RNAi Therapeutics Trends from the International Liposome Society Meeting 2009

Christmas to me means going to London to learn about what liposomes have in store for RNAi Therapeutics. As you know, liposomes have emerged as the leading platform for the systemic delivery of RNAi Therapeutics and offers considerable promise for diseases of the liver, solid cancers, and after that potentially enhanced vaccines, infectious disease and immune cell-related disorders. The fact that liposomal drugs are on the market already should further speed up its development, not least from a regulatory point of view.

The liposome field appears to be split between between cancer and vaccines about 2:1. Especially the success story of doxil (liposomal doxorubicin) has shaped the field and should partly explain why it is attracted so much towards solid cancers for RNAi Therapeutics. The EPR effect, targeted delivery, tumor vascular biology have been recurring topics and research using various advanced imaging modalities nicely informs the application of liposomal siRNAs in oncology. For example, liposomes (not necessarily unique in that regard) are not uniformly distributed within the tumor, but form a concentration gradient around the blood vessels. Strategies to achieve more uniform drug delivery may involve metronomic dosing in which the drug is administered more gradually at lower dosages such that the simultaneous onset of cell killing in proximity to the vessels will relieve intratumoral pressure so that subsequently arriving liposomes are freer to move within the tissue. Hyperthermia or low-dose TNF-alpha were two suggested strategies for increasing the leakiness of tumor vasculature, also to improve liposomal tumor distribution.

Since the highest concentration is achieved around the tumor vasculature and because there are differences between normal and tumor vasculature, targeting genes within the endothelial and supporting cells has become an attractive anti-tumor strategy. This for example is also the strategy that Silence Therapeutics is pursuing and the lipoplex formulation (siRNAs associated with liposome on the outside, not inside as in a SNALP) for some reason might have its application here.

But once within the tumor, mostly the result of the overwhelming EPR effect, the question becomes how to get the liposome into cells and then release the drug cargo into the cytoplasm in the case of siRNAs. Ligand-targeted liposomes are one answer to increase the cellular uptake of the liposomes. Antibodies are an increasingly popular means (immunoliposomes), although they present certain manufacturing challenges. It is still debated how PEG affects cellular attachment. Some claim that you cannot take up a pegylated liposome, some say you can. The answer may be that it depends on the size of the PEG. This is important for example in the context of a targeted liposome and whether to add the ligand to the PEG or directly incorporate it into the lipid bilayer as a lipid-conjugate, and when and how the PEG should leave the liposome. For efficacy and safety, triggerability of membrane lysis is of course important. Too much charge for example can be cytolytic, so you would like to expose membrane lytic activities in a pH-dependent (endosomal acidification), redox or protease-dependent manner. For the liposome field, this typically means pH-sensitive lipids.

Combined chemo-siRNA therapeutics is another trend that will become important for liposomal cancer RNAi Therapeutics. Combining ever more chemotherapeutics appears to be a game of diminishing returns with triple chemo Rx often not any better than double chemo Rx. This is because of limited mechanism of actions of chemotherapeutics and associated resistance mechanisms. RNAi Therapeutics therefore can either target these resistance pathways directly or bring to the table an entirely new anti-tumor mechanism of action.

I have always found it attractive to apply siRNAs for developing more effective vaccines. While I am not familiar with the literature around virosomes, viral ghost shells, a presentation by Swiss company Pevion Biotech caught my attention in this context. Basically, they are using flu viruses, disassemble them to get rid of the viral RNA and associate protein, and then reassemble them. During the reassembly process, they can then variably add their lipid-anchored antigen of choice and use the re-formulated virus as a vaccine. The fact that our bodies have seen flu virus before apparently helps to obtain a more favorable immunological response. By this, they believe to have broken the potency-toxicity linear correlation that seems to plague adjuvant science. While promising and based on a credible scientific presentation, it still does not appear to be among the most potent adjuvants, so I wonder whether an siRNA targeting the right immunomodulatory gene loaded into the virosome, essentially replacing the viral RNA there, could improve this equation even further. A challenge for nanoparticle-formulated siRNAs for enhanced vaccines is that although they are easily taken up by phagocyotic antigen-presenting cells, the phagocytic pathway usually destroys the siRNA and little escapes into the cytoplasm. The virosome could offer a solution since the flu virus is THE paradigm of how membranes fuse with each other and also explains how an extracellular antigen, the vaccine, can produce a favorable cellular immune reponses in this system. What I noted at the conference is the surprising lack of cross-talk between people using liposomes for vaccines versus those using them for oligonucleotide delivery which is unfortunate in cases like virosomes.

