Monday, December 24, 2007
Journal Club: Trojan Horses that Harbor RNAi to Dupe the Blood-Brain-Barrier
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.
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