDV replication may serve as a model system to understand this type of gene regulation. Finally, it is tempting to speculate whether the modulation of capped small RNAs may be utilized for therapeutic purposes.

Nucleonics in Liquidation While Benitec Shows Signs of Life

Just a day after boasting how RNAi Therapeutics continues to enjoy ample financial support in a tough economic environment, a google news alert indicates that Nucleonics is in liquidation mode.

Since its inception, Nucleonics has made more headlines with their IP battles with Benitec over DNA-directed RNAi supremacy rather than with good science, and must be filed under those early biotech companies that spent more money on administrative and legal expenses than R&D. The ultimate nail in the coffin may have occurred when a Federal Court denied Nucleonic’s wish for a declaratory judgment against Benitec’s patent claims last year, thus leaving the company and investors vulnerable to future lawsuits by Benitec.

While it has to be said in Nucleonics's defense that it wasn't solely responsible for the endless litigation, the obvious winner from this new development is Benitec. With their arch rival out of the game it may now find it easier to concentrate on drug development, although financing and IP issues remain of concern. Encouragingly, their collaborator on the HIV AIDS lymphoma program, John Zaia from the City of Hope, just recently presented at the annual ASGT meeting early interim phase I data on the successful transplantation of RNAi-modified hematopoietic stem cells in two patients (using lentiviral vector technology). It will be exciting to determine how safe and sustained RNAi expression is and whether the derived T-cells have a survival advantage compared to those derived from the unmodified stem cell fraction transplanted at the same time.

Coming back to Nucleonics, the apparent bankruptcy is also likely to affect their recent HBV RNAi clinical program involving the administration of a plasmid formulated with cationic lipid. We may never know about what happened to the first patients that received the plasmid, and maybe that’s good so. The Nucleonics experience shows that early IP battles are dangerous and costly, and that as the RNAi Therapeutics field matures it is becoming difficult to attract funding based on me-too technologies and long-shot scientific strategies.

Disclaimer: This Blog may not be based on reality and reflects my views as of today only.

RNAi Therapeutics: RNA Research Blossoms in Economically Challenging Times

As the war in Iraq continues to cannibalize investments into the US health-care system and particularly funding for basic research, and spiraling commodity prices and the bursting of the credit bubble conspire to create risk-averse capital markets in which small biotech companies are struggling to fund R&D, RNA research is blossoming while RNAi Therapeutics appears to be one of the very few areas in biotech financing where money is still readily available.

This view is not only supported by the exploding number of RNA-related research publications, not to a small degree triggered by interest in RNAi and microRNAs, but also anecdotally by the exponentially increasing numbers of official registrants of the Bay Area RNA Club meetings. An impromptu meeting for RNA researchers to gather and schmooze over RNA science every half a year or so, it drew more than 250 RNA enthusiasts this past week to the UCSF Mission Bay Campus across the street from the expanding Merck subsidiary Sirna Therapeutics in San Francisco, also the place that had just hosted the Qiagen HT RNAi user meeting the week before. At the same time, the corporate RNAi world was assembled for the Beyond Genome conference at the lofty Fairmont Hotel.

Of course, the big meeting taking place concurrently in San Francisco was the enormous ADA conference. Although RNAi was not really represented there, this should change in the coming years as metabolic disease together with other liver disease and cancer is emerging as one of the hot areas for first-generation systemic RNAi Therapeutics. RNAi Therapeutics appears to be the logical answer as the genome revolution is yielding serious therapeutic targets for metabolic disease and cancer on an almost daily basis. Recent papers studying non-alcoholic steatohepatitis with AAV RNAi (a very potent technology for persistently knocking down genes in the liver, at least in mice) or the surprising identification of transcription factor XBP1 in regulating lipogenesis in the liver are just two examples. The latter is also particularly interesting as targeting XBP1 may require tissue-specific delivery, and the availability of RNAi delivery technologies such as SNALP RNAi which can carry up to 95% of the injected dose to the liver.

Given the potential for innovation, rational drug development, and more efficient and shortened development time-lines, it is therefore not surprising that one of the very few areas in Big Pharma R&D not affected by big cutbacks is RNAi Therapeutics. Think Merck, GSK, Pfizer, Abbott, Takeda, Novartis… a list of those Big Pharma companies not participating in RNAi Therapeutics research would probably be more informative at this point.