Pevion also seems to be producing good-quality liposomes. For liposomal RNAi Therapeutics, it is probably making the jump from rodents to primates and scalable manufacturing that separates the boys from the men. There are many interesting early-stage strategies, but if the goal is translational medicine, it is of utmost importance to take manufacturing issues into account from the very beginning. Of course, if you have such know-how, this challenge also presents an opportunity/entry barrier that can be capitalized on and should also present a significant barrier for generic competition. A scientist from Gilead told the story of the attempt by a fairly large Argentinian generics manufacturer to copy Gilead’s AmbiSome formulation by just mixing the individual components together and that this went quite wrong.

In order to address that gap between basic and applied science, the opening talk advocated the creation of companies whose sole business model is to take promising academic research and transform it into industrially partnerable technologies. Certainly, this concept appears to be gaining traction and it will be important in how the compete with or complement small biotech companies. The Centre for Drug Research and Development in Vancouver is one such entity that can be put into this category, also with a liposome focus (Pieter Culles e.g.). Vancouver in general can be considered the world's capital for liposomal research, which brings us to the Tekmira, UBC/Alcana, Alnylam triangle.

Pieter Cullis, co-founder of a number of liposomal companies including Tekmira, but now apparently more closely associated with Alcana and Alnylam, gave a nice talk about the rational design of lipids for use within SNALPs. Only small changes in the lipids can have large biological and physico-chemical effects. For example, by just modulating the chemical strength of the linkage of the fatty acids with the lipid head-group, SNALPs of very different potencies are obtained. Yet another objective is the investigation of the pKa-potency relationship which again can greatly impact liposomal performance. Through this, Alcana-UBC, largely driven by two ex-Tekmira scientists have come up with around 100 new lipids which further help to refine the design rules in an interative process. Actually, the rational lipid design approach is the approach Tekmira itself has been taking- probably not surprising given this background and may explain why Tekmira is getting access to these lipids for free without having to fund their development. It also differentiates it from the ‘lipidoid’ approach by MIT-Alnylam. Maybe given the varied performances due to even only subtle changes, huge libraries of lipids are not that desperately needed as was thought just 3-5 years ago. It is also the animal model that would become the bottle neck, as the tissue culture system is of only limited utility in predictively characterizing SNALP performance and mice become the system for the screen. Still, ‘lipidoids’ may offer fundamentally new structures which can be further refined using similar rational design approaches. For the liver, at least in terms of potency, this may not be needed according to Cullis’ back-of-the-envelope calculation that predicts single digit microgram(siRNA)/kg potencies that the technology is approaching for this tissue, an area that is approaching certain theoretical limits.

In summary, the conference, has confirmed my view that liposomal delivery is a good place to be in RNAi Therapeutics at the moment. It may even be time to have a conference entirely devoted to liposomal strategies for RNAi Therapeutics. There is a need, however, to focus on those companies that operate on sound scientific principles and ideally have a track record in being able to produce high-quality/consistent liposomes in a manner that is scalable.

Lipidoids Expand Chemical Space for Cationic Liposome Delivery of RNAi Therapeutics

[Important update, see end of entry]

Following a long series of presentations and publications involving Protiva, Tekmira, Alnylam, and Merck/Sirna, PEG-stabilized cationic liposomes known as SNALPs have to be considered one if not THE most advanced systemic RNAi delivery technology to date. While efficacy was very potent at single-digit mg/kg doses, the major drawback of the first studies on SNALP-siRNA delivery were slight elevations in liver enzymes, an indicator of toxicity (Zimmermann et al. study). It was therefore important to expand on those studies and search for formulations with even better knockdown efficacies and inherently less toxicity, thus pushing the therapeutic index well into predictably clinically safe ranges.

The recent announcements 0.1mg/kg IC50 knockdown efficiencies for SNALP-like formulations by both Protiva and Tekmira (due to their upcoming merger from now on referred to as Tekmira for simplicity) support the notion that the exploration of new chemistries should facilitate the development of SNALP RNAi for clinical use, and although only the surface has been scratched, a first IND with realistic chances at therapeutic success may not be very far away.

