Tag Archives: RNA interference

Animal studies point to clinical trial of hypothermia for stroke victims

On Monday Dr Malcolm Macleod, head of experimental neuroscience at the Centre for Clinical Brain Sciences at the University of Edinburgh, joined scientists from the European Stroke Research Network for Hypothermia (EuroHYP) in urging European governments to fund a  trial of moderate hypothermia for the treatment of ischemic stroke victims.  In ischemic stroke the blood supply to part of the brain is blocked, leading to the death of nerve cells in the affected area, which can result in death or long-term disability. In an interview with the BBC Dr Macleod was reported as saying that:

Every day 1,000 Europeans die from stroke – that’s one every 90 seconds – and about twice that number survive but are disabled…Our estimates are that hypothermia might improve the outcome for more than 40,000 Europeans every year.”

This call does not come as a great surprise for me; when I was researching the role of animal research in the development of brain cooling to treat perinatal hypoxic-ischemic encephalopathy (HEI), a condition where a lack of oxygen and reduced blood supply during or shortly after birth causes brain damage, I found that all the papers I read cited animal studies of hypothermia to prevent damage in ischemic stroke. This is not surprising as in both conditions injury results from impaired blood supply.

CT image of an ischemic stroke. The dark area in top left quadrant of brain shows the damaged brain area. Welcome Images.

The publications page of the EuroHYP website lists the most important publications supporting their decision to initiate large-scale clinical trials of hypothermia in stroke. Among them is a 2010 review by Bart van der Worp, Malcolm MacLeod and Rainer Kollmar entitled “Therapeutic hypothermia for acute ischemic stroke: ready to start large randomized trials?” which highlights the importance of studies in animal models of stroke in demonstrating the potential of hypothermia in stroke, and states:

…we believe that hypothermia has been studied in sufficient detail and under a sufficiently broad variety of experimental conditions in animal models of ischemic stroke to support the translation of this treatment strategy to clinical trials”

Among the papers cited by this review is a systematic review and meta-analysis published in 2007 by a group of neurologists led by Dr Mcleod and Dr van der Worp which made a very thorough examination of over one hundred studies of different hyporhermia techniques in a range of animal models of ischemic stroke. This study is clear about the limitations of the studies, and identifies several areas where further animal studies are warranted, such as the longer term effect of hypothermia on the risk of developing pneumonia. Overall the authors conclude that:

In animal models of focal cerebral ischaemia, hypothermia improves outcome by about one-third under conditions that may be feasible in the clinic, with even modest cooling resulting in a substantial improvement in outcome. Cooling is effective in animals with co-morbidity and with delays to treatment of 3 h. Large randomized clinical trials testing the efficacy of moderate hypothermia in patients with acute ischaemic stroke are warranted”

This is important, as anyone familiar with stroke research will recognize Dr Macleod and Dr van der Worp as fierce critics of inadequate design and reporting of some preclinical animal studies, and of mistakes made when designing clinical trials due to the misinterpretation and misapplication of the results of animal studies. Quite often they found that the design of clinical trials was so different to the design of the preclinical study that it was impossible to tell whether the failure of a treatment in human patients actually contradicted the earlier success in an animal model, both outcomes were entirely plausible even if you assumed that there was absolutely no fundamental biological difference between the effects of stroke in the animal model and in human patients. For example, one problem is that the majority of neurprotective drugs evaluaded in the past few decades were shown to be effective in animal models of stroke only when administered very soon after induction of stroke – usually after less than half an hour – whereas in clinical trials there were usually long delays – four hours or more-  before initiation of treatment.  The fact that hypothermia has a neuroprotective effect in animal models up to three hours after stroke onset will make design of a clinical trial that matches the conditions under which treatment was successful easier, though as with all stroke treatment the earlier it is started the better!

It is notable that unlike animal rights campaigners who use deficiencies in some animal studies to call for a ban on it, Macleod and van der Worp understand its continuing importance to medical progress, and have worked with animal researchers to improve both the design and reporting of the preclinical animal studies that underpin the decisions to initiate clinical trials.  Initiatives such as the ARRIVE guidelines are similar in many ways to recent improvements the design of clinical trials supported by the work of the Cochrane collaboration, and the widespread adoption of standards for the reporting of clinical trials.

So the animal evidence supporting the clinical initiation of trials of hypothermia for ischemic stroke had to satisfy a very strict panel of judges, we hope that funding is provided to initiate these important trials in the very near future.

Finally, and completely off topic, there was an interesting item in Nature news on the use of RNAi to attack block viral replication in a mouse model of HIV infection. It’s an interesting application of an exciting new technology that we have discussed several times on Speaking of Research, indeed back in 2008 we discussed the work of another group who are using a mouse model of HIV to aid development of RNAi based therapies for HIV infection. It is fascinating work, though as the Nature article stresses the technique needs to be refined, re-evaluated and improved a lot in animal models before it can be tried out in clinical trials of HIV patients. I expect that Drs Macleod and van der Worp would agree with that sentiment.

