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.

Dharmacon’s Accell Delivery Technology Signals Increasing Focus on non-Endosomal siRNA Uptake Strategies

[Update May 8, 2008: Based on a recent patent application by Dharmacon, WO 2008/036825, it appears that Accell siRNAs are in fact very similar in principle to Alnylam's 2004 cholesterol-conjugated siRNA paper in Nature and for which the Australian patent office has now accepted a related patent application (Australian Patent Application No. 2004206255)].

Dharmacon (now part of Thermo Fisher Scientific), the company that rose to prominence as one of the first and trusted siRNA suppliers that understood to maintain their leading position by staying at the cutting-edge of RNAi research, just announced the introduction of Accell siRNAs.

Accell siRNAs may not be just yet another type of modified siRNA, but appear to be inherently cell membrane permeable allowing for straight-forward in vitro gene knockdown in all cell types tested, including the notoriously difficult-to-transfect primary cells, without the need for prior formulation with special transfection reagents. This is in contrast to conventional siRNAs that, due to charge and size, are not cell permeable and are therefore require formulation with delivery reagents such that they may gain access to the cytoplasm (the site of Ago2-mediated RNAi) following endosomal uptake and lysis of that compartment. This can be toxic, either because of the chemistry of the specific formulation or the triggering of cytokine responses by engaging TLR receptors in the endosomes.

It is also clear that although potent gene silencing may be achieved following endosomal uptake, typically only a fraction of the siRNA that had been delivered to the cell is actually active in gene silencing. In fact, the rate-limiting step in vivo in many instances may not be getting the siRNA to the target cell, but getting it into the target cells once in close proximity. In addition to better understanding the endosomal uptake-release mechanism, research into alternative technologies may therefore prove highly rewarding.

Although technical details were not disclosed, it is reasonable to assume that Accell siRNAs have been rendered more cell permeable either by the addition of a small lipophile to the siRNA duplex or by modifying the highly charged siRNA backbone without compromising siRNA silencing activity. Stanley Crooke, the CEO of ISIS, has e.g. noted before that it is the amphiphilic nature of single-stranded antisense nucleic acids facilitates their cellular uptake, so that it is conceivable that Accell siRNAs may have been engineered to be similarly amphiphilic.

The current Accell siRNA technology, however, has some drawbacks in that a special tissue culture media is required and therefore does not allow them to be directly translated to in vivo applications, although formulating them in ways that get them to their target cells should not represent an insurmountable challenge, with small conjugates such as cholesterol being likely candidates. The recommended concentration of 1 micromolar siRNA (about 50-fold higher of what is typically used in vitro) further indicates that the process needs to be optimized before Accell siRNAs will be widely used for large-scale and routine tissue culture experimentation. The immediate market potential should therefore largely come from work with primary cells.

Accell siRNAs reminds us that systemic RNAi delivery is a multi-step process, starting with extravasation of the siRNAs from the blood stream into the tissues to cell attachment and cellular entry, and the potential for improving each step is significant. It is fascinating to see how intensely each of these tasks are being addressed and the many creative solutions coming out of these efforts.