To exploit the chemical space available for SNALPs though requires the ability to generate new and diverse lipid chemistries as well as the ability to manufacture and formulate these chemistries to scale. The latter has been achieved by Tekmira’s spontaneous vesicle formation by ethanol dilution method allowing for the speedy manufacture of SNALP-siRNA formulations that can support late pre-clinical and clinical studies, while the long-awaited paper by Akinc and colleagues from the MIT (Langer/Anderson lab) and Alnylam Pharmaceuticals on so called lipidoids and that has now been published in Nature Biotechnology [Akinc et al. (2008): A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.], will now faciliate the efficient exploration of novel, SNALP-compatible lipid chemistries.

Rather than laboriously synthesizing and testing one lipid after the other on a hypothesis-driven basis, Akinc and colleagues developed a synthesis method that allowed them to generate libraries of cationic lipids with quite unusual and diverse characteristics to systematically evaluate them for siRNA delivery. A first library gave an indication of which chemistries worked better than others, and a second library was generated based on the characteristics of the best performing ones in the first set.

Initial tests were based on silencing reporter genes in tissue culture. It should be noted that for these high-throughput experiments, probably for speed and ease, simple siRNA-lipoplexes were used (siRNA-lipid mix), instead liposomally formulated siRNA as for later tests in vivo (siRNA captured inside liposomes), and this may be one limitation as it could have caused them to miss even more promising in vivo silencing chemistries. In any case, the best candidates were then taken forward into rodent and non-human primate studies, this time formulating the lipids together with cholesterol and PEG-lipid into cationic liposomes, essentially based on the same principles as SNALPs (stabilized liposomes containing diffusible PEG-lipids, the latter interestingly manufactured by Alnylam itself).

Overall, IC50s in the low mg/kg range were routinely observed for a number of liver targets. This was achieved without significant toxicities based on careful safety analysis, and only in some cases mild elevations, less than 2-fold, of liver enzymes were observed. Similarly, the absence of negative interference with endogenous microRNA pathways was reported last year. This is a good start and may not have employed the most efficacious siRNAs, but by optimizing the formulations further, e.g. by engineering additional fusogenic lipids and other properties into these particles, sub-mg/kg doses that would be desirable in the clinic should be achievable and still be compatible with the more scalable manufacturing technologies practiced by Tekmira (although 50nm particles and high encapsulation efficiencies were achieved, the extrusion-based method as employed in the present study may limit scale).

Beyond the liver, the lipidoid formulation showed some promise for the delivery of siRNAs to the lung as demonstrated in an RSV model. Interestingly, inhibition of RSV replication was enhanced by lipidoid-siRNAs (almost 3-log knockdown at 2mg/kg) over unformulated, naked siRNAs (1-log knockdown), in contrast to previous studies on which Alnylam’s ALN-RSV01 is based that showed somewhat less viral knockdown with naked siRNAs and that suggested no enhancement of siRNA delivery to the lung by formulation with other delivery chemistries. In addition, given the propensity of such nanoparticles to be taken up by phagocytic cells of the immune system and the largely unmodified siRNAs used for targeting RSV, follow-up studies need to look at any innate immune responses elicited by such lipidoid-siRNA combinations.

In summary, this study opens up a wide chemical space for the systematic evaluation of cationic liposome-mediated delivery of drugs, particularly siRNAs, but also microRNA antagonists (demonstrated in this study) and beyond. Following some of the recent breakthroughs and due to the triangular relationship between Alnylam, Tekmira, and the MIT, complementary in terms of both know-how and IP, progress of SNALP-siRNAs into the clinic may hopefully occur within the next few months and should be followed by next-generation chemistries.

[Update May 1, 2008: According to a report by RNAiNews , an Alnylam spokesperson indicated that the company had not given guidance on the specific liposome formulations to be used for their hypercholesterolemia and liver cancer clinical programs. This is in contrast to an earlier report by RNAiNews from last year’s Beyond Genome conference in San Francisco which indicated that Alnylam had chosen “choose lipidoids over SNALPs” for these indications (also discussed in a blog entry here). It therefore seems that Protiva/Tekmira's 0.1mg/kg IC50 liposomal nanoparticle formulations may be the current frontrunners in entering the clinic (see also a recent PR).