I even wouldn’t be surprised to see Genentech, based on their increased presence at RNAi conferences, make themselves less reliant on protein-based therapeutics and throw their support behind RNAi Therapeutics relatively soon, probably before 2010. Genentech, the pioneer and poster child of personalized cancer therapeutics, would be the logical, unnamed Big Pharma that Tekmira mentioned has been evaluating their SNALP delivery technology. I should apologize for mentioning SNALP RNAi so often, but by following the Alnylam-Tekmira, I believe one can gain invaluable insights into current RNAi Therapeutics trends.

I am confident that the intense efforts in RNA research in academia and industry will provide a fertile soil on which RNAi Therapeutics will continue to thrive, and may not only survive the very real economic downturn, but given the pressures experienced by Big Pharma and the healthcare system even benefit from it. Merck, Roche, Takeda and others are not the result of clever business development removed from science, but a consequence of such efforts.

RNAi Therapeutics: A Spoilt Market, or Waiting for Clinical Progress?

The market reaction to Alnylam’s deal with Takeda was, well, a bit disappointing. Although the argument can be made that next to the Sirna acquisition by Merck this has been the most favorable deal for the RNAi Therapeutics field yet, shares of Alnylam not only did not advance, but even declined on the news. Can the muted reaction be explained by a market spoilt by Alnylam’s RNAi mega-deals and that has come to expect an even larger cash component, or has Alnylam’s hinting at more deals given the shorts plenty of time to prepare for such an event by aggressively shorting the stock on the news as the considerable volume may have suggested? Possible. However, here I would like to consider the third possibility that for Alnylam’s stock to move to new highs, the market would like to see the deal making being complemented by scientific and clinical progress.

Actually, these platform licensing agreements are evidence for just that as the therapeutic areas covered by the non-exclusive license, namely metabolic disease and cancer, clearly indicate that the deal was driven by the maturation of liposomal RNAi delivery technology. Moreover, an important component of the Alnylam-Takeda relationship will be the exchange of RNAi delivery capabilities, suggesting that Takeda had also made progress in this area.

However, the bears would argue that what Alnylam, and the wider field, needs is further demonstrating progress with their development programs. Of course, we had the phase II ALN-RSV01 proof-of-concept data which was a great relief for everybody and should strengthen Alnylam’s position at the deal table if not on a PPS basis, but we should also remember that Alnylam had previously guided that it would advance one or two SNALP-related programs into the clinic in 2007. This has not happened and I can understand that some may have well been disappointed by that. Paradoxically, rather than reflecting science that has stalled, the delay can be explained at least partly by ongoing progress in SNALP RNAi delivery and it may not be wise to commit to a development candidate when improvements in both safety and efficacy can be made so readily as supported by recent conference presentations and publications.

Having followed biotechs for a while now, one major question I ask myself before investing in a biotech company is whether programs have been rushed into the clinic well before they have been properly validated pre-clinically. That often happens with small biotechs with acute fund-raising needs or when existing investors would like to exit and an IND is the goal, not final approval by the FDA. How otherwise could one explain e.g. Nucleonics’ decision to advance a plasmid-based, DNA-directed shRNA program for Hepatitis B into phase I when all the in vivo data consisted of some co-transfection studies (= glorified in vitro data) and when current non-viral plasmid delivery methods are unlikely to achieve the transfection rates sufficient for curing such a viral infection through direct gene knockdown. Next to Nucleonics, other early RNAi pipeline candidates, while somewhat more promising scientifically, may also serve the role of flag-waving programs demonstrating to investors that RNAi is not just a fascinating science, but also of clinical relevance.

Much has been learnt since the first candidates entered the clinic in 2004 and, like for SNALP RNAi in particular, it makes a lot of sense, also economically, to take advantage of the steep learning curve in general to increase the odds of late-stage pipeline success at the cost of slight early delays. Meanwhile, the irresistible expansion of RNAi into in vivo applications and the increasing number of RNAi-related conferences (another one, Beyond Genome, is just about to take place a little bit North of where I live) demonstrate that the field is vibrant and the science is making rapid progress.