Paul Browne

Mice, Nanotechnology, and Inflammatory Bowel Disease

Back in March I discussed a new therapy that combines nanotechnology and RNA interference (RNAi) to treat metastatic melanoma, and how basic and applied animal research has contributed to its’ development.  Now researchers at the Georgia Institute of Technology and Emory University have reported the development of another nanotechnology and RNAi approach to treating inflammatory bowel disease (IBD), and the report published in Georgia Tech Research News highlights the importance of research in a mouse model of ulcerative colitis to the development and evaluation of their novel nanoparticle drug.

Nanoparticles remain intact in healthy tissue (upper panel). In inflamed tissue reactive oxygen species break down the nanoparticle shell to release siRNA and lower TNF-alpha levels through RNAi (lower panel). Image courtesy of Scott Wilson.







IBD is a term that covers a range of conditions where a dysfunctional immune system causes inflammation in the intestine, and includes disorders such as Crohn’s disease and ulcerative colitis which can be very debilitating to sufferers, and ultimately very damaging to their health. While treatments are available they do not work well in all cases, and often have serious side effects, so new treatments that target the inflamed tissue while sparing healthy tissues are highly desirable.

The way this new nanoparticle developed by Georgia Tech and Emory targets inflamed tissue differs from that used by scientists at Caltech to target melanoma cells. The latter employs nanoparticles coated with a protein called transferrin that is preferentially absorbed by cancer cells, while the former relies on reactive oxygen molecules produced by the inflamed tissue to break down the thioketal polymer shell of the nanoparticles, releasing the siRNA payload that triggers RNAi.

What the two approaches share is that they demonstrate the vital role played by animal research in advancing the use of nanotechnology in 21st century medicine.

Paul Browne

RNAi: Send in the Nanobots!

The publication of the preliminary results of a small clinical trial of a new therapy called RNA interference (RNAi) online in the scientific journal Nature is causing quite a stir in the scientific community this week.  A team led by Professor Mark E. Davis at Caltech targeted the delivery of a nanoparticle only 70 nanometers in diameter containing small interfering RNA (siRNA) to cancer cells in three patients with metastatic melanoma, which reduced the levels of a protein called RRM2 that is required for the tumour growth.  This trial is the result of over a decade of research in organisms as diverse as nematode worms, mice and monkeys, but why is the result of this trial so noteworthy? And what is RNAi anyway?

Cancer genes in human melanomas have been switched off. Image courtesy of the National Cancer Institute

If you have ever studied biology you will probably be familiar with the “central dogma of molecular biology”; it describes how our genes encode the proteins that are the building blocks, and indeed the builders, of all the cells in our bodies. The very short version is that our genes are made up of sequences of double stranded DNA consisting of the deoxyribonucleotides A,C, G and T, and these sequences are transcribed by a protein called RNA polymerase into matching sequences of the single stranded messenger RNA (mRNA) , made from the ribonucleotides A, C, G and U. Another protein complex known as the ribosome then translates the mRNA sequence into a corresponding sequence of amino acids that when completed make up a brand new protein.  Our new protein almost invariable undergoes further processing but we needn’t concern ourselves with that here.  RNAi is the process where an assembly of proteins named the  RNA-induced silencing complex (RISC) binds short double stranded segments of RNA that in turn target RISC to particular mRNA sequences to which they are complementary.  RISC breaks down the mRNA molecule, preventing production of its associated protein and effectively silencing the targeted gene. The beauty of RNAi is that it allows an organism to target specific mRNA molecules for destruction, and it is a mechanism for regulating the flow of genetic information whose importance we are still only beginning to appreciate.

RNAi was discovered only 12 years ago by Andrew Fire and Craig Mello through their basic research on the regulation of gene expression in the nematode worm Caenorhabditis elegans, a discovery which earned then the Nobel Prize in 2006. C.elegans is a popular model organism for scientists studying gene function and development, its small size and simple structure make it relatively easy to follow the fate of individual cells, while as an animal it shares many of its genes and biological processes with mammals.  This turned out to be the case with when in 2001 it was shown that RNAi helps mice to control hepatitis B infection, and scientists began to examine whether RNAi could be used therapeutically (1). To do this scientists made siRNA, an artificial version of the short double stranded segments of RNA that target RISC to complementary mRNA sequences, and early experiments in mice demonstrated that siRNA induced RNAi could reduce the levels of target proteins in mice.  The first human trials of RNAi began in 2004 for the treatment of wet age related macular degeneration and at first seemed promising, but suffered a setback when further research in mice revealed that the “naked” siRNA injected into the eye in these trials actually stimulated an immune response that was responsible for at least some of the benefits seen in earlier trials (2). This was a worry as an unwanted immune response might lead to an adverse reaction if the siRNA was injected into the bloodstream rather than a small part of the eye.

