Liposomes Also Useful for Functional siRNA Delivery to Phagocytic Cells

The goal in developing a RNAi Therapeutics delivery technology is to achieve gene knockdown in a certain cell or tissue type. Once this is achieved, only our exploding genetic insights into disease limit the potential therapeutic applications. This is for example why we are seeing this SNALP-fuelled expansion in the clinical pipeline for indications in which gene knockdown is targeted in the liver.

In addition to the liver (SNALP), vascular endothelial cells (AtuPLEX, DACC), tumor cells (SNALP), skin (self-delivering rxRNAs), hematopoietic stem cells (lentivirus), and cells in the eye and CNS (lentivirus, AAV), RNAi Therapeutics delivery technologies have reached sufficient maturity to warrant their clinical development for knockdown in phagocytic cells of the immune system. In fact, there is already a clinical trial by Duke University that involves the ex vivo electroporation of siRNAs into dendritic cells for therapeutic cancer vaccine development (think Dendreon), and a first systemically administered example, Tekmira’s SNALP-EBOLA, is about to enter the clinic with an IND planned by year-end.

Probably fearing that the investing public has largely forgotten about such potential of Tekmira’s technology, Alnylam this week advertised two liposomal siRNA delivery studies, one published in Molecular Therapy (Basha et al.) and the other one in Nature Biotechnology (Leuschner et al.), which nicely characterized RNAi activity in phagocytic cells and provided a few tanatalizing examples of their potential therapeutic applications.

Leuschner et al: Interfering with Pathologic Phagocyte Recruitment by Knocking Down CCR2

The Leuschner at al. study, stemming from a collaboration between the Massachusetts General Hospital, MIT and Alnylam, used lipidoid lipid-formulated siRNAs to target chemokine receptor CCR2 in phagocytic cells. The lipid was the C12-200 cationic lipidoid that was shown in a paper last year to promote gene knockdown in the liver at much lower dosages compared to 1^st-generation lipidoids (Love at al. 2010).

After demonstrating that the systemically administered liposomes were taken up by various phagocytic cell populations in the bone marrow, spleen, and the circulation, the researchers then focused in, for the functional part of their study, on the Ly-6high subset of these cells from the spleen. These were chosen because of the particularly efficient liposomal uptake in this cell population and their role in the pathogenic, CCR2-dependent recruitment to sites of local inflammation.

As a reminder, while such inflammatory recruitment is a vital part of our first-line defense and response to foreign invaders and tissue damage, respectively, they often end up doing their job too enthusiastically leaving behind pathologic scars, unwittingly promote cancers, or interfere with medical interventions such as transplantation.

Leuschner and colleagues studied four settings where interfering with Ly-6high through CCR2 knockdown might be beneficial (all mouse studies): ischemia-reperfusion injury as would occur following a heart attack; inflammation at atherosclerotic lesions; pancreatic islet graft survival; and reducing the number of tumor-associated macrophages which are thought to promote cancer. In all four settings, it was found that a moderate ~50% knockdown of CCR2 was sufficient to effectively inhibit inflammatory cell recruitment to the sites of disease and was accompanied by the hoped-for outcome.

While this is certainly encouraging news and seems to open up many therapeutic avenues just based on CCR2 targeting, the degree of knockdown suggests that while it should be possible to achieve therapeutic outcomes with well-chosen target genes, further improvements in efficacy are necessary for the particular formulation to be broadly applicable to other target genes that may not be as dosage-sensitive as CCR2 appears to be.

Basha et al.: Tekmira SNALPs Promote Gene Knockdown in Antigen-Presenting Cells

Unlike the Leuschner et al. study which focused on the potential therapeutic applications of gene knockdown in phagocytic cells, the Basha et al. studied the mechanism, efficacy, and safety of SNALP delivery to antigen-presenting cells (APCs). This paper was largely conducted in the laboratory of Alnylam's new friends from Vancouver.

Evaluating four SNALP formulations differing only in the nature of their ‘critical’ lipid- DlinDAP, DLinDMA, DLinK-DMA, or DLinKC2-DMA- it was found that three of them (DMA, K-DMA, KC2-DMA) were capable of promoting gene knockdown in antigen-presenting macrophages and dendritic cells- both in vitro and in vivo following systemic administration. Taking into account safety, the authors favored the formulation with the KC2-DMA lipid, the lipid of the Tekmira line-of-research that was shown last year in the Nature Biotechnology paper to be highly efficient also for gene knockdown in the liver (Semple et al. 2010).