If that weren’t enough, the coming months should bring more than enough RNAi clinical results to contemplate. Calando just reported this past week initial results from their phase I studies on CALAA-01, an unmodified siRNA in a targeted nanoparticle formulation for the treatment of solid tumours. According to their press release, the first patient has now completed a course of 4 intravenous doses over two weeks without any show-stopping adverse events. As this is the first clinical experience of systemically administered siRNAs, unmodified at that, it therefore also marks an important and reassuring milestone for the synthetic siRNA field in general. It is easy to envision scenarios how an adverse event could have had disastrous repercussions for RNAi Therapeutics. Well done, Calando!

Then there are the SNALP RNAi programs that, once entered, could yield relatively soon clear in vivo efficacy data on systemically administered RNAi Therapeutics. Finally, Benitec’s HIV program is another program that deserves some attention as quantitative patient data may emerge relatively soon; for example, an expansion of T-cells derived from the stem cells transduced with the lentiviral vector encoding for a triple-RNA antiviral relative to unmodified cells would be very promising.

RNAi Therapeutics: Qiagen High-Throughput RNAi User Meeting San Francisco (Part 2 of 2)

After the coffee break, John Hogenesch illustrated how RNAi has revolutionized our concept of mammalian genetics. Before RNAi, researchers essentially studied two expression states of a gene: when it was fully expressed (+/+), or when both alleles had either lost their function or weren’t expressed at all (-/-), and rarely the heterozygous state (+/-). RNAi, however, now allows the functional output of a gene to be measured in virtually all the states in between, depending on the RNAi dosage used.

Hogenesch’s systems biology team is interested in how the circadian rhythm is genetically wired that that it can be robust and yet adaptable, and consequently set up a beautiful system whereby the activity of master transcription factors of this genetic program could be measured in real-time over days based on the luminescence generated by binding of the transcription factors to a reporter gene. They then knocked down known regulators of this genetic circuit to varying degrees by titrating the siRNA amount and then assessed the correlation between the degrees of knock down and functional outputs. Pleasantly surprising not only Hogenesch, but also all the researchers worrying about how the degree of knockdown affects the phenotype they are seeing in RNAi experiments, in many cases the functional output appeared to almost directly correlate with the remaining expression level of a gene after knockdown.

However, in cases where there exist paralogues, that is highly related genes that have largely retained overlapping functions, the knockdown of one gene is often compensated almost entirely by the increased expression of the corresponding paralogue. You probably may want to avoid targeting these genes with an RNAi Therapeutics, or any other therapeutic for that matter. Finally, there were also cases where the knockdown gave non-linear responses, and this is mostly when enzymatic reactions were affected.

Nevertheless, as everybody in the audience having traveled from across the country for this one-day meeting knew, genetic circuits, long-term, are usually quite robust and can adjust to external fluctuations. It is therefore also of interest to RNAi Therapeutics (and then again, all classes of drugs, too), particularly for chronic applications, that out of the ~4400 gene knockouts in mice that have been generated so far, only 1000 are lethal in the homozygous state, and just 53 in their heterozygous state, and these already are probably biased numbers (scientists like to study genes with apparent phenotypes, lethality being a very obvious one). This has implications both for chronic drug treatment in general (drug effect may diminish) as well as when we worry about adverse consequences of off-targeting (off-targeting may be tolerated better than expected).

Next up was Loren Miraglia from the Genomics Institute of the Novartis Research Foundation (GNF, San Diego), a basic research institute with preclinical capabilities to feed into the Novartis drug development pipeline. The main part of the presentation focused on the technical aspects of establishing a lentiviral RNAi library for the investigation of difficult-to-transfect cells. The upshot was that these libraries once established can be very powerful, however it takes a considerable infrastructure to realize that goal.

There were two pieces of information that may give some quite interesting insights into Novartis’ RNAi Therapeutics plans. One was an overview of the RNAi libraries being used by GNF which essentially all somewhat disappointingly were focused on the “druggable genome”, disappointing because a lot of genes may be missed that could be addressed by RNAi Therapeutics. There was one notable exception, however, and that was a library devoted to genes involved in cancer. As the time approaches for Novartis to make their move on Alnylam, this confirms my belief that cancer will be an important part of the new license agreement. With screening efforts such as these, there should be more than enough targets to keep all the Alnylam licensees busy without having them compete too much with each other.

