ALN-RSV01 Data Put Regulators in a Bind

ALN-RSV01 has been a controversial drug candidate in the field of RNAi Therapeutics not because of doubts that it has antiviral activity, but strong suspicion that it may reduce RSV levels by a non-RNAi mechanism of action, likely related to the activation of innate immunity.  The latest topline data from a phase IIb study in adult lung transplant patients are consistent with Alnylam's most advanced clinical candidate having such antiviral efficacy*.  The data show that inhalation of ALN-RSV01 in RSV-infected lung transplant patients results in improvement in a key important outcomes measure in this underserved orphan patient population: the incidence of new or progressive bronchiolitis obliterans syndrome (BOS) for which RSV infection is a major risk factor and which is associated with transplant rejection and death.

* antiviral efficacy, a secondary outcomes goal is yet to be reported

Despite of what appear to be clinically significant data- ALN-RSV01 treatment was associated with more than 50% relative risk reductions in new or progressive BOS in all reported patient groups (intent-to-treat or not)- critics will point out that, strictly speaking, ALN-RSV01 has barely missed its pre-specified primary endpoint in terms of statistical significance: p-values of slightly below 0.06 in the intent-to-treat populations (note: p-values in the arguably more relevant last-observation-carried-forward population was below the magic 0.05 mark).  This result in probably the largest clinical study of its kind (87 patients enrolled) is consistent with an earlier phase IIa study which was also strongly in favor of ALN-RSV01, but which had suffered particularly from imbalances in patient baseline characteristics which might have skewed results in favor of ALN-RSV01.

Despite the encouraging data, Alnylam made it abundantly clear that it is far from certain that it will further develop ALN-RSV01 for this patient population: having poured what must have been tens of millions into this clinical candidate which has been abandoned for development in the commercially much more attractive infant population due to the aforementioned mechanistic and resulting safety concerns, investing more in an orphan indication with maybe 500 to 1000 annual cases after running the probably largest study of its kind in this patient population would be difficult to justify in economic terms.

As a result, the company more or less said that the ball is now in the court of regulators in the US and Europe to make a tough public health decision: approve ALN-RSV01 with the present data and/or provide us with a path forward towards expanding the patient population without too much added effort (e.g. bone marrow transplant patients), or we will abandon this program and have lung transplant patients continue to be treated for RSV infection with highly questionable agents for which there is no clinical evidence of benefit (inhaled ribavirin is particularly notorious in that regard). Judging from the body language, another pivotal large or even larger phase III study certainly is not in the cards and religious adherence to p= 0.05 won't be taken lightly. 

In my opinion, the secondary outcomes from this trial, including lung function, transplant rejection, and overall survival, all of which were not disclosed yesterday, will tip the scales.  Full results are to be presented at the European Respiratory Society meeting in September of this year.  Mark your calendars.

Tekmira Reports Important Advances in Widening Applications of SNALP RNAi Therapeutics Delivery

‘We Make RNAi Work.’

Tekmira’s slogan may sound a bit cheesy as they all do, but when you consider that it is the only company that has managed to translate the theoretically very powerful unilamellar liposome technology into the clinic and ready for commercialization while essentially everybody else is being frustrated from following up on promising early-stage results with liposomal delivery, it is a very appropriate one.

This leadership position was underlined in a recent presentation by Tekmira Pharmaceuticals at the CC-CRS meeting on May 25 (see related press release here). There, the company presented the advances it has made in widening the applications of SNALP delivery for use in major additional medical and commercial opportunities. Some of these advances were made in collaboration with large pharmaceutical/biotech companies, illustrating that Tekmira views these collaborations not only as a way to simply monetize their assets, but also as a way to grow the potential of SNALP delivery to areas such as respiratory disease (after cancer arguably the highest priority area in drug development) and targeted RNAi delivery by leveraging the partners' capabilities.

However, before reporting on the company’s advances in nebulizing and tagging SNALPs with monoclonal antibodies, the presenter made the point that more potent lipids is not what is holding it, or the field back from applying SNALP technology. Using ApoB as a target in rodents (mouse and rats), the data show that many of Tekmira’s lipids developed without Alnylam, including Alnylam’s Canadian subsidiary Al-Cana, have equal or superior potency to the MC lipids that have become one point of contention in the Tekmira-Alnylam litigation as such lipids, in addition to manufacturing, have been one of the pillars of Tekmira's gate-keeping position in SNALP delivery. For example, in the case of ApoB and the 2111 lipid 10 microgram/kg SNALP-formulated siRNA triggered more than 50% target gene suppression.

The presentation then moved on to demonstrating the progress Tekmira has made in formulating SNALP for respiratory and actively targeted delivery applications. Actually, it is formulating SNALP at commercially relevant scales that is Tekmira’s most valuable competitive asset, and this is why the unbelievable disclosures in the Amended Complaint that Alnylam abused its insights into Tekmira’s technology to misappropriate and represent formulation technology as its own are so grave (I will give it a couple of more days rest before I comment on Tekmira's Amended Complaint).

Data on the Ebola biodefense program show that the US government contract has been successfully commenced and is working just as the program was intended to work: not only is the company on track to developing a treatment for Ebola infection with the goal of filing an IND in the second half of this year, but the process has proven very valuable in maturing and bringing SNALP delivery to the next level, especially in terms of one of the most critical steps in SNALP delivery: high-quality manufacturing at commercial scale. Being able to formulate 1 kilo-gram siRNA instead of just 10 grams at a time without changing critical SNALP parameters such as size, encapsulation efficiency and poly-dispersity is enormous and means that SNALP can not only be used for running clinical trials, but can also be readily commercialized.

Data on targeted SNALP delivery showed that it is possible to decorate SNALPs with antibodies using Genentech’s defined THIOMAB antibody conjugation technology, again without compromising on critical SNALP parameters. Tissue culture data show that highly specific, targeted uptake and gene silencing can be achieved using these antibody-decorated particles. Such liposomes will have particular utility for applications outside the liver where delivery does not rely on ApoE (apparently initially an insight of Tekmira, not Alnylam as that company claims- more on this in that other blog post). I look forward to learning more about the performance of these particles in animal models, especially for cancer applications.