Importantly, the present data showed that increased efficacy did not come at the cost of safety. Safety is all the more important here since, similar to the lipidoid formulations described above, the delivery efficacy is still much less compared to their use in functional SNALP delivery to the liver, meaning that higher dosages would have to be administered. Since hepatotoxicity could be a dose-limiting toxicity for these formulations, the demonstration that by increasing particle size, gene knockdown in the liver could be abolished without compromising gene silencing in APCs, suggests a strategy to increase the safety of APC-targeted SNALP applications.

The Basha et al. Study Employed Tekmira-Owned Lipids and Formulations and was Partly Funded by Tekmira

While the specific results were not really surprising in light of what has already been known about liposomal delivery, what really confused me was the fact that it was Alnylam that did all the advertising of the Basha et al. study with no word whatsoever from Tekmira. After all, the paper acknowledged Tekmira as a funder of the study and based on the Responses by Alnylam, including their Canadian subsidiary Alnylam-Canada (aka AlCana), to Tekmira’s allegations, neither Alnylam nor AlCana contest that all four lipids are owned by Tekmira. In fact, KC2 is the latest in the series and, as we know, the Nature Biotech paper in early 2010 was advertised by both companies (see here and here).

The acknowledgement:

‘The authors would like to acknowledge support from the Canadian Institutes for Health Research (CIHR) under U0P grant FRN 59836, as well as support from Tekmira Pharmaceuticals and Alnylam Pharmaceuticals. P.R.C. has a financial interest in Tekmira and receives grant support from Alnylam.’

Sure, Alnylam has acted a bit irrational and confused when it came to Tekmira and its general understanding of what constitutes ownership. However, advertising a lipid for which there has been little controversy that it belongs to Tekmira and a study that was in part paid for by Tekmira, without so much as an acknowledgement of Tekmira sets a new standard. Perhaps as perplexing is the fact that Tekmira has not responded at all to these developments.

I entertain two explanations for this, explanations that I consider equally likely. The first one is that things have gotten so much out of control that Alnylam does not care any more about the liabilities of such press releases. If Tekmira needed to find an example of where Alnylam falsely advertised Tekmira’s technology as its own, one of Tekmira’s allegations, Alnylam just provided it with the most striking one.

The other explanation is that maybe things are pointing towards a settlement or merger of the two companies. This would explain why Tekmira seems to co-operate with Alnylam by not protesting about last week’s press release. It is also of note that Alnylam, as of October 8, is now represented by a new lead counsel in the ALN-VSP02 patent Interference case. Initially represented by Rothwell, Figg, Ernest & Manbeck, it is now the attorneys from Wilmer, Cutler, Pickering, Hale, and Dorr that have taken charge of the Interference for Alnylam…you guessed correctly, the same law firm representing Alnylam in the Big One with Tekmira. An alignment of these two cases would have always made sense. At this advanced stage of the Interference, however, the change seems somewhat surprising, but would make sense if it is now critical to have the two cases strongly aligned so as not to jeopardize a mutual understanding that may have been reached.

100-fold Improvements in Liposomal siRNA Delivery Potencies in just 2 Years

By applying new cationic lipids to the basic SNALP/LNP formulation developed by Protiva (now Tekmira), liposomal siRNA delivery has now reached IC50 potencies of around 10microgram/kg in mice and non-human primates, a roughly 100-fold improvement over the last 2 years alone. This has profound implications for the types of clinical uses of this leading RNAi systemic delivery approach, particularly by predicting a critical improvement in the therapeutic index and lowering cost of goods and length of administration. Tekmira’s SNALP-ApoB program, for the chronic condition of severe hypercholesterolemia, is a good example where these types of improvements could prove critical for clinical success.

Before I go into the nitty-gritty of what will also include the politics behind liposomal siRNA delivery, a topic that I am sure is of interest to many readers here, let me state unambiguously: these are the types of scientific discoveries that could make a big difference to the clinical trajectory of RNAi Therapeutics and are extremely positive. All this can easily be forgotten when going into scientific and commercial details.

