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.

Preventing Heart Disease with RNAi Therapeutics

High blood cholesterol, a major risk factor for heart disease, already is an enormous public health problem that is likely to worsen given current lifestyle and demographic trends. Fittingly, statins, a class of small molecules that inhibit cholesterol synthesis and therefore have a wide range of effects that together lower the risk of developing heart disease, are probably the best-selling drugs on the market today. However, many patients still do not achieve their cholesterol goals and there is significant demand for new innovative treatment options.

An RNAi Therapeutic alternative, due to the ability to target almost any gene, is particularly interesting because of well validated targets, but which have proven refractory to targeting by the existing drug classes. These genes are not directly involved in cholesterol synthesis and targeting them should be synergistic with statins. Currently the most interesting gene targets are ApoB100 and PCSK9. ApoB100 is the sole protein component of “bad” LDL cholesterol and is produced in the liver. Notably, targeting ApoB100 by RNAi in the using cholesterol-conjugated siRNAs in 2004 by Alnylam scientists was also the first demonstration of gene silencing following systemic administration of siRNAs. This study not only showed considerable reductions of ApoB mRNA and protein, but also the hoped for decrease in blood LDL cholesterol. I should add that clinical data from phase I and II trials conducted by ISIS Pharmaceuticals using antisense oligo technology further document the promise of ApoB100 as a target for hypercholesterolemia. It will be interesting to follow their further clinical progress, but I expect siRNAs to do even better, because of increased specificity and potency thus allowing for lower amounts of nucleic acids to be administered.

Two years later after the demonstration of systemic RNAi in mice, Alnylam scientists then reported even enhanced ApoB-100 silencing and improved lipid profiles in monkeys, this time using liposomal formulations originally developed by Protiva Biotherapeutics. These and similar liposomal formulations have proven to be very efficient for liver gene knockdown in general and are now being pursued by a number of companies in preparations for the first systemic RNAi clinical trials. Unfortunately, however, their promise has also led to legal haggling as to who owns the IP behind this delivery technology. Companies involved in this dispute involve Protiva, Inex Pharmaceuticals, and Sirna Therapeutics/Merck and I hope that legal issues will not do further damage to the development of this promising delivery technology. No matter who owns the commercial rights to the technology, Protiva scientists have to be credited with this major achievement.

Interestingly, despite their publication record on ApoB100, Alnylam decided to target PCSK9 for the treatment of hypercholesterolemia. Although I cannot exclude that this move is partly due to a deal with one of their collaborators in siRNA delivery, PCSK9 has a lot riding for it. In fact, they are pursuing this program in collaboration with scientists from UT Southwestern Medical Center that arguably are world-leading in the genetics of hypercholesterolemia. PCSK9 itself is a protease that degrades LDL-receptors (LDL-R). LDL uptake by the liver is important for clearing LDL in circulation and it is expected that increasing LDL-R levels by suppressing their inhibitors should lower LDL cholesterol. Indeed, data presented at this year’s Keystone Meeting support this thesis. However, it should be kept in mind that many drug development projects fail, not because the drug fails to reach its target, but because of side-effects. Side-effects are a particularly important consideration for drugs that have to be taken chronically as is often the case for hypercholesterolemia. So one of the major questions here is whether long-term downregulation of PCSK9 can have adverse consequences. Here, the genetics of PCSK9 are compelling: Naturally occurring mutations in the human population that increase PCSK9 activity have been shown to increase LDL and lead to hypercholesterolemia, while those that inactivate it lower LDL dramatically- without any obvious detrimental consequences! Of course, compensation mechanisms cannot be excluded, but this is probably as good as you can get with choosing a target based on human genetics.

In summary, due to the availability of excellent “non-druggable” targets and the ability to knockdown genes in the liver with current delivery technologies, RNAi Therapeutics are a promising strategy for treating hypercholesterolemia. Alnylam is expected to initiate phase I studies in the second half of this year, and I would not be surprised to see further studies being initiated in the near future by Protiva or Inex (mere speculation here though). The major obstacle for these trials that I see are side-effects due to the liposomal formulations, and my advice would be to carefully characterise them in animal models before committing to phase I instead of simply bowing to investor expectations.

RNAi and Bird Flu

Every year labs around the world are racing to get vaccines ready for next year’s flu season. Even if the logistical nightmare can be managed and complicated vaccines are manufactured in time, this is no guarantee that the vaccine protects against the actual flu strain that season. This is because even as we observe the new strains emerging from East Asia, once it has reached our shores, the virus is likely to have changed his outer surface making it more difficult to be recognised by the immune system. That’s what viruses have learned to do anyway. If seasonal flu is challenging enough to predict, then developing a vaccine for a pandemic flu strain that had been generated by considerable genetic drift, is a daunting task.