Another area where antibody-targeting could have utility is in the delivery of siRNA to the respiratory epithelium following nebulization. As we know, Alnylam’s ALN-RSV01 has been overshadowed by concerns that some, or even most of the antiviral activity of the nebulized naked and unmodified siRNAs may be due to innate immune stimulation. More generally, while I am convinced by now that high concentrations of naked siRNAs on the epithelial lining of the respiratory tract can achieve some target gene knockdown, to my knowledge, there has been no convincing report of RNAi knockdown through inhaled siRNA delivery robust enough to warrant clinical development.

Tekmira’s success in nebulizing SNALP particles therefore is the first critical step in opening up the respiratory space for inhaled RNAi Therapeutics (there are some strategies such as cationic lipoplexes or PEIs that can deliver to parts of the lung following systemic administration). Again, the key to success will likely come from proper formulation/manufacturing.

Using traditional LNPs, nebulization leads to gross changes in morphology, uniformity, size, and dramatic loss of encapsulation efficiency (from mid 90%s to 10%s). However, with undisclosed changes to Tekmira’s SNALP formulation, it is now possible to nebulize SNALP without changing these parameters. Tissue culture data confirm that these particles retain unchanged knockdown activity.

Altogether the presentation emphasizes that not only has Tekmira/Protiva been leading SNALP technology in the past but is continuing to do so…by a distance. It seems that in order for it and shareholders to capitalize on this leadership, more than getting the technology right, it is preventing Alnylam from using partnership status, money and PR from misappropriating the technology and representing it as its own.

Comment on referring to Tekmira’s delivery technology as SNALP versus LNP

Stable nucleic acid lipid particles (SNALPs) are a form of liposomal nanoparticle (LNP). The reason why I prefer to keep using the narrower term ‘SNALP’ when referring to Tekmira’s liposomal delivery technology instead of adopting the broader term ‘LNP’ as both Tekmira and Alnylam have done, is that the liposomes in clinical development by Tekmira and Alnylam currently and in the foreseeable future are still based on the original formulation (cationic/ionizable lipid+neutral helper lipid+PEG-lipid+siRNA), whether the cationic/ionizable lipid is a different one or whether a ligand is added or not. The term ‘SNALP’ is therefore a reminder of the innovator behind the technology, Protiva (now part of Tekmira), as they coined the term.

In a way, you would think that such terms that lack precise scientific definition anyway should not matter that much. However, in Alnylam’s efforts to marginalize Tekmira’s role in liposomal delivery, first changing from ‘SNALP’ to ‘LNP’, and then calling the newer formulations ‘second-gen LNPs’ has been an effective way of hiding ownership of the technology with the goal of eventually not only denying Tekmira contractually owed milestones and royalties, but by-passing Tekmira altogether (incl. manufacturing) also to appease partners such as Novartis and Takeda. ALN-PCS01 looks set to be the first battleground.

ALN-RSV01 Update: The Drug is Safe- and Efficacious?

Alnylam provided this Monday an update on their phase II study results of ALN-RSV01 in lung transplant patients naturally infected with respiratory syncytial virus (RSV). RSV infection in lung transplant patients is linked to irreversible damage of the lung, decrease in quality of life, and sometimes even death. With no proven drug for the treatment of RSV, this remains an area of high unmet clinical need.

The 90 day data follow results announced in June that showed the drug to be well tolerated and adds to the growing evidence of safety obtained with this drug in various phase I and II studies. While safety was the primary objective, based on the initial 30 day data report no conclusions could be drawn on the efficacy of the drug as measured by either reduction of RSV titers or symptom scores due to differences in baseline characteristics between the drug-treated (N=16) and placebo control-treated populations (N=8).

The 90 day data confirmed the safety of the drug that was administered 3 times daily via inhalation. The longer time period also allowed for monitoring lung function. Remarkably, while lung function was significantly impaired in about one third of both the drug and control populations at the start of the study, most likely as a result of the RSV infection, at 90 days after drug treatment only 14% of the ALN-RSV01 patients experienced an FEV1 value 20% below baseline compared to 38% in the placebo group. While this did not reach statistical significance in this small study, when another related measure of lung health was considered, namely the incidence of new or progressive BOS (bronchiolitis obliterans syndrome), ALN-RSV01 did significantly better than control (1 in 15 patients for ALN-RSV01 versus 4 in 8 patients of placebo).

Taken together, the drug was shown to do no harm and there is intriguing evidence for clinically relevant efficacy of the drug in a randomized, double-blind trial, albeit somewhat tainted by the baseline differences. The difficulty of running such a trial is illustrated by the fact that 13 institutions around the world were involved and I cannot see a regulatory body requiring a 200 person lung transplant trial when there are only about 2000 lung transplants a year and only a fraction of those actually becomes infected with RSV. The results are certainly better than the standard of care today, ribavirin, which has unproven efficacy at least in this setting and is well known to be toxic. Transplant surgeons certainly would love to have another treatment option, even if only supported by such data. Therefore, is there a case for Alnylam and their co-development partners to talk to the FDA about approving ALN-RSV01 in this orphan patient population (compassionate use argument), maybe after further follow-up confirm the positive trend in improving lung function which has to be the ultimate measure of treatment success?

It would be ironic for the FDA e.g. to deny such a request due to the difficulty in interpreting the RSV biomarker results while the medically relevant outcome, lung function, showed positive results. Normally, the agency rejects drugs that although the biomarkers were positive (e.g. blood sugar and cholesterol), more direct clinical outcomes were not.

Needless to say, such an outcome would be unexpected, but it does not cost much to ask. In any case, for the wider application of RNAi Therapeutics in lung disease it is comforting to see that the inhalation of unmodified siRNAs even in this fragile population was so well tolerated and it will be interesting to see how the RSV program, sometimes criticized for its seemingly labyrinthine course, will unfold.

RNAi Therapeutics and Innate Immunity- Keeping the Field Honest

As part of the RNAi Therapeutics review series in Human Gene Therapy earlier this year, former Protiva scientists Adam Judge and Ian MacLachlan (both now with Tekmira following the Protiva-Tekmira reunion) made some rather bold statements with regards to the interpretations of a number of pre-clinical RNAi Therapeutics validation papers (‘Overcoming the innate immune response to small interfering RNA’). As part of the same reviews series, in the risk section of “The Business of RNAi Therapeutics”, I also cautioned that some of the first RNAi Therapeutics candidates may show clinical efficacy, but not necessarily for all the right reasons.

