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.