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.

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.