The reason for this should not come as a surprise to anybody in the oligonucleotide therapeutics field: long known from the experience with antisense and other oligonucleotide therapeutics classes, oligonucleotides such as siRNAs have the potential to induce innate immune responses which can have antiviral and anti-angiogenic activity independent of their gene knockdown capacity. In fact, there are significant efforts to harness this biological property for therapy in its own right, particularly the TLR responses. Furthermore, the potential for inducing innate immune responses by synthetic and DNA-directed RNAi has been well documented since 2003 and many of the pathways involved elucidated. Nevertheless, one should not ignore the fact that while RNAi Therapeutics may actually be able to take advantage of such activity as part of synergistically acting immunostimulatory RNAi Therapeutics, the risk is that the oligo-dependent immune responses are quite complex and therefore often difficult to predict and in the worst case may cause serious adverse events.

Since many of the early RNAi Therapeutics validation papers involved antiviral and anticancer applications, it was therefore reasonable to suspect that some of the studies misinterpreted therapeutic effects as the result of RNAi gene knockdown when, in fact, innate immune responses accounted for the majority of the activity. In support, the Tekmira researchers now report that almost all of the unmodified siRNAs reported in a sample of such papers were immunostimulatory whereas a single siRNA that, somewhat disturbingly so, was used as the control siRNA in many of the studies proved to be the exception having no such detectable activity. I should add, however, that the assay conditions were rather stringent (types of cells used and high siRNA concentrations) and just because an siRNA may induce immune responses under these conditions does not prove that these were actually responsible for the treatment effect seen in each of the cited studies. Also, if TLR therapeutics history is any guide, oligonucleotides that elicit immune responses in small animal models, do not necessarily do so in primates.

Given its potential as a whole new class of therapeutics, the scientific and clinical bar for RNAi Therapeutics is set particularly high and reports like the effect of TLR3 stimulation by siRNAs on preclinical models for wet AMD and the present paper by Tekmira tend to get quite a bit of press. While they remind us of the complexities involved in establishing a functional new drug discovery platform, they should also be regarded as promoting that process. In fact, the handful of bona fide RNAi Therapeutics groups, pure-plays and Big Pharma subsidiaries alike, are already taking oligo-induced innate immune responses very seriously and have taken advantage of the rapid progress in the field by applying best practices for identifying and correcting these responses (modification, siRNA structure) in developing the latest crop of RNAi Therapeutics candidates.

The acquisition of former TLR therapeutics company Coley Pharmaceuticals by Pfizer for example may be interpreted as Pfizer investing in solving siRNA-induced innate immune responses as one of the main challenges for RNAi Therapeutics they had identified. Similarly, Sirna Therapeutics and Protiva in their prominent 2005 Nature Biotech paper on RNAi delivery in a mouse model of hepatitis B recognized the potential of unmodified siRNAs to elicit non-specific viral suppression and solved the issue by appropriately modifying the siRNAs. Around the same time, Alnylam somewhat quietly generated IP related to double-strand RNA immune stimulation that it then exclusively licensed to Tekmira. Clearly, the main players in the field have not chosen to ignore the issue, but have invested considerable efforts with tangible results.

But what about the current RNAi Therapeutics clinical candidates that have already entered the clinic? There are one phase III (Opko Health) and two phase II (Sirna/Merck-Allergan and Quark-Pfizer) siRNA candidates for the treatment of wet AMD that obviously have naturally come under increased scrutiny. As far as I am aware, all three of these are ‘unformulated’, intravitreally injected siRNAs with one of them, Opko’s, being an unmodified siRNA. While it is not clear how well the mouse TLR3 studies translate into humans, they certainly raise the concern that non-specific responses might be responsible for any thus far clinically observed therapeutic effects, particularly since in the recent Nature study gene knockdown by this route was very limited at best (cholesterol-conjugated siRNAs, however, administered by the same route were shown to mediate functional gene silencing in the same study).

As is the case with Alnylam’s lead candidate ALN-RSV01 for the treatment of RSV infection which has raised similar concerns, it will be important to be forthcoming in the interactions with the regulatory agencies such that safe trials can be designed based on our best understanding of the mechanisms of action of the different siRNAs. While I haven’t read the documents, it certainly wouldn’t be the first time if such non-specific effects were noted as potentially contributing to treatment. In the future, it would not surprise me at all to see openly declared immunostimulatory siRNA drug candidates enter the clinic. If, however, these issues are not addressed upfront, and should adverse events occur as a result, this could easily backfire and future trials rendered much more onerous- something that should be in nobody’s interest. As for the prospects of the individual drug candidates in question, even if non-specific effects contributed to the therapeutic efficacy of these candidates, as long as they are safe and well tolerated they may very well be viable drugs.

Finally, it is curious as to what exactly motivated Tekmira to re-test an entire battery of published siRNAs for their potential of inducing non-specific effects. It is possible that Tekmira has evaluated siRNA therapeutics for a number of the same applications like flu and wet AMD and were frustrated to see publications come out that according to their experience should have been artefacts (scientists tend to measure themselves by the number of publications and their impact factors and don’t like to see their own published work de-valued this way). Another part of the answer may also have been to keep the field honest at this early stage of RNAi Therapeutics drug development before long-term damage is caused: “However, surprisingly few of the reported studies have adequately tested, or controlled, for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.” By setting a rigorous new standard, Tekmira also signals their expertise not only in RNAi delivery, but also in siRNA chemistry and safety (like Coley, Tekmira has a long-standing interest in the use of immunostimulatory oligonucleotides for therapy). Supporting their claim, Tekmira/Protiva’s publications on abrogating TLR7/8 responses and SNALP RNAi delivery have proven to be extremely reproducible in many different laboratories.

The road to RNAi Therapeutics reality won’t be smooth. As much as it is important to tackle the scientific hurdles head-on, investors and the press should also make an effort to discriminate between ‘good’ and ‘bad’ science.

