SNALP Structure Reconsidered

SNALP delivery technology has not only been the subject of a heated fight over ownership and control, but also continued mechanistic, chemistry, and structural investigations.  

In terms of structure, I used to think of SNALP as simple, unilamellar liposomes with RNAi triggers captured in their aqueous interiors.  A patent application by Tekmira (WO2012/000104A1) and a recent paper by the Cullis/AlCana group (Leung et al., 2012) convincingly challenge this view.  Accordingly, SNALP particles are highly electron-dense entities in which pockets of RNAi triggers surrounded by positively charged lipid micelles lipids fill a lipid-enclosed vesicle.  

This new view should guide the future development of the technology, especially the ratio space of its components, targeted delivery approaches, and cytoplasmic release.

Numbers hinting at need for new model

One metric of Tekmira’s results that has always impressed me were its RNAi trigger formulation efficiencies, often well above 90%.  As Leung and colleagues note in their paper, this is at odds with the old simple aqueous encapsulation model which would predict that the likelihoods of an RNAi trigger to be encapsulated or not are roughly equal.  

It now seems that Tekmira’s manufacturing method of rapidly mixing lipids and nucleic acids is at least one critical factor allowing for this remarkable concentration effect.  Other groups largely failed to reproduce such data at least partly because the published method called for the use of costly amounts of reagents.  This is changing, however, due to the use of microfluidic formulation methods as was also practiced by Leung and colleagues. This method likewise allows lipids and nucleic acids to be mixed vigorously and consequently also generates electro-dense structures, but using much smaller volumes.    

Limit-size particles smaller than anticipated

A consequence of the new model is that SNALP LNPs may be as small as 15nm in diameter, whereas previously I subscribed to the view that 40-50nm was the limit.  While a 15nm spherical particles would still have problems in passively getting out of the vasculature in most tissues, it is in a dimension where it might become of interest for additional tissue targets than those that we have assumed to be suitable SNALP targets. 

While it is nice to think that SNALP LNPs has more tissues that it might be able to address, it seems that the more tangible, near-term value of the new insights is in how the particles can be designed to target the existing low-hanging fruit tissues such as liver, sites of inflammation, phagocytes and other cells in the blood/lymph, solid tumors (by systemic delivery), and local applications such as lung epithelia by inhalation.

You could for example imagine that this model makes the post-insertion method of adding a targeting ligand more attractive compared to a co-formulation one (both of which have been considered). Similarly, the model changes the space of lipid and RNAi trigger ratios that should be explored.  It also requires new models for the cytoplasmic release of the RNAi triggers from the endosomes to be considered.

Who was first?

While I do not wish to carry on with pointing out potential tensions between Tekmira, Alnylam, and AlCana, the fact that both Tekmira and the Cullis laboratory (‘AlCana’) came out with essentially the same discovery does not leave me much choice but to briefly comment on the coincidence (note that this clash was set in motion before the settlement).

In the patent application by Tekmira, the priority date is June 30, 2010 2011.  As this is also the international filing date, the invention/discovery must have occurred at least a few months, if not at least a year earlier.  The Leung et al. paper was received by the the Journal of Physical Chemistry approximately 2 1 year later (April 5, 2012).  This suggests that the Tekmira were the first to have these insights.  This, of course, could also be critical for the patent application.  If Tekmira were able to get patent protection for such ‘non-lamellar’ LNPs, this could be an important one and somewhat replace in importance the Semple/Wheeler patent estate which are about to expire over the next years.

The new findings illustrate that SNALP LNP delivery continues to be an area a rapid progress.  One would make a mistake to assume that the MC3 formulations currently in the clinic are as good as it gets and that other somewhat overlapping, and therefore competing approaches such as Arrowhead’s DPCs are about to catch up.  Here’s hoping that with the leading delivery technologies having so much obvious room to mature, 2013 will be at least as exciting if not more so than 2012 for RNAi Therapeutics.

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.