The sometimes imprecise use of the terms SNALPs and lipidoids may be partly to blame for the confusion. SNALP refers to a liposomal formulation technique, while lipidoids are a new class of lipids generated by combinatorial chemistry which can now be evaluated for liposomal drug delivery. As such, lipidoids could be used within the context of SNALPs, and both of these approaches are therefore complementary to each other.]

Journal Club: Therapeutic RNAi Delivered via Vitamin A-Coupled Cationic Liposomes Reverses Liver Cirrhosis in Rats

Illustrating how the basic concept of liposomal RNAi delivery can be modified to target specific cell types at low dosages, a study by Sato and colleagues published last week in the online version of Nature Biotech shows how the addition of vitamin A to cationic liposomes carrying siRNAs against Hsp47 can not only efficiently knock down the gene in hepatic stellate (HS) cells and block disease progression, but that this can actually reverse cirrhosis in a number of rat models for this disease [Sato et al. (2008). Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nature Biotech doi:10.1038/nbt1396].

It is known that by adjusting their stability and therefore pharmacokinetics, liposomes can be adjusted to shift their relative accumulation in tissues such as liver, tumour, or lung. For example, recent improvements in formulation methods now allow for the delivery of around 90% of the injected dose to the liver. While for liver applications, this already minimizes the exposure of non-target organs to the RNAi therapeutic, the ability to knock down genes in specific cell types at low dosages should further improve its therapeutic index.

Adding small conjugates such as sugars (e.g. Mirus Bio’s DynamicPolyConjugates to target RNAi to either hepatocytes or Kupffer cells) to a basic delivery formulation appears to be particularly promising. The authors of the present paper reasoned that since the HS cell which is central to liver cirrhosis efficiently takes up vitamin A, adding this vitamin to liposomally formulated siRNAs may facilitate their uptake in HS cells, in addition to endowing the nanoparticle with favorable pharmacokinetics by binding to retinol binding protein in circulation.

Indeed, while some non-specific uptake was observed into phagocytic cells, the modified A-liposomes efficiently entered HS cells in a vitamin A-dependent manner while largely avoiding other cell types of the liver, including hepatocytes. Actually, delivery was truly targeted since overall drug uptake was greatly enhanced in cirrhotic rats versus non-cirrhotic rats, likely a reflection of the HS cell activation/proliferation state.

Importantly, knockdown of Hsp47, required for collagen production and therefore a potential target for treating cirrhosis, was achieved at the clinically relevant low dosages of 0.1mg/kg to 0.75mg/kg, resulting in the survival of rats concomitant with resolution of the cirrhosis both by histology and normalization of liver enzymes.

The paper also shows that as we are learning more about the molecular biology and pharmacological properties of synthetic RNAs, the scientific standards for publishing papers on therapeutic RNAi have risen. Consequently, the sequence-specific nature of the therapeutic effect was demonstrated by the use of two additional Hsp47-targeted siRNAs. Cytokines and interferon-alpha were also looked at, including IL-12 which has recently gained notoriety
as a TLR3-related pro-inflammatory cytokine. Reassuringly, no IFN-alpha, TNF-alpha, or IL-12 induction was detected at the reasonably early 7 hour time-point with this unmodified Dicer-substrate (Dicer substrates are longer and therefore should be more prone to immune recognition). However, some non-specific elevation in the apoptosis of rat liver HS cells was observed following treatment with VA-liposomes carrying a control siRNA. Albeit small, it remains to be determined whether this was related to sequence-specific cytokine induction, or liposomally or vitamine A-induced cytotoxicity.

It is likely that the systematic screening for improved VA-liposome chemistries and optimized siRNAs should considerably improve the therapeutic index of this interesting variation on the cationic liposome for siRNA delivery. Similarly, vitamin A derivatives that are still taken up by HS cells, but do not cause potential vitamin A-related toxicities may be warranted for actual clinical use.

Liposomal RNAi Delivery to the Liver Making Progress

[Disclosure: I chose to own both Tekmira and Alnylam stock, of which particularly Tekmira should benefit considerably from progress on liposomal RNAi delivery due to their IP position on cationic liposomes and relationship with Alnylam.]