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.

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.

Alnylam Grants Takeda RNAi Therapeutics License for Cancer and Metabolic Disease and Anoints Asian Partner of Choice

As if executing from a script, Alnylam today announced its first major Asian licensing deal giving Japanese pharmaceutical company Takeda access to the US biotech's RNAi Therapeutics IP for cancer and metabolic disease. The deal also provides Takeda with the preferred option of co-developing Alnylam product candidates for the Asian market. In return, Alnylam gets a sizeable $100M in immediate upfront, and another $50M in "near-term technology transfer payments". Adding the typical development and commercialisation milestones, field area expansion payments, and royalties built into these deals, the eventual value may well exceed one billion US dollar.

The agreement is also another validation as to how important SNALP RNAi has become for the current valuation of RNAi Therapeutics, as both metabolic disease and cancer are the two major near-term opportunities for SNALP RNAi. Remember, Roche’s therapeutic areas also included metabolic disease and cancer, as well as certain liver diseases and respiratory disease.

That this deal was announced one day before the close of the Tekmira-Protiva reunion should also be no coincidence and the exact timing may relate to the interests of various Tekmira and Protiva shareholders. Furthermore, my expectation is that the announced $50M in near-term technology transfer payments refers to the final closing of Tekmira-Protiva as well as finalization of an R&D collaboration agreement for SNALP similar to the ones that Tekmira currently has with Alnylam and probably soon with Roche as well. It also reminds us that there are at least another 3 Big Pharma and biotechs that are currently evaluating SNALP RNAi with Protiva-Tekmira. So plenty of more news to come and auction dynamics to set in.

Takeda’s move once more emphasizes the thirst of Big Pharma for innovation. Takeda itself has just propped up its sales numbers and cancer franchise with a headline-grabbing $8.8billion purchase of Millennium Pharmaceuticals and adding to that another $150M for planning more long-term may not be bad for balance. And on the same business trip at that. This is because Millennium is also Alnylam’s Cambridge, MA, neighbor and former employer of Alnylam’s CEO John Maraganore. In fact, it is rather curious that John Maraganore was chosen by the Boston Globe to voice his views on the Millennium deal, to which he responded that “the deal could potentially free Millennium from worries about making every quarter's profit figures, allowing it to invest more money in drug development.” Well, with the $150M in added cash, and ~$400 already stashed in the bank, he has just gotten one step closer to that dream.

Takeda has just had a high-profile failure for an cholesterol-lowering drug candidate, and may now be keen more than ever on exercising its right to partner Alnylam’s PCSK9 program for Asia. ALN-RSV01 interestingly is the only program that Takeda does not have first right of refusal, and may indicate yet another deal in the Alnylam pipeline.

Takeda is also strong in the diabetes area, and it will be interesting as to whether Takeda will pursue such programs with RNAi soon and for which liver-expressed genes may be targeted. This may particularly benefit Alnylam since, unlike in the Roche deal, it now has also the opportunity to opt into Takeda’s RNAi Therapeutics programs in a 50:50 relationship.

Based on statements such as "We believe this alliance will accelerate our initiatives to establish the foundation for RNAi drug discovery" in the presss release by president of Takeda, Yasuchika Hasegawa, it also appears that Takeda has been quite a bit active in RNAi Therapeutics already. Moreover, the deal provides for a cross-licensing of delivery technologies, and it will be exciting to learn what these are.

Novartis, Roche, and now the Takeda deal by yet another major foreign company may illustrate two points. One is that the US currency has fallen so low that foreign companies cannot resist the opportunity to secure their rights to US innovation, in this case to RNAi Therapeutics at bargain exchange rates. The other is that Alnylam may want to keep US rights to itself for the time being.

Finally, the deal is yet another validation that if you have money and are serious about developing RNAi Therapeutics, Alnylam is the company to partner with for access to fundamental IP and, thus far, enablement, too.

Alnylam’s ALN-RSV01 Clears the Human Proof-of-Concept Bar

[Update following this morning’s conference call: 1) Importantly, statistical analysis showed that the reduced infection rate following ALN-RSV01 administration was independent of the level of pro-inflammatory cytokines, and therefore further indicates an RNAi-based mechanism of action, instead of an siRNA-triggered non-specific cytokine response; 2) The dosages were at the upper end based on previous phase I nasal administration safety studies. There were two dosages used, a 75mg group (8/88 subjects) and a 150mg group (80/88). This number was too small, and probably too close together, to determine dose-response. 3) while I labeled the virus in my summary as a “laboratory strain”, it was stressed that the virus was a relatively fresh clinical, wild-type isolate from an RSV patient and therefore not attenuated; 4) the slides from John De Vincenzo’s presentation in Singapore will be available on the company’s website this weekend.]

Concurrent with a presentation at the respiratory disease conference in Singapore, Alnylam has just released more detailed data from the phase II experimental infection study of ALN-RSV01, an unmodified siRNA for the treatment of RSV infection via RNAi. The study, termed GEMINI, was designed to demonstrate the safety and antiviral activity of intranasally administered ALN-RSV01 in adult volunteers artificially infected with a laboratory strain of RSV virus. As such, it could therefore represent statistically validated proof-of-concept for RNAi activity in humans.

According to the press release, there were no obvious adverse effects attributable to ALN-RSV01. Although this may have been expected based on the previous phase I intranasal safety study results, considering the inflammatory potential of some siRNAs (which were apparently excluded during the pre-clinical siRNA screening process) , an siRNA in the context of a viral infection could have conceivably triggered unforeseen safety issues, even worsening rather than treating the viral infection.

On the efficacy side, ALN-RSV01 statistically reduced the infection rate across a range of laboratory parameters. When given 5 times daily, 2 days before and 3 days after viral administration, the number of volunteers remaining infection free almost doubled, from 12/42 to 24/43 treated with placebo and ALN-RSV01, respectively. Measures of viral dynamics in those patients in which viral infection took hold showed a trend towards improvement with ALN-RSV01, although they did not reach statistical significance. Similarly, symptom scores were not much different between ALN-RSV01 and placebo (at least they were not worse as may have been expected for a siRNA-triggered inflammatory response!).