The second insight was Miraglia’s presentation of a new “gene X” that when mutated had been found to lead to an increase in LDL-R, the new star in the hypercholesterolemia field. When tested whether RNAi knockdown would also elevate LDL-R, this was indeed the case with a nice correlation between degree of target knockdown and LDL-R elevation and subsequent cholesterol lowering, just what you would like to see in an RNAi Therapeutic. So, like for Takeda, cancer may be well joined by metabolic disease as one of the main initial therapeutic fields for Novartis’ RNAi Therapeutics efforts and driven by current delivery capabilities.

Systems biologist Sumit Chanda (Burnham Insititute, La Jolla) praised RNAi screening as an important tool to learn more about the vast part of the genome that is essentially left unexplored. He bemoaned the fact that it is paradoxically genes of which so much is already known about, think of p53 and TNF-alpha, that get the bulk of the research funding.

Chanda’s laboratory used RNAi screening to discover host factors involved in HIV replication, which could therefore also be potential targets for drug intervention. As a virologist/molecular biologist myself, I also see targeting host factors as a very promising approach for treating viral infection. Unfortunately for Chanda’s group, another group from Harvard just published the results of a similar RNAi library screen, meaning that they were “scooped”. Nevertheless, it appeared that there was only limited overlap between the results and one could explain this by the slightly different assay conditions employed or the quality of each screen. In any case, the point was well made that one screen will never answer all the questions and should be complemented by additional ones. Moreover, the importance for multiple negative controls and multiple redundant siRNAs towards a single gene (to confirm the sequence-specificity of the hit) was emphasized.

Natasha Caplen from the National Cancer Institute (NCI; Rockville, MD) picked up the personalized medicine theme for cancer therapy. As noted before, while monoclonal antibodies have changed the way cancer is treated today, it is often only few patients that respond to them. Moreover, many of the drugs’ mechanism of action converge on the few same signaling pathways, meaning that the mechanisms of actions are still limited and prone to mutational escape. RNAi Therapeutics could then either play a role in exploiting new mechanisms of actions or for sensitizing towards existing drugs. Caplen’s group chose the latter approach and focused on devising new strategies for the use of the bacterial L-Asparaginase enzyme (L-ASP) that has been used to selectively starve acute lymphoblastic leukemia (ALL) cells. One problem associated with L-ASP, however, has been the quite variable response to this treatment.

Caplen’s group suspected that cancers that do not respond well to L-ASP treatment may overproduce the human asparagine synthetase (ASNS) to compensate for asparagine deprivation by L-ASP. To test their hypothesis they first screened a library of cancer cell lines for L-ASP responsiveness and, indeed, L-ASP negatively correlated with ASNS expression levels. To demonstrate a causal relationship, they then knocked down ASNS which greatly increased the sensitivity of the cells to L-ASP. The sensitization was so unbelievable, up to 500-fold, that Natasha Caplen made her co-worker repeat the experiment again and again to convince her of the veracity of the results.

ASNS expression levels could now be used to select patients for L-ASP treatment. Alternatively, ASNS knockdown may be a viable way to increase L-ASP responsiveness. It more and more appears therefore that cancer drug sensitization, and not just targeting novel oncogenes appears to be a very promising avenue for future cancer RNAi Therapeutics. Similar patient selection and drug sensitization strategies were subsequently discussed for the topoisomerase inhibitor camptothecin (Caplen) and the Wnt signaling pathway by Paul Kassner (Amgen).

Finally, I’d like to summarize the talk given by Xiao-Dong Yang from Intradigm (Palo Alto, CA) on the delivery of RNAi Therapeutics to cancer. Using siRNAs provided by Qiagen, it appears that Intradigm screens for siRNAs essentially based on potency. I am somewhat skeptical whether this is sufficient and consequently raises some questions as to their findings that were largely centered on targeting the VEGF pathway in mouse cancer models.

