Tuesday, July 1, 2014

Leveraging New RNAi Trigger Chemistries for Gene Knockdown in Phagocytic (and Other) Cells

Much of the achievement of solid RNAi gene knockdowns in hepatocytes (liver) by non-LNP means (Arrowhead DPCs and Alnylam’s 2nd gen GalNAc-siRNAs) has involved the use of heavy modifications that render the RNAi triggers highly stable. This is because nucleic acids that are not protected by the delivery chemistry itself would otherwise be subject to rapid degradation in extracellular body fluids.

In the case of Alnylam’s GalNAcs, they can even function in the absence of an explicit endosomal release chemistry.  Moreover, GalNAcs and DPCs have shown more sustained gene knockdowns than is achieved with LNP delivery which historically has relied on minimally modified RNAi triggers. 
All that is required is receptor-mediated uptake into the endosomal-lysosomal pathway which is an immensely degradative environment (esp. the lysosomes).

Considering the extended duration of silencing and degradative environment, it seems as if the RNAi triggers have to be able to survive for long enough in late endosomes/lysosomes so that when they get an opportunity to escape by as yet undefined mechanism(s), they are still there ready for gene silencing action.

Such chemistry progress may also be particularly useful for RNAi gene silencing in phagocytic cells of the immune system.  This is because there have been multiple reports, especially concerning the use of LNPs (e.g. Novobrantseva et al.)  where some, but not the very robust, hepatocyte-type of gene knockdown have been obtained.  It is the ability to confidently achieve robust knockdowns that opens the gate to a flood of therapeutic applications, and this is why pushing borderline-technologies over the edge is so tremendously valuable.

Uptake into phacocytes is usually not the problem as these have evolved to scavenge for foreign particles and macromolecules.  In fact, phagocytic uptake is often a nuisance in RNAi delivery both because it may cause off-target toxicity and because it can make pharmacology less predictable.

In the case of untargeted nanoparticles such as LNPs, phagocytosis is the likely uptake mechanism.  Similar to endosomal uptake, phagosomal uptake involves the fusion with lysosomal compartments meaning that phagosomal contents are also exposed to a highly hostile environment.  Since the normal endosomal escape chemistries and mechanisms do not appear to be very effective in phagosomes, one strategy besides of possibly tailoring existing mechanisms to the phagosomal environment (e.g. lipid pKas) is to simply use the same ultra-stable RNAi trigger chemistries that are showing promise in the liver.

It is also possible that ligand-targeted conjugate approaches will be useful here, whether they enter the cells via phagocytic mechanisms or not.  In fact, Arrowhead Research (then Mirus Bio) in their seminal publication on DPCs (Rozema et al.) have demonstrated efficient uptake of DPC-conjugates into liver phagocytes (Kupffer cells) by using mannose as the targeting ligand (they haven’t tested and/or shown the corresponding RNAi knockdown though and this may relate to their using much less nucleic acid modification back in 2007). 

Since the mannose receptor is expressed on phagocytes throughout the body and not just in the liver, more extended circulation times promises the application of this conjugate-targeting strategy more generally.

Similar principles may apply to the targeting of other ‘frontier tissues’ for RNAi delivery.  However, given the degradative and differing nature of phagocytosis which means that other release mechanisms are not readily applied to this process, RNAi in phagocytes should particularly benefit from the new RNAi trigger chemistry developments.  I look forward to seeing the results.


Anonymous said...

Doesn't Tekmira use Marina triggers? Are't they stable?

Dirk Haussecker said...

UNAs are expected to stabilize, yes. Would have to see how extensively they could be used in a given RNAi trigger and as such could be part of the solution.

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