While these developments have been anticipated by comments made in conference calls and presentations at academic conferences by Alnylam and its collaborators from the MIT, Tekmira, and the University of British Columbia, the first scientific paper on this has just been published in the Proceedings of the National Academy of Sciences (Love et al.: Lipid-like materials for low-dose, in vivo gene silencing). This paper is a continuation of the so-called ‘lipidoid’ theme first

published on 2 years ago (Akinc et al.: A combinatorial library of lipid-like materials for delivery of RNAi therapeutics) in which Alnylam and their collaborators at the MIT have been screening new lipids that had been generated in a high-throughput fashion by combinatorial chemistry for liposomal siRNA delivery in vivo.

In the 2008 Nature Biotech paper, a library of alkyl acrylates or acrylamides coupled to amines yielded ‘98N12-5’ as the lead lipid with an IC50 in mice of around 1mg/kg. In that paper, the authors noted that unlike the SNALP formulation by Alnylam-Protiva/Tekmira published in Nature in 2006, the 98N12 system required only 1/3 of the lipid content and that it could be used in vivo without the need for an

additional helper lipid typically employed in SNALP formulations. Certainly, the less lipids, including the number of lipids in the mix, the simpler and better. Moreover, extrusion technologies which are difficult to scale up for clinical utility were used in that study. As we will see, the formulation technology as well as the lipid mix can make a huge difference in what liposomes are actually generated and somewhat takes away from the allure/necessity of high-throughput approaches to lipid discovery such as practiced here.

The new paper, however, is not a direct continuation from the initial screen, but is based on an entirely new library of amino-alcohols generated by epoxide chemistry. The stated motivation for doing so was the comparatively cleaner nature of the chemistry, circumventing the need for laborious purifications for initial tissue culture screens. While the 2008 library consisted of around 1200 compounds, this one was an order of magnitude smaller. Like before, the positively charged lipids were first screened in tissue culture by simple direct complexation with the siRNAs, i.e. not as a liposomal formulation. 12 of the most potent lipids from that screen were then carried forward into mouse studies. Although it soon became apparent that there is only little predictive value of a tissue culture screen for in vivo studies, there were 3 formulations that achieved almost complete Factor VII ablation at 3mg/kg in mice. A dose-response revealed that behind the complete knockout data at relatively high dosages were concealed vastly different IC50s of 0.01 (C12-200), 0.3, and 1mg/kg. It therefore appears that the researchers got a lucky break that they included C12-200 in the 12-compound in vivo screen, a level of experimentation that can arguably be considered to be the real bottleneck for liposomal delivery research.

Since Alnylam is increasingly talking about second-generation liposomal nanoparticles (LNPs), including with cationic lipids, and regularly refers to Tekmira’s SNALPs as first generation in this context [example from press release on PNAS paper.: “ALN-VSP and ALN-TTR both utilize a first generation lipid nanoparticle formulation known as stable nucleic acid-lipid particles (SNALP), developed in collaboration with Tekmira Pharmaceuticals Corp.”], as do the ‘lipidoid’ papers contrast their formulations with the SNALPs, it is worth going into the formulation details here again.

For the initial in vivo screen, again no helper lipid was noted (cationic lipid/'lipidoid' +cholesterol +PEG-lipid only). As to the formulation method, the following can be found in the methods section: “siRNA at a concentration of 10 mg∕mL in 50 mM sodium acetate was added to empty liposomes at a weight ratio of 10:1 total lipids:siRNA and the mixture was incubated at 37 °C for 30 min. Formulations were then dialyzed…” While no mention is made of extrusion, they reference the 2008 Nature biotech paper for further details on that formulation method. If not, I would almost have to come to the conclusion that this description would lead to Silence Therapeutics-type lipoplexes and not liposomally encapsulated siRNAs[update 15Jan10: I have looked up the incubation method, and it does appear to result in encapsulating liposomes with the right pHs and EtOH concentrations]. Be that as it may, the 3-lipid formulation and method differs from that used by Tekmira. In the case of C12-200 this yielded a 140nm particle for the initial in vivo screen that had the IC50 for the liver-expressed Factor VII of ~0.01mg/kg.