It therefore appears that at least in this setting efficacy was largely an all-or-none and once viral infection took hold there was little stopping it. For a drug candidate that is designed to treat, but not prevent RSV infection (note that there are very effective preventive neutralizing antibodies for RSV on the market), this may appear disappointing at first glance.

However, there are a number of factors that complicate how predictive these results are for naturally infected patients. One unknown to me is the dose used in the study and whether they were on the conservative or aggressive side, which could have made a big difference in antiviral efficacy (but also safety, of course). Another factor is that in order to achieve reliable experimental infection, the nasal epithelium was probably overwhelmed with viral loads that would not be encountered at early stages of a natural infection. Moreover, although the experimental infection should have initially been largely restricted to the nasal epithelium, the odd survivors may be able to establish reservoirs in areas of the respiratory tract not reached by the nasal administration of the drug, at which point the route of administration became limiting in the ability of ALN-RSV01 to stop RSV replication. This may be addressed with the use of aerosolized versions of ALN-RSV01, or even in conjunction with nasal administration, in future studies.

It will also be interesting to find out whether the all-or-none response was due to viral escape mutants that have changed their sequence at the siRNA target site, as had been observed in a number of pre-clinical antiviral RNAi studies before. In that case, co-administering two different siRNAs, similar to Benitec’s HIV and HCV RNAi strategies, should considerably lower the likelihood of such an event.

Today’s results are probably almost all one could have hoped for. They are the culmination of a very well-planned and executed scientific program involving challenges such as the not-so-trivial task of establishing the experimental infection model itself. But there will be little time to rest on their laurels. The eyes are now on the design of the phase II natural infection study slated to start in the first half of this year. The all-or-none response seen here strengthens pre-clinical results suggesting that early detection and treatment will be important for the success of ALN-RSV01 of such studies, and ultimately in the clinic. With increasingly rapid nucleic acid-based diagnostics coming online, hospital-acquired cases of RSV may be the low-hanging fruit. Tomorrow’s conference call may give many of the answers, possibly linked to the enigmatic post-hoc naming of the trial as “GEMINI”.

In the larger scheme of things, the results may also have implications for the treatment and prevention of other respiratory viral infections. In terms of human proof-of-concept, with RNAi and viral infections one always has to take into account the possibility that stimulation of innate immune responses rather than specific gene silencing may have caused the antiviral effect. However, based on the preclinical studies and the fact that the drug was apparently well tolerated, one would probably have to give human proof-of-concept a pass.

Sunday, March 2: The Singapore presentation is now available on the company’s website.

Disclosure: I hold stock in Alnylam Pharmaceuticals.

Alnylam Reports “Positive” Data from RSV Experimental Infection Trial: Human Proof-of-concept for an RNAi Therapeutic?

Alnylam got observers scratching their heads today as they announced “statistically significant anti-viral activity” for ALN-RSV01, an RNAi Therapeutic against RSV infection, in an early phase II trial, but without providing further details.

In this experimental infection trial, an siRNA delivered to the nasal epithelium is tested for activity against a non-pathogenic strain of RSV. The trial has two main purposes: firstly, to provide the first demonstration of RNAi activity in man (human proof-of-concept; HPOC); and secondly, to inform the design of late-stage phase II trials in naturally infected adults slated for the first half of this year, although in that trial delivery will be to the lung by nebulizer (see 13Dec07 Blog: Breaking News: “Alnylam Reports on the Safety of Inhaled RSV-01”).

The question then arises why did they not present any hard data? It is possible that, as often the case in biotech, initial press releases and top-line data are exploited for PR purposes while the warts-and-all data turn out to be less than impressive. However, given their usually well executed PR strategy with which they have earned themselves considerable credibility, I doubt that they would start playing around with the meaning of “statistically significant”.

One possibility is that they are still crunching numbers as to viral titers by PCR and plaque assay as well as symptom scores in the different groups, to then comprehensively present the whole data set at the International Symposium on Respiratory Viral Infections meeting next month in Singapore. Some meetings also do not like it when data are presented beforehand in press releases. In any case, I hope the company makes the full data set available on their website once they are presented. I am particularly curious as to whether they allow insights into the mechanism of action of this drug.

Another maybe more intriguing possibility for the strange lack of any concrete numbers may have to do with the rather equally unusual post-hoc christening of the trial as GEMINI. Assuming that they would not want a failed trial to be well remembered by giving it a name, one that could be maliciously interpreted as to mean “ambiguous”, the naming of an early stage trial AFTER the results are announced may have a deeper meaning. If the results hold up, GEMINI could be used in the future to refer to HPOC as another momentous de-risking event in the history of RNAi Therapeutics. Equally tantalizing is the possibility that the full data presentation of GEMINI may coincide with the announcement of a co-development partner. Although the initial adult market for ALN-RSV01 may be limited, this program should attract considerable interest as it will give the partner a pioneering role in RNAi Therapeutics, potentially associated with first dips for future respiratory disease programs.

So stay tuned on GEMINI as we are likely to hear it being referred to quite more often in the future. January 23, 2008, may have been another important milestone in the development of RNAi Therapeutics. In the meantime, the prize goes to he/she who can figure out first what the acronym GEMINI stands for*.

PS: I would guess that today’s results will have triggered milestone payments from the Novartis and Roche collaborations.

[* Update May 2, 2008: It is becoming clear now that GEMINI stands for the intention of the study to both show a) proof-of-concept for RNAi efficacy in man, and b) further evaluate the safety and efficacy of ALN-RSV01 for the RSV antiviral development program.]

Breaking News: Alnylam Reports on the Safety of Inhaled ALN-RSV01

The eyes of the RNAi Therapeutics world are on Alnylam as they are approaching critical clinical milestones for their lead development program ALN-RSV01 for the treatment of respiratory syncytial virus infection.

Before initiating phase II studies in naturally infected adults, the company seeks to gather more information first on the pharmacodynamics of RSV-01 in an experimental infection model where the siRNA is administered by nasal administration (top-line data expected early 2008; company reported patient enrollement complete now), and second from the safety of an inhaled version of RSV-01, the eventual route of administration, in healthy adult volunteers. Results from the latter phase I trial were just reported at the 18th Annual Drug Delivery to the Lungs meeting in Edinburgh, Scotland.