Following my blog on the RSV-01 experimental infection results, I received a number of emails noticing my apparently less-than-usual bullish assessment of this Alnylam result, a concern heightened by a falling Alnylam share price.

The problem with calling the results a definite proof-of-concept for RNAi in humans is that this program targets a virus, and it is well documented that particularly unmodified siRNAs can have profound antiviral effects independent of their gene silencing activity, i.e. by inducing cytokine responses. However, I acknowledge that Alnylam and their clinical collaborators have done almost all humanly possible to (statistically) exclude the influence of various parameters such as inflammatory cytokines on the antiviral efficacy, although it is not clear e.g. when and where these markers were measured.

I guess I am just too much of a trained scientific skeptic here when I can agree with proof-of-concept for a clinical effect of an RNAi Therapeutic in man, but remain cautious on proof-of-concept for gene silencing in man. Maybe proof-of-concept should not be seen as a one-time event, but something that will emerge over time.

Especially with some of the other early clinical RNAi trials, one has to be aware that these were entered into the clinic before the entire scope of non-silencing activities of unmodified siRNAs were known (and some of the trials lacking supporting sound scientific data, btw). For those that would like to learn about this topic, I would warmly recommend the freely available, and very critical review article by Protiva’s Adam Judge and Ian MacLachlan from Protiva on the immune-stimulatory potential of siRNAs, including their non-specific antiviral and antiangionic (e.g. relevant for wet AMD applications) potentials. It is important to note that critics, also called “shorts” in stock market lingo, may exploit these issues to portray them as fatal short-comings of the RNAi Therapeutics platform, but like delivery, there is every indication that these will be overcome in a timely manner and the field should not shy away from openly addressing them.

As I indicated in my 2008: Year of the Liver , I believe that the demonstration of RNAi gene silencing in the human liver may very well steal the thunder from the RSV program this year. It was gratifying therefore to hear at today’s Alnylam’s R&D Day that by working together with Tekmira and applying a systematic, industrial-type evaluation of different liposomal nanoparticle formulations (LNPs, aka SNALPs), the potency of these particles has been improved such that only 0.1mg/kg dosages can effect over 50% knock downs. This is very important since these are now concentrations that should be well below the concentrations previously associated with toxicities by these cationic liposomes and likely the cause for delays in filing for the much anticipated liver INDs. Not only was it then confirmed that an siRNA administration led to silencing over an entire month, but equally important was the demonstration that repeat-administration of these particles over 3 months was able to cause sustained gene silencing, meaning that neutralizing antibodies, typically facilitated by concomitant inflammatory cytokine induction, are not generated to blunt repeat administration.

As a result, Alnylam today gave the best indication thus far that they are now ready to enter the clinic with such a systemic RNAi formulation, most likely for treating hypercholesterolemia. In addition to demonstrating safety, this phase I study may indeed provide the most impressive evidence for RNAi gene silencing activity in humans simply by measuring the level of circulating PCSK9, the gene target of this program.

Btw, comparable 0.1mg/kg efficacy and repeat-administration data with the essentially same underlying SNALP technology was also reported on Protiva’s website which I give an ‘A’ for being scientifically engaging, and possibly an ‘A+’ when Protiva and Tekmira can finally come to the realization that it does not make sense to have two companies operating in the same city on the same RNAi delivery technology, while diverting money and attention on lawsuits fighting each other.

As you can see, despite the broad progress in delivering RNAi to many other organs and tissues, based on the available data, RNAi delivery to the liver (not only by SNALPs, but also an increasing number of competing technologies) appears to be the most advanced in terms of efficacy and should be one of the major drivers of the valuation of the RNAi (and also microRNA) Therapeutics space in the next 1-4 years by filling the clinical pipelines with high-quality, promising drug candidates.

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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Nature Publishes Reassuring Study by Alnylam on the in vivo Delivery of siRNAs and their Effect on the Endogenous microRNA Pathway

Given that therapeutic RNAi takes advantage of an endogenous biological pathway, the introduction of very high levels of small RNAs certainly has the potential to interfere with related small RNA pathways, such as microRNA function.

