Oligonucleotide Strategies beyond the Liver

As the first major wave of pivotal trial successes involving gene knockdown in the liver has reached the shores, oligonucleotide therapeutics are quickly establishing themselves as the dominant modality in this important target organ for new drug development.  While the industry is milking this organ for therapeutic applications, my thoughts are directed towards the next tissue opportunities that should further feed this revolution in drug development we are witnessing now.

After not having been part of the conference circus for 2 years, this week’s Oligonucleotide Therapeutics Society annual meeting in Bordeaux, France, was just what the doctor ordered for me to get a clear perspective on this issue.  Importantly, lessons learned from GalNAc-targeted oligonucleotide delivery to hepatocytes, but also LNP delivery to the liver prior to this, now allow the field to take the next step in extending delivery beyond the liver.

Chemical stability

All these efforts essentially share their use of highly modified oligonucleotides which have particularly changed the philosophy around the RNAi modality.  This allows the oligonucleotide to not only reach the target tissue intact, but also to remain trapped in endosomal compartments which serve as slow-release depots for long duration of action.

Chemical modification also has greatly reduced the immunogenicity of RNAi triggers and obviated the need for protective nanoparticle formulations.  These often came with the added liability of amplifying the immunostimulatory potential of these molecules.  Consequently, decade-old approaches are now being revisited with more fully modified RNAi trigger versions (e.g. self-delivering RNAi trigger structures as pursued by RXi Pharmaceuticals and the Khvorova group at UMass).

Ironically, after all the song and dance by RNAi bellwether Alnylam about the utility of exotic modifications in their conference presentations (one can also call it willful misleading of the field- not really the purpose of scientific conferences), the strong trend is towards maximizing 2’-O-methyl content, in addition to some 2’-F and phosphorothioation at the RNAi trigger termini.

PK enhancers

One of the reasons why the liver became the first major oligonucleotide target organ is that it is readily accessible from the blood.  This allows it to soak up oligonucleotides before they get removed by renal filtration.  In an effort to fight this tendency, the use of PK enhancers, in particular lipophilic groups is frequently seen.  Ionis and Alnylam are testing these for example for getting better distribution to the muscle and potentially also better functional uptake.

Receptors

PK enhancers, however, are largely about shifting around biodistribution, but it is hepatocyte ASGPR-type receptors that are the most valuable assets the industry is striving to identify.  My highlight of the conference therefore was a talk on a collaboration by AstraZeneca and Ionis demonstrating strikingly selective and effective targeting of beta cells in pancreatic islets. Think diabetes! 

It is the GLP1-receptor that does the magic here and which can be targeted by GLP1-peptides for effective oligonucleotide uptake.  While the in vivo validation was limited to rodent models, including an elegant GLP1-receptor knockout mouse model, I am convinced that the findings will translate to larger animals and humans.  It is one of those things you just know when seeing such data.

This example illustrates the value of knowing your target cell type really well, as this may allow you to identify additional ASGPR-type receptors which had been thought of elusive.  But even if they are lacking in some tissues, a nice, yet simple strategy to overcome this was illustrated by MPEG LA and Axolabs: by linking more than one RNAi trigger to a small scaffold, they were able to show that cellular oligonucleotide uptake capacity can be increased beyond the limits of receptor amount on the cell surface.

 

While the liver certainly does not need this strategy, it should definitely be applied to new targets like the beta cells.  Let free market competition do its magic and have oligonucleotide therapeutics solve diabetes now that you can effectively reach hepatocytes, adipocytes, and now also beta cells. Yes, I love the free markets, but I digress… 

After beta cells, it was a collaboration between Alnylam and Johnson & Johnson on overcoming the long-held dream of oligonucleotide therapeutics addressing gene regulation in cancer cells.  Here, small and stable ~2nm peptide scaffolds referred to as centyrins were coupled to the RNAi trigger and directed towards different receptors like PSMA and EGFR.  Perhaps the most striking aspect of centyrin-siRNA conjugates was their effective tumor penetration where prior RNAi delivery attempts like LNPs had fallen short.

Endosomal release

Sometimes getting to the endosomes alone is not enough when the rate of cytosolic release therefrom is insufficient.  So despite of the DPC fiasco last year and despite of aborted arginin-based endosomal release attempts prior to this, active endosomal release is still embraced in some delivery efforts.  Most notably, Sarepta has shown dramatic increases in dystrophin exon skipping in non-human primates with new peptide-PMOs (PPMOs) compared to their unconjugated parent molecules.

Of course, everybody now wants to know what the therapeutic window really is.  While the Sarepta representative at OTS was a bit cagey when asked about it, Sarepta’s CEO noted in a recent investor presentation that the filing of an IND by the end of the year would be a major positive signal in that regard.

Finally, all of the above developments are aided by more general progress in technologies interrogating biology such as single cell technologies (cell type isolation from complex tissues like the kidney), reduced chemistry costs allowing for much larger numbers of oligonucleotides to be screened, and ubiquitous low-cost and high-throughput sequencing.

 

Sometimes I pinch myself asking whether all this is real and not just a figment of my imagination, but at least in my mind the stars just keep aligning allowing for RNAi and oligonucleotide therapeutics to take the next step up the value ladder.