Monday, December 10, 2012

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.


Anonymous said...

Well,thats a surprise,another bullish comment on Tekmira.

Anonymous said...

This blog has never been a technological orientated blog – so one should not expect a deeper insight into this really good and interesting paper, without any doubt a great piece of work!

But when you are arguing, „who was first?“ you have to mention this:

Cullis et al. / UBC
filing date 04. November 2010
priority date 04. November 2009

Furthermore, for those who are interested in such lipoplex morphologies, stabilization of HII phases etc. please have a look to e.g. ...

Shi, Engberts, Hoekstra et al. „Interference of poly(ethylene glycol)-lipid analogues with cationic-lipid-mediated delivery of oligonucleotides; role of lipid exchangeability and non-lamellar transitions“ Biochemical Journal, 366 (2002), 333-341.

... already 10 years ago, pretty small and stable particles and with lipids that are expected to favour such dense structures.

Best regards,

Dirk Haussecker said...

Fair point, I did not see that. What allowed the particular dense core LNPs discussed here to be formed was the rapid mixing, either by the T-shaped mixing device or microfluidic mixing. This would seem to set it apart from the 10-year old report that focused on chemistry alone, esp. the impact of PEG-lipids.

I'm therefore curious whether any issued claims will call for certain manufacturing methods or not. If the two patent applications should indeed clash, under the old regime in the US at least the question of first inventorship would come down to idea and subsequent diligence/reduction to practice (this may be traced back in Tekmira's case back to the 2004ish invention of the T mixing device)...

In any case, in the event that Cullis/AlCana prevailed, Tekmira should have access to the patent via the cross-license, although there could be restrictions on sublicensing.

RNAI inv said...

It will be interesting to see if ALNY's new PCSK9 partner will use the old MC3 formulation, or a newer one.

Dirk Haussecker said...

Funny that you mention PCSK9 today,, it's been on my mind as well. My guess is that the subQ conjugate would be part of the PCSK9 partnering package. ALNY has had a nice run these days, this Friday is the subQ webinar, and January has always been a deal-heavy month in RNAi Therapeutics.

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