This study included 109 subjects, 71 of whom received siRNA either in a single dose (0.1mg/kg to 3mg/kg) or multiple doses (0.01mg/kg to 0.6mg/kg once daily for 3 days). The safety data were encouraging as there were no serious adverse events reported. Nevertheless, the press release mentioned a “mild to moderate flu-like adverse event” at the higher dose group in the single-dose arm. While this is pure speculation, this reminds me of the fact that RSV-01 is an unmodified siRNA and should therefore be more prone to elicit cytokine responses, an area Alnylam by the way is taking quite seriously as they actively seek to recruit scientists working in the field of immunostimulatory nucleic acids, which by the way may be accelerated by the recent acquisition of Coley by Pfizer. Indeed, I would not be surprised if part of the remarkable antiviral efficacy of RSV-01 is based on the siRNA acting as an isiRNA, the term with which Gunther Hartmann from Bonn describes small interfering RNAs with immunostimulatory properties.

In order to find the best therapeutic window, Alnylam has also been working on comparing the pre-clinical efficacy of single-dose versus multiple dose RSV-01. Gratifyingly, for the same amount of total siRNA administered, multiple dose administration was significantly more potent. This means that a good strategy of increasing antiviral efficacy while at the same time decreasing the risk of eliciting flu-like effects may be to choose multiple dose administration for the phase II studies of naturally infected patients.

Another interesting point mentioned in the press release is that siRNA delivery as evidenced by siRNA plasma levels was remarkably efficient compared to some pre-clinical data. Well, I guess had they not observed siRNA in the plasma, then the press release would have stated that avoiding systemic exposure was an additional safety feature of RSV01. This is how the phase I nasal data were interpreted. In any case, this is reminiscent of recent data for systemic siRNA administration to the liver where the efficacy and duration of RNA silencing in non-human primates was above expectation based on small animal experience and may have in fact contributed to the side-effects observed at the higher doses. But before actual plasma levels are reported, it is premature to speculate whether systemic siRNA administration by inhalation should be considered for other indications.

Overall, while the results indicate that the development of RSV-01 is not without risk, Alnylam continues to demonstrate that by conducting a wide-ranging scientific program supporting the compound, it will allow them to choose the most promising development path. The apparently efficient delivery efficacy via nebulizer makes me quite bullish about the antiviral efficacy of ALN-RSV01 so that safety should be the focus of future studies.

Impressions from the 3rd Annual Meeting of the Oligonucleotide Therapeutics Society: Day 2

The second day of the Meeting was filled with cutting-edge science related to RNAi drug development with emphasis on RNAi mechanism, delivery, and cytokine responses triggered by various nucleic acid classes.

Tuschl kicked off the day by presenting data on the systemic identification of Argonaute interaction partners (mostly published data) and a new approach towards identifying microRNA targets. In contrast to microRNA target identification algorithms which heavily rely on sequence conservation, Tuschl and colleagues identified RNAs pulled down in Argonaute immuno-precipitations and then sequencing. Validating the approach, these RNAs were enriched for sequences with seed targets of the most abundantly expressed microRNAs in the tested cell line (1.8x enrichment over random). He finally reported on small molecule screens aimed at identifying inhibitors of microRNA maturation, however, with less success. It appears to me that he should stick to his guns and work at inhibiting microRNAs and their precursors instead by nucleic acids, such as antagomirs, which have proven to be much more potent and specific inhibitors of microRNA activity.

Ingo Roehl (Roche Gmbh, former Alnylam Europe) gave a nice presentation on progress in the development of analytical chemistries to support DMPK/PD (Drug Metabolism and Pharmacokinetics/dynamics) studies of RNAi Therapeutics. It was encouraging to see significant improvements that now allow siRNA and endogenous microRNA quantitations in the picomole-femtomole range in biological samples at high-throughput. In contrast to methods used by other groups, their coupled HPLC mass-spec setup allows for the discrimination between intact siRNAs and their degradation products. However, further improvements in sensitivity are desired as the potency of siRNAs means that biological activity can be observed long after siRNA levels drop below the level of quantitation.

Peter Linsley from Rosetta Inpharmatics (a subsidiary of Merck), which gained early “notoriety” for being the first group to point out the problem of off-targeting by siRNAs some years ago, highlighted the dilemma posed by the fact that the off-target signature of a given siRNA is extremely different in mouse and man. This means that mice will not function as a safety model for off-target toxicity studies. Furthermore, by limiting oneself to cross-species specific siRNAs for the sake confirming the treatment in the animal model, many siRNAs with better potencies and off-targeting signatures in humans may ultimately be missed. Also of interest is the fact that by leveraging its genomic expression profiling capabilities, Rosetta Inpharmatics has now established microRNA overexpression signatures for over 150 microRNAs, raising the question when Merck will officially announce its entry into the microRNA therapeutics arena.

Following on from his identification of SID-1 in being required for systemic RNAi in worms by acting as an siRNA channel, Craig Hunter (Harvard University) presented data to support a model in which siRNAs enter and exit cells via SID-1 by diffusion, whereas other proteins such as SID-2 are involved in binding RNAs so as to increase their concentration close to the siRNA channel. That this is relevant for the development of RNAi therapeutics is highlighted by the recent Nature Biotech paper by Dr. Stoffel from the ETH Zurich and Alnylam where data suggested a role for SID-1 in taking up siRNAs following docking of siRNA-studded lipoprotein particles to their cellular receptors. In addition to walking his audience the published data-heavy paper, Stoffel stated that he saw no reason why it should not be possible to create artificial lipoprotein particles containing siRNAs and targeting agents.