When it came to the monkey studies, however, the formulation and physical nature of the C12-200-containing particle was changed dramatically. The 2008 advantage of having only 3 lipids in the mix, was given up in favor of the addition of the now industry-standard helper lipid DSPC. Moreover, for the formulation method itself the rapid mixing-ethanol dilution method was employed with the T-shaped apparatus- both invented by Protiva/Tekmira. If anything, the what is referred to now as non-SNALP ‘lipidoid’ formulations are starting to look more and more like gold-standard SNALPs both chemically and formulation technique-wise. Also not insignificant is the fact that while the crude 3-lipid nanoparticles were 140nm in size, the SNALP formulation that was then used for the non-human primate studies was only 80nm in size (smaller is better here), a huge difference and complicating translational studies. Scale-up needs to be considered from the early stages.

Nevertheless, despite the big differences in the physical properties of the particles, the SNALP formulation containing the C12-200 cationic lipid silenced the monkey TTR gene by 70% at 0.03mg/kg following a single low-volume 15-minute infusion (note: Alnylam has filed for an TTR IND at the end of last year using 'first generation SNALP technology'). It thus appears that a potent C12-200 cytoplasm-accessing ability for hepatocytes can mask variable SNALP shapes. In an attempt to elucidate the mechanism of this, in vitro cell uptake studies were performed, although it is unclear from the paper which exact formulation mix was used for this purpose. Based on these studies, the authors argue for a macropinocytic uptake mechanism distinct from classical endosomal uptake thought to be relevant for other types LNP/SNALP formulations. While the data are consistent with this, I would not classify them as proof that this is indeed the productive uptake mechanism, and a side-by-side comparison with a ‘SNALP’ would have been helpful when arguing for unique uptake mechanisms. This also reminds me of a presentation by Tekmira's CSO Ian MacLachlan on a visit to Stanford where only slightly changed SNALP formulations exhibited vastly different biodistributions within the liver. It therefore appears that while the variability of uptake mechanisms is not a property of ‘lipidoids’ per se, they may be one (of several) factor that can account for the greatly varying knockdown potencies of these formulations.

This paper and similar conference reports on the rational design of lipids (= approach taken by Tekmira and Alnylam's collaborators at the University of British Columbia and AlCana) demonstrate that the technological development of RNAi Therapeutics is still on a steep trajectory which should help reinvigorate interest in the field by the financial markets, especially as the worst has been avoided and the longer-term comes into focus again. The next steps will be to further characterize the pharmacology of these formulations and the application of scalable formulation methods. Both of this and the further development of liposomal delivery for receptor-targeted and (gain-of-function) immune applications as highlighted in the JP Morgan presentation by Alnylam CEO John Maraganore, should be sped up by good relationships between Tekmira and Alnylam, at a time that I feel Alnylam is more and more distancing itself from and down-playing the importance of Tekmira when in fact Alnylam is increasingly adopting methods invented by Protiva/Tekmira. The JP Morgan Healthcare meeting where both companies are present should be a good opportunity to patch up relationships. One potentially mutually beneficial solution may be for Alnylam and Roche to take a majority stake in Tekmira with a simultaneous cash infusion, but let Tekmira operate independently to keep their minds sharp, similar to the Roche-Genentech example.

Note added in proof: January 2010 Nature Biotech biotech paper by Tekmira and Alnylam on significantly improved SNALP formulations through the rational design of lipids (link and press release).

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.]

2008: The Year of the Liver

It is true that next year could bring the first human proof-of-concept for an RNAi therapeutic. But since results from the experimental infection studies for ALN-RSV01 were originally expected in late 2007 and the results would be an overhang of work done in ’07, I find it more appropriate to pick a fresh candidate. It is also true that the lung may rival the liver in the number of programs moving into the clinic in ‘08 given recent data that suggest delivery of siRNAs to the lung by nebulizer to be fairly innocuous, and the fact that AstraZeneca and GSK should be busy working in that area following their agreements with Silence and Sirna Therapeutics, respectively.

