Less Is Sometimes More: Roche Brain Shuttle Technology for Drug Delivery into CNS

After Roche and ISIS Pharmaceuticals announced last April that a co-development effort to apply Roche’s Brain Shuttle technology for the systemic delivery of antisense oligos would form part of their Huntington’s collaboration, I started to pay some attention to this technology.  This is because despite the already enormous promise of Oligonucleotide Therapeutics for CNS disorders (think of all the neurodegenerative diseases) with the direct administration of single-stranded oligonucleotides into the CNS, its value could be further enhanced with a systemic delivery approach such as the Brain Shuttle tech.

This is not only because intravenous administration methods would be preferable over the more invasive direct intra-CNS injections/infusions, but also because a systemic delivery approach promises a more uniform drug distribution and would minimize the importance of diffusion.  It is the limited diffusion of current RNAi delivery techs that is holding back RNAi Therapeutics in this important therapeutic area.

Monovalent antibody binding to transferrin receptor allows for efficient transcytosis

The fundamental principle behind Brain Shuttle is actually not that novel at all. Companies like Armagen have long attempted to target receptors such as the insulin receptor and the transferrin receptor on brain capillary endothelial cells as in normal physiology these receptors function to shuttle transferrin/iron and insulin across the notoriously recalcitrant blood-brain-barrier.

Based on newly published data by Roche scientists (Niewoehner et al. 2014), a conventional antibody approach does not work.  To wit, a conventional antibody consists of two binding sites (divalent) and such engagement apparently causes receptor trafficking to be re-directed to the degradative lysosome compartment of the brain endothelial cells, at least in the case of the transferrin receptor tested.  By contrast, when receptor targeting occurs via monovalent interaction, the normal receptor physiology, including transcytosis, is maintained.  Using this strategy, it was shown that the intra-parenchymal delivery, i.e. delivery into the brain proper, of an antibody against beta-amyloid (related to Alzheimer’s disease) that had been tethered to the monovalent transferrin receptor antibody was enhanced on the order of 50-fold.

The way how this research can be translated into RNAi Therapeutics is to simply append an RNAi trigger instead of the beta-amyloid antibody to the transferrin receptor antibody.  Or you could do away with proteins altogether and replace the transferrin receptor antibody with an aptamer-RNAi trigger combo (e.g. a Dicer-substrate in the spirit of tomorrows IPO by Dicerna Pharmaceuticals).  Hence, the irony of the ISIS-Roche delivery collaboration is that ISIS could- to put it just slightly hyperbolically- be shoveling its own grave by eroding the current advantage of phosphorothioate antisense over RNAi for gene knockdown in the CNS.

Journal Club: Trojan Horses that Harbor RNAi to Dupe the Blood-Brain-Barrier

An interesting paper relating to the delivery of RNAi Therapeutics to the brain via systemic administration was recently published by the Pardridge group at UCLA in the journal Pharmaceutical Research (Xia et al.: “Intravenous siRNA of Brain Cancer with Receptor Targeting and Avidin-Biotin Technology.”).

The Pardridge group has been working on so called Trojan Horses to get pharmaceutical agents across the blood-brain-barrier. This may be achieved by coupling the active drug ingredient to a monoclonal antibody that targets receptors on the vascular endothelium of the brain which can then ferry the entire complex through the endothelial cell layer and into the brain.

In this study, Xia and colleagues combine streptavidin-antibody fusion proteins to biotinylated siRNAs which form tight complexes mediated by the extremely high affinity of streptavidin for biotin. The antibodies recognize the transferrin and insulin receptors which are the most widely studied receptors in this apparently understudied area of research. After demonstrating that it is possible to generate reasonable amounts of pure Trojan Horses, the authors use them for knock down studies in tissue culture with 75-85% knockdown efficacy which was further dependent on the antibody. Importantly, the biotin-streptavidin interaction did not impair knockdown efficacy and both 5’ and 3’ biotinylation of the passenger siRNA strand was tolerated, in support of the flexibility of this technology.

They then move on to an in vivo tumour model where rat glioma cells expressing a reporter gene (luciferase) are implanted into the brains of rats and grown for 5 days. At this point, the animals were intravenously administered with the Trojan Horses incorporating an siRNA against the reporter gene. Remarkably, at a dosage of as little as 270ug/kg siRNA (this does not include the protein component of the formulation though), luciferase levels stabilized and were 4 fold less compared to animals injected with saline control alone, and no obvious toxicities were observed.

One critical control that I would have liked to see is a Trojan Horse with a control siRNA as the assay did not distinguish whether the relative decline in luciferase activity was the result of actual gene knockdown or due to some cytotoxic effect, so that one could rule out that binding of the monoclonal antibody to transferring receptors on the cancer cells itself was the cause for the reduction in luciferase activity. Another nice experiment would have been to target a cancer-related gene and look at survival and other therapeutic measures. In any case, it will be interesting to follow the progress of this technology which appears to be partnered with the Californian biotech company ArmaGen Technologies.

PS 1: In my 19 June 07 Blog: “New Breakthrough in the Systemic Delivery of RNAi for the Brain” I described a related study published in Nature where a rabies peptide facilitated the transfer of an siRNA across the BBB. In regards to that study, Xia et al. make the following cryptic remark: “The RVG peptide is hypothesized to cross the BBB via receptor-mediated transport on the brain capillary endothelial nicotinic acetylcholine receptor. However, activation of brain microvascular AChR causes BBB disruption.” Although temporarily disrupting the BBB may be a way to get drugs into the brain, it seems to raise safety concerns as the BBB serves to keep bad stuff, such as viruses out of the brain for a reason.

PS 2: Last week, Santaris signed a nice collaboration agreement with GSK for the development of LNA-based antisense drugs valued at up to $700M, further highlighting Big Pharma's interest in RNA-based therapies. While I have much to learn about the safety of LNAs, I have little doubt that they can be quite efficient in binding to their targets and are perhaps particularly attractive for antagonising microRNAs since the simple act of sequestering a microRNA would already be effective. Unfortunately for retail investors, the same week it announced another private funding round for M20 Euros. Santaris should find sufficient interest should it decide to go public.