Instead of Dinah Sah or David Bumcrot presenting Alnylam’s progress on cancer RNAi therapeutics, Rachel Myers stood in to give a general overview of the approach Alnylam takes to RNAi-based drug development. Since little new primary data was presented, I thought it is noteworthy that both Ingo Roehl and Myers gave scientific credit for the development of the hotly contested SNALP development to Protiva Biotherapeutics and that the delivery technology used in last week's Nature paper on microRNA competition by siRNAs was called "Alnylam proprietary”. Also, it was a great relief for me to hear that Alnylam had reproduced all of Sailen Barik’s data on the RSV-RNAi treatment paradigm, including efficacy of siRNAs after RSV infection. Indeed, siRNAs reduce viral titers up to 3 days after infection in a mouse model where maximal viral burden is seen by day 4 of the infection. Moreover, illustrating that the upcoming proof-of-concept studies for RSV in the experimental infection model is not merely an academic RNAi therapeutics de-risking exercise, the patients that Alnylam expects to treat with ALN-RSV01 will have infection in the upper respiratory tract with little or no exposure in the lung. Consequently, ALN-RSV01 will be a combination of treating upper respiratory tract infection and prophylaxis for the lung. Everybody, of course, is quite restless now to learn about the outcome of the phase II experimental infection results which appear to be on track and from which will be presented in detail early next year without precluding a PR on the results in December.

Before the session on RNAi/oligonucleotide delivery, a number of speakers spoke about recent findings on the immunogenic properties of various forms of RNAs, including siRNAs. The importance of considering the expression pattern of the main oligonucleotide receptors (the endosomal TLR-3,7,8,9 and the cytosolic RIG-I and MDA-5), their localization, and their exact ligands became evident and will inform modification strategies aimed at avoiding the induction of unwanted cytokine responses following siRNA administration which is something that Rachel Myers noted Alnylam is still trying to get a better handle on (see the delay in the pandemic flu program). Other groups, however, such as Gunther Hartmann’s group from Bonn, embrace the immunogenic properties of some, particularly unmodified siRNAs and would like to combine it with their silencing activity for treating cancer and viral infections. Some encouraging data in that regard were presented.

Song Li (Pittsburgh) presented the use of neutral lipids for delivering a variety of oligonucleotides to the pulmonary circulation. Like others at the meeting, this approach illustrates a trend away from using cationic liposomes which have been associated with interacting with components of the blood and may trigger certain toxicities. Delivery to endothelial cells of larger vessels and capillaries was particularly efficient, and his group is currently collaborating with ISIS on targeting endothelin-1 for treating hypertension. Encouragingly, he was able to report on good gene knockdowns and quite impressive in vivo efficacy data, such as the reduction of hypoxia-induced right ventricular hypertrophy.

Similarly impressive in vivo efficacy data were then reported by Klaus Giese from Silence Therapeutics, mostly located in the host city of Berlin. Without showing all the controls, tumor burden, metastasis, cell proliferation were all strongly reduced following systemic delivery of siRNAs in a number of mouse tumor models. Although I cannot agree with his repeated claims on the uniqueness of their Atu-siRNAi design and related IP claims, the Atuplex delivery technology certainly deserves more credit. Unlike other lipid-based delivery methods, siRNAs here associate with the liposomes externally thus allowing liposomal charge to be modulated from cationic, via neutral, to anionic. Of practical importance, these particles can be lyophylised for storage and shipping and then resuspended without loss of silencing activity. Progress has also been made on lung delivery and a number of pre-clinical programs are being pushed forward into the clinic.

The founder and CEO of the nucleic acid delivery company Novosom, Steffen Panzner, continued the string of impressive in vivo efficacy data in a mouse model of RA (inflammation of the paws) following delivery of their proprietary siRNA-loaded Smarticles, a charge-reversible liposomal delivery technology. One problem of using the more desirable, in terms of safety, neutral and anionic lipids, is their reduced siRNA binding affinity. Novosome’s amphoteric liposomes circumvent this problem by binding siRNAs at low pHs, at which point they are positively charged, and rapidly shifting them to higher pHs for closing the liposomes. During this process, most of externally bound siRNAs are shaved off, with the additional benefit of consequently minimising endosomal exposure of the siRNAs which may trigger TLR7-mediated cytokine responses.

The scientific day was closed by a talk from Alan Sachs of Merck who set out to emphasise that despite a dearth of recent information on Sirna’s/Merck’s preclinical and clinical RNAi therapeutics pipeline, that they were more than ever committed at developing RNAi therapeutics. Unfortunately, this was not followed by a presentation of primary scientific data and specific examples, but rather how Merck thinks about RNAi drug development in general and repeated appeals of please partnering with Merck, particularly in the area of targeted siRNA delivery. It appears to me that Merck would like to rival Alnylam in accessing the best minds in the RNAi drug delivery field and is offering to send out their siRNAs for free so that others can formulate them and report back on their findings without any strings attached as to the use of these results. Sachs feels that Merck, through their expertise on the genomics and genetics of gene expression through Rosetta Inpharmatics, that it knows best which targets are most suitable to go after for drug development. Since siRNAs allow essentially any gene to be targeted, he like other feels that this is the natural drug development platform to be harnessed and thinks about 21 month development timelines from gene identification to preclinical proof-of-concept given the availability of suitable biomarkers. While searching for more targeted delivery solutions and collaborators, Merck is meanwhile establishing a platform for siRNA delivery to the liver and, encouraged by ISIS' 301012 results, is clearly motivated in targeting ApoB100 with siRNAs for the treatment of hypercholesterolemia. I would not be surprised at all, to see them join the PCSK9 frenzy as well. Being second in a $50-60B should not be that bad.
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Can RNAi Therapeutics do a Better than Monoclonal Antibodies in RSV Infection?

Arguably, the next main proof point that looms for RNAi Therapeutics is proof-of-concept that RNAi can be made to work in humans. Alnylam takes its first shot at this in their randomised, placebo-controlled, double-blinded phase II study for the treatment of respiratory syncytial virus (RSV) infection. In this study, volunteers infected with an attenuated form of RSV will be treated with ALN-RSV01, an siRNA targeting a conserved region of the nucleocapsid (N) protein mRNA, and its effect on viral load and symptoms evaluated. Results from this trial, initiated last month (see Blog from 25 June 07: “Alnylam Progresses RSV RNAi Program into Phase II Clinical Studies”) are expected by the end of this year.

Currently, the only effective drugs in addressing RSV are neutralising antibodies that were developed by MedImmune (now AstraZeneca). These monoclonal antibodies (MAb) are directed against the F-protein on the surface of RSV and block cellular entry of the virus. Importantly, whereas these MAbs are used for the prevention of RSV infection in a small at-risk population, premature infants, ALN-RSV01 is geared towards the treatment of RSV.