Here, using RNAi, and in principle other nucleic-acid based therapies for that matter, offers a number of considerable advantages. Significantly, rather than going after a moving target, RNAi allows us to aim at flu genes that are not as mutable given the structural constraints of their encoded proteins for viral replication. This means that even if a re-assorted bird flu might look quite different on the surface, many parts inside of the virus will look very similar between even divergent flu strains. These conserved parts, or better their underlying RNAs, can be computationally predicted, and it is then very straightforward to design small interfering RNAs (siRNAs) against these RNAs well before we even know that there will be a bird flu pandemic. This can be done with high statistical confidence.

Of course, this strategy is not only applicable to bird flu, but any other emerging viral threat. A number of high-quality scientific papers have been presented documenting the feasibility of this approach. Intradigm e.g. has shown in 2005 in a timely manner that siRNAs could protect monkeys from severe lung damage due to SARS. Research teams from Nastech and Alnylam have similarly documented the potential of RNAi for flu in animal models. The US government is funding much of the research and may eventually decide to stockpile siRNAs for pandemic bird flu. Concomitantly, manufacturing capabilities are also being developed.

It is further encouraging that the lung which has been exploited for eons by viruses to infect the host now emerges as a good organ for drug delivery. Specifically in the case of RNAi, it came as a rather pleasant surprise that even unformulated siRNAs could readily get into lung cells and silence their target genes. RNAi therefore not only holds promise as a rapid response to viruses, but also other diseases of the airways such as asthma and COPD. As I write, many of these conditions are the subject of pre-clinical RNAi programs and one program by Alnylam for RSV (respiratory syncytial virus) has already reached the clinic. Data from their viral challenge studies in the second half of this year are widely anticipated, and if successful, would present the first proof-of-concept for a human RNAi Therapeutic.

The RNAi Patent Landscape

Patents are essential for drug development as they protect the significant investments that have to be made in order to obtain marketing approval from the relevant regulatory agencies. Common estimates put the cost of successfully developing a drug at around $1 billion. A whole technology platform such as RNAi would suffer and be unable to attract sufficient funding if the scientific discoverers and institutions failed to file patents and license them to organisations capable of taking RNAi to the clinic. Luckily, fundamental patents have been filed and have been either exclusively or non-exclusively licensed. What are they?

According to folklore, Fire and Mello, the discoverers of RNAi in worms, had to be “encouraged” by the NIH to spend the time to file a patent for RNAi. This resulted in the Fire and Mello patents that can be non-exclusively licensed by almost whoever wants to for a nominal fee. While their science was impeccable, commercially their patents suffer from the fact that originally the dsRNA inducer length was defined as 25bp and above. Additionally, even if efforts to bring this size down to the relevant 21 and upward size succeed, this will not give licensees automatically the freedom to operate as it does not claim the use as a therapeutic. For this, the Tuschl patent series are essential. These are based on Tuschl’s seminal and non-obvious discovery that small double-stranded RNAs (siRNAs) of 19-23 base-pair length are the mediators of RNAi gene silencing. It appears that Alnylam, of which Tuschl is a co-founder, can claim rights to most of the claims in the series, although the Tuschl I series can be claimed by Alnylam, Sirna Therapeutics (now a Merck subsidiary), and CytRX alike. This is because one of the four academic institutions involved in the licensing of Tuschl I decided to go it alone and license it to CytRX and and Sirna in addition to Alnylam. It seems strange to me that one institution alone can do this without the apparent support of the other parties, but I will leave this to the lawyers. The Kreutzer-Limmer patents, covering double-stranded RNAs for the purpose of gene inhibition, may also turn out to be an important piece of the puzzle and were acquired early on by Alnylam through its acquisition of Ribopharma AG.

There are a number of other pending and granted patents in the RNAi field, including siRNA manufacturing and modification patents that will be important in the actual drug development process. However, I view them as secondary albeit important and meritorious patents when measured on an innovation scale. Efforts are also being made to circumvent the need for prototype siRNAs by using either their biological precursors or even variants such as blunt-ended small double-stranded RNAs (apparently it works!), however their exact merit remains to be evaluated. In this context, I am encouraged by recent Supreme Court decisions that emphasise the importance of innovation and non-obviousness in a patent.

Note: The discussion did not cover DNA-directed RNAi that do not involve synthetic siRNAs.

In Focus: FDA allows Nucleonics to start phase I trial for HBV RNAi

Today, privately held Nucleonics announced it has received permission from the FDA to start phase I clinical trials using RNAi for the treatment of Hepatitis B Virus. I will take this as an opportunity to highlight potential merits and disadvantages of that particular program, introducing some general terms along the way.