In recent years scientists have been developing technologies that allow injected siRNA to evade the immune system and target only those tissues where RNAi activity is desired,  reducing the quantity of siRNA that needs to be injected and also the risk of adverse  effects due to RNAi affecting off-target tissues. Mark E Davis, a professor of chemical engineering at Caltech and one of the scientists leading these efforts, uses polymers that assemble with siRNA to form a nanoparticle that resembles a tiny ball with siRNA at its centre.  The nanoparticle shell protects the siRNA from being broken down while it is circulating in the bloodstream, and then interacts with the cell membrane to help the siRNA enter a cell so that it can do its job.  Of course he didn’t want the nanoparticle to release its siRNA payload into any old cell so he attached a protein called transferrin as a targeting ligand to the nanoparticle. Tumour cells express far more of the transferrin receptor on their surfaces than normal cells, and the hope was that the nanoparticles would bind to tumour cells in preference to normal cells.  To test whether this would work Prof. Davis team injected the nanoparticles, containing a siRNA that targeted a cancer gene, into mice that had metastatic Ewing’s sarcoma(3). They observed that the transferrin labelled nanoparticle delivered the siRNA to the tumour cells, knocked down the activity of the target cancer gene and dramatically slowed tumour growth, and when the transferring ligand was removed his effect not seen.  They also observed that the nanoparticle did not stimulate the immune system or affect any of the major organs of the mouse, indicating that their method had solved safety problems seen in earlier RNAi trials.

The targeted nanoparticle used in the study and shown in this schematic is made of a unique polymer and can make its way to human tumor cells in a dose-dependent fashion. Image courtesy of Derek Bartlett and the California Institute of Technology.

Prof. Davis and his colleagues next needed to identify an appropriate target for human trials of their nanoparticle siRNA delivery system, and decided to target the M2 subunit of Ribonuclease reductase (RRM2), a protein that is required for cell division and which has recently been the subject of a lot of research as a target for anti-cancer drugs.   They first used in vitro studies to identify a siRNA sequence that effectively targeted the RRM2 mRNA, which they named siR2B+5, and then demonstrated in mice that this siRNA could block the production of RRM2 and reduce the growth of tumours (4).  As a final safety evaluation prior to human trials they injected different doses of their nanoparticle  containing siR2B+5 and labelled with transferrin to cynomologus monkeys, whose RRM2 mRNA is targeted by siR2B+5 in exactly the same way as in humans,  and found that it was safe and did not produce any unwanted effect on the immune system (5).

The human clinical trial reported this week confirmed that transferrin-labelled nanoparticle injected into the bloodstream were safely delivered siR2B+5 to the tumours of metastatic melanoma patients, and that the siRNA knocked down the production of RRM2 protein by RNAi (6).  Of course this is only a preliminary result, at this stage we don’t know to what extent this experimental treatment will reduce tumour growth in these patients, let alone if it will cure their cancer. If it is a success it will probably need to be combined with other anti-cancer drugs to be fully effective, so it is good to know that thanks to animal research other nanotechnology based drugs such as Lipoplatin are in clinical trials that offer more potent anti-cancer activity with less toxicity than existing anti-cancer drugs. Nonetheless to focus on this uncertainty would be to miss why this small trial is causing such excitement; for the first time scientists have shown that it is possible to target RNAi therapy to a particular tissue type within the body, and that is a breakthrough that opens up a whole new area of medicine. The era of RNAi medicine has begun!

Paul Browne

1)      McCaffrey A.P., et al. “RNA interference in adult mice.” Nature Volume 418, pages 38–39 (2002) DOI: 10.1038/418038a

2)      Castnotto D. and Rossi J.R. “The promises and pitfalls of RNA-interference-based therapeutics” Nature Volume 457(7228), pages 426-433 (2009) DOI:10.1038/nature07758

3)      Hu-Lieskovan S. et al. “Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma.” Cancer Re. Volume 65 (19), Pages 8984-8992 (2005) DOI:10.1158/0008-5472.CAN-05-0565

4)      Heidel J. et al. “Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo” Clin. Cancer Res. Volume 13(7), Pages 2207-2215 (2007) DOI: 10.1158/1078-0432.CCR-06-2218

5)      Heidel J. et al. “Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA.” Proc.Natl Acad. Sci. USA Volume 104(14), Pages 5715-5721 (2007) DOI: 10.1073/pnas.0701458104

6)      Davis M.E. “Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles”  Nature Advance Online Publication 21 March 2010 DOI:10.1038/nature08956