Gentle Endosomal Escape to Expand Reach of Oligonucleotide Therapeutics

One of the attractive aspects of oligo-mediated ADAR RNA Editing is that it can piggyback on decades of oligo delivery R&D.  New breakthroughs in oligonucleotide delivery are therefore instantly applicable to and greatly enhance the value of the platform. 

In order to make extrahepatic target engagement of oligonucleotide therapeutics as robust as in the liver, various cell-targeting ligand conjugates are being attempted. This includes transferrin receptor-1 in the muscle or various lipid conjugates for the CNS.  The goal is to get highly stabilized oligonucleotides being taken up into the endolysosomal compartments of target cells from which they would somehow escape into the cytoplasm.

Increasing their release through the addition of endolysosomal disrupting agents is highly effective, but it may not be surprising that spilling the insides of endosomes and particularly lysosomes into the cytoplasm in addition to the intended oligonucleotides is a dangerous proposition.  This is illustrated by (not just) the experience with Arrowhead’s DPCs, but also various cationic polymers before it. 

Certain liposomal nanoparticle (LNPs) chemistries are better tolerated.  This may be related to how they release their payloads into the cytoplasm and which may involve complex lipid bilayer dynamics that provide oligonucleotides the opportunity to slip through.

Trapping Oligonucleotides in Metastable Endosomal Compartments

Trapping stabilized oligonucleotides in a similar metastable endosomal membrane environment is what Finicle and colleagues (in a collaboration with Ionis Pharmaceuticals) sought when inhibiting both endosomal recycling and lysosomal fusion with the synthetic sphingosine analog SH-BC-893.

Indeed, ‘893-treated tissue culture cells accumulated ~4 times the amount of a model phosphorothioate gapmer and there was a 9x oligonucleotide increase in the desired cytoplasmic-nuclear compartment.  This in turn lowered the IC50, the concentration of gapmer oligo necessary to inactivate 50% of target RNAs, by over 100 fold.  The IC50 of a model RNAi trigger was similarly lowered 20 fold.

As expected, crude endosomolytic agents proved extremely effective when added to tissue culture cells after the addition of oligonucleotides, but highly toxic.  And when compared to gentler oligo delivery enhancers reported before (AZD8055, 6BIO), ‘893 turned out to be significantly more effective.

Importantly, the sensitization benefit translated to the in vivo setting in a mouse model. Here, ‘893 was given orally around the time of subcutaneous oligonucleotide administration. 

While no gene silencing was really observed when a gapmer antisense oligo was hybridized to a cholesterol-conjugated complementary oligo (which was supposed to aid CNS delivery) and then subcutaneously administered, ‘893 led to noticeable, albeit still quite modest target engagement in some areas of the CNS.

This means that either the approach is optimized and ‘893 can be used in a subcutaneous oligo delivery setting when invasive intrathecal administration is prohibitive, or that ‘893 may be better suited for use along with intrathecal administration of oligos with the goal of achieving liver-like robust target engagements.  For highly severe and relatively rare diseases, the latter would be my preference.

To generalize the in vivo benefit of ‘893 following systemic oligo administration, greatly enhanced RNA silencing was seen in the mouse lung.  Again, I would have liked to see it compared to the benefit that ‘893 might bring to local, in this case aerosolized delivery.

 

Safety of Approach

That this endosomal kinetic entrapment strategy may have clinical legs is supported by the apparent safety of ‘893.  Indeed, this compound is already being considered for cancer and obesity-related indications.  So who knows, you may end up taking your oligos along with ‘893 and similarly acting agents that have anti-ageing activity.  And even if ‘893 should reveal negative side effects following chronic dosing, as a PK enhancer of oligonucleotides it may only have to be given infrequently, depending on how often the oligonucleotides need to be administered.

 

It is nice to see oligonucleotide deliveries making continued inroads throughout the body.  After the liver and CNS, the lung and muscle are becoming fertile hunting grounds for Oligonucleotide Therapeutics developers.  Because of its novelty and differentiation, RNA Editing will be busy with liver- and CNS-related applications alone for some time to come, but when there is robust clinical target engagement by oligos in other organs, RNA Editing can instantly jump on those opportunities.  Next up in terms of extrahepatic delivery is Arrowhead Pharmaceuticals with a number of early clinical readouts for RNAi gene silencing indications of the lung.