Tag Archives: RNAi

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

From the bench and the bedside; how animal research is taming Multiple Sclerosis

Multiple sclerosis (MS) is one of the most common diseases of the central nervous system – the brain and spinal cord – affecting about one person in every thousand in the USA. It is an inflammatory condition, where the immune system attacks the myelin sheath that surrounds the axons of nerve cells. Myelin is a fatty material that insulates nerves, acting much like the covering of an electric wire and allowing the nerve to transmit its impulses rapidly. It is the speed and efficiency with which these impulses are conducted that permits smooth, rapid and co-ordinated movements to be performed with little conscious effort. Loss of myelin interrupts these impulses, and the nerve cells themselves are also damaged and eventually die. 

The consequences for people with MS can be devastating, and MS is associated with a wide variety of symptoms, including muscle weakness, spasms, ataxia, problems with speech and vision, acute and chronic pain, and fatigue.  MS is a very variable disorder, and the rate at which it progresses varies considerably from one patient to another, but a defining characteristic of it is the lesions that are visible by MRI where the myelin has come under attack. The relapses, attacks of worsening neurological function that are often found in MS, are closely associated appearance of new lesions in the CNS, although not all new lesions cause a relapse.

Until about 20 years ago there were no treatments available that could prevent relapses or slow the progression of MS – known as disease modifying treatments – but thanks to the efforts of scientists working around the word this situation has begun to change.   A number of effective disease modifying treatments are now available, the most recent to receive FDA approval is Fingolimod (known as FTY720 during its development), a drug whose immunosuppressant properties in reducing transplant rejection and as a treatment for MS were evaluated in a range of animal models during its development.

These drugs may soon be joined by another. A couple of years ago I wrote about the crucial role of studies in mice, rats, and dogs in the development of a new disease modifying treatment called Laquinimod, which safely -though relatively modestly conpared to other new therapies – reduced the number of relapses, while slowing progression of disability more that current disease modifying drugs in a Phase III clinical trial. This is good news, and one more step towards turning MS form being an incurable disease to being a manageable disease.

One reason I say manageable rather than curable is that while these treatments are effective in reducing the number of relapses for many patients they do not work for all patients and all forms of MS (particularly for primary progressive MS), and can sometimes have serious side effects that prevent patients from continuing treatment. That is why scientists are continuing to study the biological mechanisms in MS, a disease whose origin is still not fully understood, though clinical and animal research indicates that both genetic and environmental factors play a role, their ultimate goal is to develop treatments that can stop relapses altogether.

Another reason for not referring to disease modifying treatments as “cures” is that they do not directly repair the damaged myelin sheath at the lesions. Spontaneous repair of the damaged myelin sheath in MS lesions does happen and plays an important role in limiting neurological damage, but until now the molecular basis of myelin regeneration by cells called oligodentrocytes, in the central nervous system (CNS) has been poorly understood. The Guardian reports on how scientists at the University of Cambridge have discovered how to promote remyelination in MS lesions by activating a population of stem cells in the CNS called oligodentrocyte precursor cells (1).

The team led by Professor Robin Franklin generated a comprehensive transcriptional profile of 22,000 genes during the separate stages of spontaneous remyelination that follow focal toxin-induced demyelination in the rat CNS, and found that the level of retinoid acid receptor RXR-gamma expression was increased during remyelination. Cells of the oligodendrocyte lineage expressed RXR-gamma in rat tissues that were undergoing remyelination, in both active lesions and in older remyelinated  lesions. By examining post-mortem brain samples from MS patients, they were able to show that RXR-gamma expression was also elevated in oligodendrocyte precursor cells at the active lesion sites, supporting a general role for RXR-gamma in remyelination. Interesting as these findings were they did not demonstrate that RXR-gamma is actually required for remyelination, so they next performed studies to determine whether blocking the function of RXR-gamma would prevent remyelination.

Rats are crucial to many areas of MS research. Image courtesy of Understanding Animal Research.

Knockdown of RXR-gamma by RNA interference or RXR-specific antagonists severely inhibited the differentiation of oligodendrocyte precursor cells into mature oligodendrocytes in culture. In mice that lacked RXR-gamma, adult oligodendrocyte precursor cells efficiently repopulated lesions after demyelination, but showed delayed differentiation into mature oligodendrocytes. The next question was whether increasing the activity of RXR-gamma would speed up remyelination. Administration of the RXR agonist 9-cis-retinoic acid to demyelinated mouse cerebellar slice cultures and then to aged rats in vivo after focal demyelination caused an increase in remyelinated axons. Focal toxin-induced demyelination was used to produce the lesions, rather than an immunity mediated model of demyelination such as experimental autoimmune encephalomyelitis, in order to determine that the increased remyelination was due to promotion of oligodendrocyte differentiation rather than to the anti-inflammatory effects of 9-cis retinoic acid.