Indeed, competition with the microRNA pathway in vivo which in some cases caused the death of mice, were first reported in a study by Grimm and colleagues in the journal Nature last year. Ironically, rather than a demonstrating a failure of the viral delivery system used in that study, it was the extreme efficiency with which small RNAs could be expressed with the double-stranded AAV vectors that allowed the competition to be observed. In what is an often overlooked aspect of these studies, compared to non-viral delivery methods, silencing efficiencies of over 95% can be easily achieved with these vectors at doses that have no adverse effects on either the microRNA pathway or the viability of mice.

In an ideal world, the Grimm et al. studies would have been embraced as an opportunity to study the dose-limiting steps of therapeutic RNAi to inform future RNAi therapeutics strategies. Instead, as the Press lives from feeding the public the simple messages, rather than reporting complicated truths, it decided to label the studies as yet another example of the dangers of gene therapy, and - somewhat understandably- caused some companies involved in developing RNAi Therapeutics to distance themselves from DNA-directed RNAi for political reasons.

As the trusted leader of RNAi Therapeutics, Alnylam was therefore given the platform to reassure the RNAi community this week in Nature that, unlike AAV-RNAi, liposomally delivered siRNAs had no obvious adverse effects on the endogenous microRNA pathway (John et al., 2007). The study further highlighted that liposomal siRNA delivery has advanced to a point where around 80% gene silencing in hepatocytes can routinely be achieved following systemic administration of therapeutically viable doses of siRNAs, including their repeat administration.

Although I welcome Nature’s decision to document progress in the important area of RNAi therapeutics, and understand Alnylam’s desire to publish in the highest profile journals, I would like to take this opportunity to address a few misconceptions about the studies. One important misconception is that delivering RNAi with AAV per se is more toxic. To make this point, a direct comparison of the intrahepatic levels of small RNA levels following both routes of administration would have been necessary. Given the >99% transduction efficiency of double-stranded AAV in mice and the consequently extremely high gene silencing efficiencies, it is quite likely that double-strand AAV vectors are currently the most potent delivery system to the liver in terms of small RNA delivery and gene knockdown.

I would therefore not be surprised at all to see similar competition with microRNA function following administration of very high siRNA dosages. This is supported by numerous studies that have shown competition for gene silencing when very high levels of two or more siRNAs were introduced simultaneously into tissue culture cells. However, given the ability of hundreds of microRNAs to function in a given cell at any time, such observations represent only extreme cases and suggest a wide therapeutic index. Unfortunately, the relatively small range of doses used in the John et al. studies (2mg/kg to 5mg/kg) did allow for a careful evaluation of related competition in vivo and concomitant dose-limiting toxicities.

I guess the purpose of this Blog really is my plea to the field of RNAi Therapeutics to keep learning from each other, instead of letting the Press and uninformed “analysts” play on the fears of investors, through their indiscriminate use of buzzwords, thereby polarising and separating what really belongs together. In this spirit, I would like to stress that this study is yet another proof-point of the viability of RNAi for therapy leading up to the possibly first proof-of-concept gene silencing results in Man to be revealed in the coming months- once again by Alnylam.


PS: Although a combination of liposomal delivery methods were used in these studies, the details were not disclosed. Apparently, another study on lipidoid-delivered siRNAs, a technology developed by the Langer and Anderson groups at the MIT, has been submitted to Nature Biotech and is about to be published. Lipidoids differ slightly from the Tekmira-owned SNALP technology, and looks likely to be the technology used for Alnylam’s first clinical systemic RNAi program (liver cancer or hypercholesterolemia), for which an IND is expected by the end of 2007. More than knockdown efficiency, we should be looking for the toxicity profile as I regard this to be the big unknown that will determine the success of this program, particularly if it turns out to be for hypercholesterolemia.

Alnylam Chooses Lipidoids over Cationic Liposomes for their First Systemic RNAi Clinical Studies

[Important update at end of this entry]

In an interesting twist, Alnylam announced at the Beyond Genome conference held last week in San Francisco the use of MIT’s lipidoid technology for their first systemic RNAi programs. These formulations will be used for knocking down PCSK9 for the treatment of hypercholesterolemia and the dual siRNA cocktail ALN-VSP01 for liver cancer. This was somewhat surprising, following a proof-of-concept Nature study last year that demonstrated efficient systemic RNAi delivery in primates using cationic liposomes. These were developed by Protiva/Tekmira, and Alnylam consequently established a broad alliance with Inex Pharmaceuticals, now Tekmira, that comprised an exclusive license to Alnylam to Tekmira’s liposomal delivery IP estate. It was therefore largely expected that Alnylam would use Tekmira’s cationic liposome SNALP technology in their systemic RNAi programs which are scheduled to enter the clinic by the end of this year.