Nevertheless, I feel that most systematic progress has been made in delivering RNAi to the liver with more progress likely to follow. There have now been a fair number of publications that clearly show target-specific RNAi knockdown in the liver, unlike maybe some of the lung studies where there were occasional data interpretation issues. Of course, in declaring 2008 ‘The Year of the Liver’, I should disclose that I am somewhat biased due to the hepatotropic focus of the laboratory that I work in.

For siRNA-mediated RNAi in the liver, I currently see three front-runners, although it is possible that among the considerable work that is ongoing and not yet published, others have reached a similar stage of technological maturity. These are the in-famous SNALPs, Mirus Bio’s “Dynamic PolyConjugates”, and MIT’s lipidoids. SNALPs, being worked on by Protiva, Tekmira, Alnylam, and Sirna/Merck, are probably 12-18 months ahead of the game and best characterized, whereas the data on PolyConjugates and lipidoids are very promising, but still somewhat spotty.

SNALPs, stable nucleic acid lipid particles, are set to enter the clinic in 2008 with programs in hypercholesterolemia and liver cancer (both Alnylam) and possibly a clinical candidate chosen by Tekmira. The hypercholesterolemia program is also an opportunity to get an early measure of therapeutic efficacy, possibly in ‘08. Protiva’s and Sirna/Merck’s intentions are less clear and I believe that Big Pharma often chooses to keep phase I programs secret for competitive reasons, so that it is theoretically possible that Sirna has already entered the clinic or is about to do so with the long-anticipated SNALP RNAi for Hepatitis C.

It is obvious that there were some delays in bringing SNALP RNAi to the clinic, and I believe that this is largely due to dosing and safety issues. Both of these concerns may come down to the propensity of SNALP liposomes to be taken up by immune cells such as Kupffer cells in the liver and plasmacytoid dendritic cells which a) function like a sink for the liposomes when they enter the liver so that SNALPs become available for entering the desired hepatocytes only after the sink is saturated, something that complicates dosing; and b) increases the risk of triggering unwanted cytokine responses. I am optimistic, however, that by varying the composition of SNALPs, safe and efficacious formulations may be found, particularly if the liposome uptake mechanism by the professional immune surveillance system were to be different from that by the hepatocytes, which I think is quite reasonable to assume. I therefore hope that the fact that so many scientists are working on SNALP RNAi is a sign of its promise rather than desperation. As such, the number of R&D staff at Tekmira has more than doubled from 17 to 39 in the year ending September 2006 largely due to work on SNALPs.

As I have written before, I am much taken by Mirus’ PolyConjugate work, although this is based on only a single paper published in the middle of ‘07 (see 24 July 07 Blog: “Mirus Scientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes”). The neat aspect of that work was that it showed that it should be possible to avoid the Kupffer cells in the liver and specifically target hepatocytes for knockdown with the help of carbohydrate ligands (I am curious whether similar targeting ligands would also work in the context of other formulations). As we know, this work is partnered with Pfizer, and I wonder whether the upcoming loss of exclusive marketing rights for their wonder-drug Lipitor will spur them into action here.

I am still waiting for more data on lipidoid-mediated RNAi which hopefully will become available soon as was indicated in the footnotes of an October 2007 paper on the effect of siRNA delivery on microRNA function. From both a business and scientific perspective, it will be interesting to see whether Alnylam may choose lipidoids over SNALPs for its first liver RNAi programs and the overlap of SNALP and lipidoid both in IP and manufacturing terms.

The liver may get further attention from the targeting of microRNA-122 for the treatment of hypercholesterolemia and Hepatitis C, and other programs on targeting certain microRNAs for the treatment of hepatocellular carcinoma. There are various programs by Santaris, Regulus and others that have progressed into larger animals and we may even see a first IND being filed for one of these applications by the end of 2008. Development in the antisense field, particularly the partnering of ISIS’ mipomersen, should also generate heightened awareness for RNA-based therapeutics of liver disease.

Other predictions for 2008:

1) Unpredicted delivery technologies
2) Pfizer finally makes its move
3) News on RNAi for wet AMD- good and/or bad?
4) Protiva-Tekmira dispute resolved (wishful thinking)

For a nice presentation on the powers of SNALPs by Protiva chief scientist Ian MacLachlan, please visit: http://mms.technologynetworks.net/RNAi06Presentations/maclachlan/Player.html

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.