Unless most other companies that develop RNAi therapies using synthetic small interfering RNAs (siRNAs), Nucleonics employs DNA-based vectors that direct the expression of so called hairpin RNAs that are then further processed by the endogenous RNAi machinery into small RNAs that are functionally identical to the synthetic siRNAs. This approach, also known as DNA-directed RNAi (ddRNAi), may be advantageous in that it potentially allows for a longer treatment effect due to the potentially longer activity of a DNA vector. RNA, by contrast, is a more short-lived molecule. In the case of Nucleonics, the "naked" DNA is delivered to the liver formulated with cationic lipids. Since hairpins are very short in gene-terms, a plasmid may harbor multiple hairpins and Nucleonics' has 4 of them. As each hairpin targets a different RNA of HBV, the multipronged approach should help minimise drug-resistance which is often seen with viral therapies based on inhibiting a single target. Indeed, other RNAi companies are likely to pursue similar multi-target approaches in their viral programs, Alnylam's flu pre-clinical program being one example.

So far the theory looks promising. However, I have a number of concerns with Nucleonics' program (these were also recently highlighted during a pre-IND meeting with an FDA advisory panel). The major problem is that the company did not have convincing data about in vivo efficacy in animals. They argued that this is due to a lack of appropriate pre-clinical animal models, but I would argue that those, e.g. mice that carry HBV in their genome, exist and should have been used for this purpose. Efficacy studies were consequently limited to tissue culture experiments and in a co-transfection "in vivo" model which really is nothing more than a glorified in vitro system. This means that although HBV silencing in such a model may approach 100%, this is simply because the HBV and ddRNAi vectors tend to go into the same cells during co-transfection. It appeared from their data, however, that only a small fraction of the liver cells received the ddRNAi (and HBV) plasmid, unlikely to be enough to have a therapeutic impact in a patient that carries HBV in a much larger and non-overlapping fraction of cells in the liver. It is likely that this is because in order to be active the DNA needs not only to get into the cell but also the nucleus, which is generally inefficient with non-viral DNA vectors. SiRNAs, however, have the added delivery advantage in that they are active in the cytoplasm and do not have to reach the nucleus. Their smaller size compared to DNA may also help.

In summary, while a lot of the scientific rationale for the trial appears sound, I see delivery as a huge hurdle for this particular RNAi program and am therefore quite skeptical.
For those interested in learning more about Nucleonics' strategy, please visit http://www.nucleonicsinc.com/products/hepb.html

RNAi and the Eye

One of the criticisms one encounters when evangelising about a hot new technology from the lab is that it hasn’t been proven yet ‘in the real world’. Given the glut of medical breakthrough announcements in the daily press, investors know all too well that much of those won’t ever have a clinical impact. Validation is the key, and while RNAi has seen many pre-clinical, and some clinical validations, a drug approved by the FDA has to be the gold standard. So when will we see the first RNAi drug on the “market”? Unless the government started stock-piling siRNAs against bioterrorism threats such as Ebola or a bird flu pandemic (note there is considerable effort ongoing in this area), my conservative guess would be 2010-2011 with the application being age-related macular degeneration (AMD). Currently, it is a race between 3 drugs in phases I to III for the glory of being first to have an RNAi-based drug approved.

That the eye and wet AMD should be the subject of so much early interest should not be surprising. AMD is considered to be a low-hanging fruit as the target cells are relatively easily accessible, the eye as an immune-privileged organ, and the existence of well validated targets. Notably, there are two other nucleic acid-based therapeutic classes of which the only approved drugs is one for a condition of the eye each: the antisense oligo Vitravene of ISIS Pharmaceuticals for CMV retinitis in people with AIDS, and the RNA aptamer Macugen of OSI Pharmaceuticals recently approved for wet AMD. Like Macugen, one of the siRNAs by Opko (formerly Acuity Pharmaceuticals) targets VEGF. However, more recently monoclonal antibodies by Genentech targeting VEGF have become a more popular and effective treatment option. This illustrates how fast the competitive landscape may change and the commercial risk from competing technologies. RNAi companies reacted in different ways to this situation. Realising that the market for AMD is becoming increasingly crowded, the Alnylam (www.alnylam.com) management made the brave and early decision to stop its VEGF-AMD development program and instead focus their precious early efforts on so called non-druggable targets. These targets are well validated in biology, but either because of their localisation or structure cannot be reached by non-RNAi drug classes. The next strategy is exemplified by Sirna Therapeutics. Instead of going after VEGF, they took the decision to go after another protein in the same pathway, namely the VEGF receptor 1. This target may have increased potency as it is thought to be required for the functioning of yet another signalling molecule implicated in AMD, the placental growth factor(PIGF). Also because it is a target different from VEGF itself, VEGF-R siRNAs may act synergistically with the now established VEGF therapies. In general, RNAi may be used synergistically with other drug modalities due to its unique mechanism of action. Sirna then went one step further and partnered with Allergan, a company with experience in treating eye-related diseases. In order to better position their drug, they put considerable effort into addressing one of the short-comings of existing therapies, namely the need for relative frequent needle injections into the eye. Each needle injection carries the risk of treatment-related complications, including blindness. Indeed, RNAi may be able to provide a solution for this as is indicated by early phase I pharmacokinetic data from Sirna and studies of RNAi knockdown in the liver by Protiva and Alnylam scientists that show long-lasting RNAi silencing effects following a single application of siRNAs. When combined with a slow-release formula, this property may eventually allow for once-every-6-months injection treatment regimens. The last strategy that I would like to highlight here is to go after completely new targets- really one of the great strengths of RNAi after all. Although carrying a higher development risk, Quark Biotech embraced this philosophy and is now targeting REDD1, a gene that they identified as playing a pivotal role in the progression of AMD.