As I pointed out in some of my earlier blogs, I was concerned that Alnylam’s plans to move into systemic clinical trials so quickly were too aggressive. This concern was largely based on the considerable, albeit transient elevation in liver enzymes, a measure of liver toxicity, at the 2.5mg/kg dose range reported in last year’s Nature study. Moreover, combined with the non-linear dose response for cationic liposomal siRNA delivery that Alnylam reported at the Keystone RNAi meeting earlier this year, this made choosing the right dose range for human studies less than certain.

Interestingly, at the same Keystone meeting, Dan Anderson together with Rob Langer, a world authority on drug delivery and scientific advisor for Alnylam, presented impressive systemic delivery data using so called “lipidoids”. “Lipidoids” were discovered as part of a library approach as an apparently new class of lipid-like molecules that were very effective in delivering siRNAs systemically to the liver and were structurally sufficiently distinct to conventional lipids and cationic polymers to give it a new name. Only a few months after that, Alnylam and the same groups at MIT announced a broad systemic RNAi delivery initiative in which Alnylam would fund 10 post-doctoral researchers for 5 years in return for exclusive rights to the lipidoid technology and an exclusive option for any new RNAi delivery technologies resulting from the sponsored fellowships.

Importantly, the tiny lipidoid formulations were reported to have a favourable safety profile and showed dose-dependent gene suppression without compromising RNAi knockdown efficacy.

In hindsight, it is very reassuring to see that Alnylam did not go out on a limb by promising an aggressive systemic RNAi timeline and gamble an early program on a potentially unsafe delivery technology. It speaks to the quality of the management and Alnylam’s reputation as the leader in RNAi Therapeutics that it always had valid options outside cationic liposomes.

Where does this leave the Tekmira-Alnylam alliance? Although “lipidoids” appear to be somewhat distinct to cationic liposomes, it is certainly a good insurance to be covered by Tekmira’s important IP estate in the field of liposomal drug delivery. Moreover, as part of the alliance Alnylam invested in Tekmira’s manufacturing capabilities, and it appears this investment will pay off as Tekmira is manufacturing the lipidoid-siRNA formulations for the PCSK9-hypercholsterolemia and liver cancer trials. According to David Bumcrot of Alnylam, IND-enabling studies for these programs are well underway.

PS: In another interesting twist, David Bumcrot noted that part of Alnylam’s decision to target PCSK9 in their hypercholesterolemia program is due to a fatty liver problem seen in targeting the former front-runner ApoB100. This appears to be a target-specific phenotype as this phenomenon has been seen with a number of siRNAs targeting ApoB100. Interestingly, ISIS Pharmaceuticals which has an ApoB100 antisense compound in late phase II trials, but has not reported on that problem has followed Alnylam’s lead in targeting PCSK9 in a new hypercholesterolemia program (see May 9, 2007 post). Once again, Alnylam has demonstrated a characteristically circumspect development approach that includes bringing together the best scientists in a given disease area to carefully characterise RNAi knockdown phenotypes.

[Update May 1, 2008: According to a report by RNAiNews , an Alnylam spokesperson indicated that the company had not given guidance on the specific liposome formulations to be used for their hypercholesterolemia and liver cancer clinical programs. This is in contrast to an earlier report by RNAiNews from last year’s Beyond Genome conference in San Francisco which indicated that Alnylam had chosen “choose lipidoids over SNALPs” for these indications (also discussed in a blog entry here). It therefore seems that Protiva/Tekmira's 0.1mg/kg IC50 liposomal nanoparticle formulations may be the current frontrunners in entering the clinic (see also a recent PR).

The sometimes imprecise use of the terms SNALPs and lipidoids may be partly to blame for the confusion. SNALP refers to a liposomal formulation technique, while lipidoids are a new class of lipids generated by combinatorial chemistry which can now be evaluated for liposomal drug delivery. As such, lipidoids could be used within the context of SNALPs, and both of these approaches are therefore complementary to each other.]