Intradigm’s RNAi Nanoplexes consist of a cationic polymer to which the siRNA is complexed, a hydrophilic steric polymer such as PEG, and optionally a cell targeting ligand such as an RGD peptide. The Polytran polymer-based system is particularly interesting as it is a polypeptide composed of branched histidine and lysine residues. While the positively charged lysine condenses the siRNA, histidine only becomes positively charged once internalized into the endosome as they suck up the incoming protons. This supposedly leads to an increase in osmotic pressure causing the rupture of the endosomes to release the siRNA into the cytoplasm.

The Polytran system consists of fairly uniform 100nm particles that are slightly positively charged. Like many RNAi nanoparticle systems, of the Nanoplexes that make it into a tissue following administration, many are either found in the liver, the spleen, or tumor tissue. Many of them are also taken up by phagocytic cells such as macrophages which may pose challenges with dose prediction and requires that siRNAs are carefully characterized as to their potential to activate the innate immune system. Unfortunately, this aspect of their work was little discussed although unmodified siRNAs, prone to elicit innate immune responses and could therefore cause non-specific anti-tumorigenic effects, were used. With this in mind and the fact that only one control siRNA sequence was used, quite potent mouse antitumor effects were found at low mg/kg dosages with apparently knockdown efficiencies of up to over 90%.

While these approaches are valid for the development of an RNAi delivery system, the presentation reminded me of the fact that for companies like Intradigm it would really make sense to collaborate on the siRNA chemistry side with one of the established RNAi operations rather than committing insufficiently characterized siRNAs into the clinic.

On the way home, I was once again amazed by the fact that RNAi has emerged as the only gene knockdown technology that can be applied in high-throughput to explore the depths of our genomes. This both speaks to the potency and specificity of RNAi as well as the ability to translate our understanding of how RNAi silencing works on a molecular level into bioinformatic tools to separate the wheat from the chaff, as Sumit Chanda put it.

Thanks a lot to Qiagen for organizing a day full of excellent scientific talks in a pleasant Mission Bay setting, and I’d recommend everybody running qPCRs Qiagen’s robust and reliable SYBR Green QuantiTect reagents.

RNAi Therapeutics: Qiagen High-Throughput RNAi User Meeting San Francisco (Part 1 of 2)

An army of RNAi enthusiasts descended today onto the UCSF Mission Bay Campus in San Francisco, just across the street from Sirna Therapeutics/Merck, to discuss the latest in the use of RNAi for high-throughput screening. Illustrating the ever increasing popularity of RNAi, 175 scientists registered for this year’s one-day meeting, some from as far as Japan, compared to 75 when it was held for the first time two years ago in Boston. It was therefore fitting that a number of presenters noted as an aside that the discovery of RNAi and microRNAs not only transformed molecular biology, but also a number of lab personnel from bench-weary scientists to those that live and breathe RNAi every day. In the next two posts, I will try and highlight a few points from the meeting seen through my RNAi Therapeutics filter.

Carl Novina (Harvard and scientific advisor to mdRNA/Nastech) gave the keynote address with an introduction to microRNAs and their molecular mechanism of action. In a paper earlier this year, his lab had developed a biochemical model system to study microRNA repression that led them to conclude that microRNAs repress translation through the inhibition of the joining of the large 60S ribosomal subunit to the mRNA-bound 40S subunit of the protein translation apparatus. In today’s presentation, he expanded on that work by describing an RNAi-based screen for the discovery of genes involved in microRNA function. For this, his lab created luciferase cell lines in which one of the reporters contained a target site for an endogenously expressed microRNA and was therefore repressed. Knocking down a gene contributing to microRNA-mediated repression should therefore lead to an increase in the signal intensity from that reporter gene.

Encouragingly and attesting to the high specificity of RNAi, the screen uncovered many of the known microRNA pathway components such as Drosha, Dicer, and Argonautes 1 and 2. In addition, the screen making use of an siRNA library targeting the “druggable genome”, that is the ~7000 genes in our genome encoding for kinases, cell surface receptors etc. that are amenable to small molecule and/or monoclonal antibody inhibition, uncovered a number of proteasomal components. This raises the intriguing question whether an old hypothesis, namely that microRNAs may induce co-translational protein degradation may indeed be part of the complicated reality of the molecular microRNA repression process.