Mirus Scientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes

The ability to systemically administer siRNAs and functionally modulate gene expression in tissues of interest is considered by many an important step to opening up the therapeutic potential of RNAi to a wide range of diseases. The liver is arguably the best example where intravenously administered siRNAs have already been shown to potently knockdown target genes from mice to non-human primates.

The most promising delivery technologies so far (next to gene therapy vectors such as AAV which I will not discuss here) involve liposomes such as the SNALP particles which were first pioneered by Protiva scientists and subsequently became the subject of intense legal battles involving Protiva, Tekmira, Sirna Therapeutics, and Merck. While facilitating highly efficacious gene knockdown, toxicities such as liver enzyme elevations and non-linear relationships between knockdown and siRNA dosage have been repeatedly reported with these chemistries. While the toxicities were observed at relatively high dose levels, non-linear dose responses complicate the choice of the right dose for entering clinical trials.

One step in the right direction was taken when Daniel Anderson from the MIT reported earlier this year at the Keystone meeting the identification of a slightly different class of compounds which they termed “lipidoids”. Importantly, this class of chemistries appear to efficiently promote RNAi gene knockdown with little if any apparent toxicities and linear dose responses.

The just released paper by Rozema and colleagues from Mirus Bio Corporation (Rozema et al. PNAS Early Edition 24 July 2007: Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes) sheds light on some of the issues above and holds out a new paradigm for achieving safe and efficacious therapeutic RNAi knockdown in select cell populations of the liver. Rather than regarding drug delivery to the liver as a passive process given that the bulk of intravenously injected drugs will pass through it with a relatively high chance of entering resident cells there, the present approach involves attaching a simple galactose-derived ligand thereby actively targeting asialoglycoprotein receptors (ASGPr) displayed on hepatocytes.

Knockdown of ApoB100 and microscopic analysis of fluorescently tagged small double-stranded nucleic acids confirmed that hepatocytes were indeed efficiently transfected. Equally important, however, was their observation that Kupffer cells, a major population of macrophages in the liver that are implicated in many cases of drug-related liver toxicities, did not take up the siRNA mimicks, whereas non-ASGPr targeted particles were able to transfect surrounding cells, including Kupffer cells.

Taken together with the SNALP and lipidoid data, this study supports the hypothesis that Kupffer cells may act as a sink for certain siRNA formulations such as SNALPs, thereby not only causing non-linear dose responses, but also an immunogenic response and related toxicities. It also shows that it should be possible to design simple and small siRNA nanoparticles for RNAi delivery from relatively cheap materials. Although lipidoids appear to be a clinically viable technology already, it is comforting to know that alternative routes exist and gratifying to see almost daily improvements being made in the RNAi delivery field.

PS: An interesting aspect of this publication was data the authors obtained on ApoB knockdown. In addition to serving as a target for early proof-of-concept studies for systemic RNAi delivery, ApoB has been a favourite for treating hypercholesterolemia using both antisense and RNAi. ISIS Pharmaceuticals in particular has an anti-ApoB antisense compound in late phase II clinical trials. However, only a month ago, Alnylam reported that in their hands ApoB knockdown led to unacceptably high levels of fat accumulation in the liver (fatty liver phenotype), a finding that was confirmed in the present study. Moreover, both reports indicate that this was a siRNA sequence specific effect and was achieved using two different delivery methods. This physiologic response makes a lot of sense, since ApoB’s main role is in the export of cholesterol and triglycerides from the liver. Failure to export them should accumulate them in the liver.

This is a good example where RNAi can serve both as a target validation tool AND a platform for developing innovative drugs. Consequently, I fully support Alnylam’s decision to target PCSK9 in their hypercholesterolemia program instead, which is a genetically well validated target for reducing LDL-cholesterol and heart disease (see also Blogs from 6 May, 2007: “Preventing Heart Disease with RNAi Therapeutics”, and 9 May, 2007: “ISIS Copies Alnylam’s Heart Disease Strategy“). It is curious then that ISIS maintains and has published the absence of such a phenotype. For the sake of patients and stakeholders in ISIS, I can only hope they are right. The explanation for the apparent discrepancy? I cannot really offer a good one except to speculate on a fortuitous ISIS 301012 antisense off-target effect.

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.]