Numerous studies have shown that the effect of RNAi, and probably any type of drug, on viral replication is most potent when given around the time of infection. I therefore wondered why ALN-RSV01 should succeed in the treatment of RSV when other drug classes such as MAbs have failed. Indeed, my own literature research confirms that MAbs have been tested in animal models for the treatment of RSV, but were found to lack sufficient therapeutic activity.

A study by Mejia et al. [Antimicrobial Agents and Chemotherapy 49: 4700 (2005)] compares 50mg/kg of the latest generation of anti-RSV MAbs when given either before or after viral infection in mice, and finds that on almost all accounts (viral load, inflammation, lung pathology) MAbs were only effective when given shortly (24 hours) before infection. The only assay that showed an effect when MAbs were given 48 hours after infection was a viral plaque forming assay which may reflect the presence of neutralising antibodies in the assay.

Bitko et al. [Nature Medicine 11:50 (2005)] on the other hand showed in an almost identical mouse model that intranasally delivered siRNAs had a profound effect on RSV replication even when given after viral infection. Moreover, 3.5mg/kg doses already proved very effective. Importantly, siRNAs were able to limit viral replication even when given up to 5 days after viral infection, the time when the acute phase of RSV peaks in this particular model. This is crucial in the clinical setting where the treatment benefit will likely be optimal if RNAi therapy can be initiated before acute infection has peaked. The authors then go on to show that on a number of counts (respiratory rate, pathology score, leukotriene production), anti-RSV siRNAs almost abolished any pathological signs of the disease.

These results suggest that while current MAbs are potent in reducing the initial infection by neutralising the interaction of the virus with the host cell, they are ineffective in preventing the subsequent spread of the virus. This could be due to the kinetics of viral re-infection in close proximity to the next host cell. By contrast, unless they target host surface receptors, siRNAs will not be able to prevent viral infection. The can, however, prevent and limit the ability of the viral genomic RNA to replicate and/or inhibit virion formation. Although Bitko et al. have not measured viral RNA levels directly, it is very likely that these were also reduced, and treatment with siRNAs even after the acute phase of infection may have a clinical benefit on RSV co-morbidities such as asthma/wheezing later in life.

Alnylam Progresses RSV RNAi Program into Phase II Clinical Studies

Alnylam Pharmaceuticals, arguably the leading company in RNAi Therapeutics, today announced the start of phase II clinical studies for ALN-RSV01, an siRNA for the treatment of Respiratory Syncytial Virus (RSV) infection. RSV infection is the leading cause of infant hospitalization in the US and a significant risk factor for the immune-compromised and elderly. Although a neutralising antibody exist for the prevention of RSV infection, no drug has been shown so far to be effective in the treatment of RSV infection.

Today marks another milestone in the rapid, but at the same time circumspect development of ALN-RSV01 from the test tube to the clinic. It demonstrates just how quickly it is possible to develop RNAi as antivirals. This is because the viral sequence alone allows us to start designing and testing siRNAs for their antiviral activity. It is not surprising therefore that the NIH and Department of Defense is interested in fostering this technology for the fight against bioterrorism and pandemic flu. Indeed, Alnylam may leverage their experience in RSV and translate it into their pandemic flu program for which an IND is planned by the end of the year. Another company, Nastech, of Bothell, Washington, is also in the discovery phase of an RNAi therapeutic for pandemic flu, as was Sirna Therapeutics before their acquisition by Merck.

Alnylam’s first phase II study is an experimental challenge study in which volunteers are nasally infected with an attenuated strain of RSV and ALN-RSV01 given either before or after infection. The goal of this randomised 90 patient double-blind, placebo controlled study is to first establish safety, but more importantly antiviral efficacy as measured by incidence of infection, viral titer, and symptomatic differences. If successful, this would constitute human proof-of-concept of a human RNAi Therapeutic and represent another major de-risking event on the path to establishing RNAi as a whole new platform for innovative drugs.

PS: In another RNAi pipeline event, Intradigm, an RNAi delivery company based in Palo Alto, California, announced today that they have officially selected ICS-283 as a development program for cancer. ICS-283 is designed to target cancer angiogenesis and phase I studies are expected to start in 2008.

In Focus: Alnylam Establishes RSV Experimental Infection Model

Only one week following the announcement by Nucleonics of its intention to start HBV RNAi phase I studies, Alnylam presented further pipeline progress from their phase I Respiratory Syncytial Virus (RSV) RNAi program. As more and more programs reach the clinical stages, expect to hear about clinical progress of RNAi Therapeutics with increased frequency.

Alnylam and their collaborators from the University of Tennessee and Meridian Life Science derived a non-pathogenic RSV strain in high enough amounts so that it could be used to experimentally infect healthy adult volunteers. They showed that infection could be achieved in 72% of the subjects with incubation times and duration of infection that should allow the investigators to test the antiviral activity of ALN-RSV01. Drop-out rates were excellent with 35 of the 36 volunteers completing the study and no major adverse event reported. The company consequently announced that it would begin phase II experimental challenge studies this quarter.

The experimental infection studies are part of a wider well designed and innovative development program that places emphasis on feasibility in the early, therefore less expensive stages. Importantly, today’s results show that RSV infection can be quantified reliably across a number of platforms. This offers the prospect of obtaining statistically significant efficacy data already by the end of this year. The results from the planned phase II studies will be watched closely by the whole field as they would represent first human proof-of-concept of an RNAi Therapeutic. For those interested in investing in this area, expect such data to be a major value driver for Alnylam’s share price and beyond.

Ultimately, however, ALN-RSV01 will have to show safety and efficacy in the lower respiratory tracts of RSV infected infants. While the soon to be started experimental challenge studies will test an siRNA formulation nasal spray in the nose/upper respiratory tract, aerosolised siRNAs will have to be used later. In addition to mastering delivery, one problem particularly relevant in the treatment of RNA viral infections is the emergence of escape mutants. It is of note therefore, that although ALN-RSV01 was highly effective in reducing viral titers in tissue culture, knockdown efficiency was not compromised following repeat administration of the siRNA and no mutation around the siRNA target site was found.