Watching the drugs navigate through the complicated drug approval process will be fascinating. Let’s hope that these pioneers will be rewarded for the risk taken, but also caution them not to take any short-cuts in order to be the first to claim RNAi glory. For me meanwhile, there is little doubt about RNAi drugs in the future. Such a potent mechanism to regulate genes beckons to be used in the clinic, the only question therefore is therefore how many and how soon.

Tip: For those interested in investing in RNAi companies, the message board on Investor Village about Alnylam (symbol: Alny) is a great resource and discussion forum: http://www1.investorvillage.com/smbd.asp?mb=569&pt=m&clear=1

RNAi Therapeutics Companies

There are a few dozen companies now, small and large alike, that have started developing drugs based on RNAi. While most of them are in the pre-clinical stages, in 2004 Acuity Pharmaceuticals (soon to be known as Opko) was the first to start phase I trials for age-related macular degeneration (AMD). Since then 5 more programs have entered the clinic: 2 additional ones for AMD (Sirna Therapeutics, acquired by Merck in 2006 for the handsome sum of $1.1 billion; and Quark Biotech), and one each in RSV (Alnylam Pharmaceuticals), diabetic macular edema (Acuity), and acute kidney injury (Quark Biotech).

Due to reasons discussed in the previous post, almost all of these programs are Direct RNAi programs, i.e. the siRNA is administered close to the diseased site. All except for Acuity's program are also with siRNAs that have been chemically modified (to enhance stability etc), and it remains to be seen whether Acuity's strategy to plunge into the clinic first was a wise one. Sirna Therapeutics soon followed suit, but I think took the right decision to invest time to carefully think about how to position their potential product in the more and more crowded AMD market. Sirna Therapeutics was also the first public company based on RNAi. Formerly known as Ribozyme Pharmaceuticals, they leveraged their experience with RNAs to build an operation that had all the tools to to build a decent IP portfolio and quickly enter the clinic. This IP portfolio is mostly based on chemistry and targeting many genes one by one such that it could claim exclusivity for targeting those genes with RNAi. It remains to be seen, however, how this brute force approach will hold up in the patent courts. Also, ISIS pharmaceuticals may contest some of their chemistry claims. However, the strategy has paid off extremely well at least for those that engineered it with Merck's takeover of the company last year for about 50x the price it had been valued before it committed to RNAi. SR Pharma, known as of today as "Silence Therapeutics", of the UK, looks as if it was seting itself up for a similar sale with an almost 8-fold price appreciation since last year.

The brightest star in the sky of RNAi Therapeutics by far, however, is Alnylam Pharmaceuticals. This is the company whose scientific founders were seminal in the development of RNAi and microRNAs, particularly in humans. Consequently, the company sits on an unparalled exclusive IP portfolio that gives them freedom to operate and pursue highly lucrative deals with Big Pharma and Biotech. These deals should help it through the development phase and have brought in already well over $100M. All this is managed by a seasoned team, led by CEO John Maraganore, that has considerable experience in developing succesful biotech companies (many of them hail from Biogen). It is also the company mentioned by the Karolinska Institute in their explanation for the 2006 Nobel Prize Award to Fire and Mello, as having demonstrated the therapeutic potential of RNAi through ground-breaking studies. These studies represent important de-risking events that continue to attract academic and indrustrial collaborators and important investments, including the RNAi Therapeutics field as a whole.

Other notable companies involved in RNAi Therapeutics include Benitec, a pure-play Australian company that uses DNA-based RNAi vectors to tackle a range of viral diseases, Nastech Pharmaceuticals (not a pure-play; focuses on siRNA delivery and Dicer-substrate technology), and large pharmaceuticals such as Merck, Novartis, GSK, and Pfizer, and probably many more knocking on the doors of Alnylam and co.