As an aside, Carl Novina noted that one problem with working on RNAi-related aspects in Boston is the considerable demand for talent there by the many RNAi companies located there. A nice problem to have for the many hungry post-docs at a time when public funding for basic research has stalled to pay for apparently more important projects overseas.

One recurrent theme of the conference was the role of small RNAs in the new era of personalized medicine, particularly for cancer, which not coincidentally also was the loud message heard from last week’s ASCO mega meeting. The case was made by Lynne Bemis (University of Colorado) as she compared the costs of today’s monoclonal antibody cancer therapeutics that run at about $30k per annum with clinical responses in only a subset of patients. To avoid patients wasting precious time on ineffective therapies and also to reduce healthcare costs, it is therefore important that these treatments are accompanied by diagnostics that can predict the response of a patient to the therapy, by the way an area first pioneered by the local Genentech.

The striking conclusion from her research was that microRNAs were by far emerged the best biomarkers for this purpose, far better than single protein or mRNA diagnostics. When compared to proteins this is likely due to the fact that microRNAs function as master regulators, often with direct and critical functions in cancer itself, while a single protein may only reflect a much smaller aspect of a much larger picture. When compared to mRNAs, it is the relatively high stability in most tissue preservation methods and their compatibility with formalin fixation that adds to the competitive advantage of microRNA diagnostics.

As an example, EGFR tyrosine kinase inhibitors (TKI) are now widely used for cancer therapy, yet only 10% respond to treatment. Lynne’s team therefore looked at whether EGFR protein levels, EGFR mutations, or genomic amplification levels of the EGFR gene would be good predictors of drug response. Unfortunately, all of them either failed to do so (EGFR levels) or were not practical (EGFR mutations). Running out of options, they then turned to microRNAs. First they made use of microRNA target prediction software to discover two microRNAs that might target the EGF receptor mRNA. They then hypothesized that in cancers where EGFR had been aberrantly activated one of these microRNAs was lost. As an interesting technical aspect, unlike most other microRNA diagnostics currently being developed by Asuragen, Rosetta Genomics, Exiqon and others, they did not make use of qRT-PCR or microRNA microarray for the detection of microRNA expression levels, but the even simpler pPCR from DNA isolated from tumor tissue microdissected from formalin-fixed samples.

Amazingly, from a sample of 60 tumors of the lung, the miR-128b gene dosage was able to nicely segregate those patients that responded to TKI treatment versus those that did not. None of the other methods tested came anywhere close. As, if not even more spectacularly, it turns out that miR-128b is located on a region of the short arm of chromosome 3 that had been previously implicated and was frequently deleted in lung cancer (around 90% of late-stage lung cancers), but of which the identity of the underlying gene had long been a mystery.

In another elegant study, her group employed mRNA microarray analysis to look for genes either over- or under-expressed in a panel of cancer cell lines. Once identified, microRNA target prediction was used to come up with potential clusters of genes being targeted by the same microRNA. Demonstrating yet another promising method of discovering microRNA diagnostic candidates, 5 out of the top 7 overexpressed genes shared a common microRNA signature, and sure enough, this microRNA alone was more predictive of drug responsiveness than any of the proteins alone.

Eric Lader from the host Qiagen rounded up the session on microRNAs with an overview of products for the quantitation and inhibition of microRNAs sold by Qiagen. Qiagen’s qRT-PCR quantitation method allows for the one-step conversion of all microRNAs into cDNA so that many individual microRNAs can be measured from this one cDNA sample thereby reducing the use of precious clinical material. As to the microRNA inhibition technology, a number of nucleotide modifications were tested in the antagomirs and it was interesting that while a number of them were able to inhibit microRNA function, depending on the modification, they probably did so at various steps of microRNA biogenesis and function and included both microRNA degradation and competitive binding. LNAs and other chemistries with high melting temperature generally worked best while RNase H-dependent antisense did not appear to be an effective microRNA inhibition technology.

In my next post, I will summarize the rest of the meeting including more RNAi screens, cancer, and systems biology. Stay tuned…