The results indicate that RXR-gamma plays an important role in endogenous oligodendrocyte precursor cell differentiation and remyelination, and might be a pharmacological target for regenerative therapy in MS. The discovery that 9-cis-retinoic acid, a compound already in limited clinical use, can be used to stimulate myelin regeneration raises the possibility that within the next decade treatments that repair the neurological damage in MS will begin to enter clinical trials.

For people with MS these scientific and clinical advances are a great source of hope for a better future.

Paul Browne

1)      Huang J.K. et al. “Retinoid X receptor gamma signalling accelerates CNS remyelination” Nature Neuroscience Published Online 05 December 2010 DOI: 10.1038/nn.2702

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

Mice, rats, and the secrets of the genome.

It’s just over a decade since the completion of the first working draft of the human genome was announced, and seven years since the publication of the complete sequence, but in that short time the impact of this new knowledge on all areas medical research has been immense. Sequencing the human genome was a huge achievement, but having got the sequence an even greater task confronts scientists – working out what it all means.  To do this scientists have studied the natural variations that exist between individuals, and have also sequenced the genomes of a wide variety of species, some closely related to us, others separated from us by hundreds of millions of years of evolution. Scientists can analyze the similarities and differences between the genes of different species, and examine how changes to the structure or regulation of these genes are reflected in physiology.  In many cases it is also possible to use genetic modification to study the function of conserved genes in other species in ways that are just not possible, for technical and/or ethical reasons, in humans. A study published a couple of weeks ago in the scientific journal Nature provides an excellent example of how animal research contributes to our understanding of the human genome.

Blood flowing through the heart, but the cholesterol building up in an artery can cause a heart attack. Image courtesy of the British Heart Foundation

As the cost of technology such as DNA microarrays has fallen genome-wide association studies (GWAS) have become an increasingly popular way of examining the relationship between genetic differences between individuals and particular diseases. In a GWAS the whole genome of many individuals is screened for variations, and then any association between those variations and particular phenotypes or diseases is determined.  Tanya M. Teslovich and colleagues (1)  analysed the genomes of over 100,000 people who had been enrolled in 46 separate clinical studies, and identified 95 genes that have variants associated with increases in blood lipid (fat and cholesterol) levels.  One of the problems with GWAS studies is that while they are often good at identifying genes that are associated with a disease, they are not so good at identifying which genetic variations actually cause disease, or explaining how the genetic variations contribute to disease.  This is where Tanya Teslovich and colleagues scored highly; they were able to show that 14 of the 95 lipid-associated genes were also associated with the development of coronary artery disease, supporting the proposition that elevated blood lipids contribute to coronary artery disease. They also found that overall the effect of the variants was additive, the more risk variants of these 95 genes you have the greater your chance of having elevated blood lipids.

So that established that the gene variants were associated with elevated blood lipids, but to use that information to develop new treatments you need to know how the particular gene affects lipid levels. As you might expect many of the 95 genes identified were already known from previous studies to be involved in the regulation of blood lipids, and in several cases their precise role has been thoroughly studied. However, several of the genes had not been implicated in regulating blood lipids before, and the team decided to use genetically modified mice to investigate their function.  They injected viral vectors into the liver of the mice that contained either an extra copy of the gene being studied, to increase expression of the gene, or a short-hairpin RNA, to target the gene for knockdown via RNAi. This allowed them to discover that one gene, GALNT2, decreases levels of high-density lipoprotein cholesterol (HDLC), the so-called “good cholesterol”, while two other genes, Ttc39b and Ppp1r3b, increase  HDLC.

Another associated paper (2) in the same issue of Nature takes the analysis even further. Several studies, including the GWAS performed by Tanya M. Teslovich and colleagues, had demonstrated that variations in a particular region of chromosome 1 known as 1p13 were associated with high levels of Low-density lipoprotein “bad” cholesterol (LDLC) in the blood and heart disease, but that these variations were not within the coding sequence of any genes, so they would not affect the structure of any proteins. They first show through genetic studies of human subjects and human liver tissue culture that variations at 1p13 affect the expression of several genes –  and hence the amount of protein produced by those genes – and that one particular variation creates a binding site for the transcription factor C/EBP. Transcription factors are proteins that regulate the expression of genes, and this particular site altered the levels of a gene named SORT1. But what does SORT1 do? To answer this they again turned to GM mice, using virus vectors that specifically reduced or increased the levels of SORT1 in the mouse liver. Reducing or eliminating SORT1 expression in the mouse liver led to a reduction in the levels of LDLC in the blood, and that this was found to be due to SORT1 regulating the production of very-low-density lipoprotein (VLDL), a precursor to low-density lipoprotein, in the liver. As a result of this work a whole new pathway for the regulation of blood lipids has been uncovered, one that may offer new opportunities to scientists developing treatments for hypercholesterolemia.

As a BBC news report indicates, the identification of these genes and the elucidation of their function may aid the development of better diagnostic tools to identify those at risk of heart disease, and ultimately the development of better treatments.

These studies illustrate how important animal models, particularly GM mice, are to efforts to decode the human genome. As the biosciences move towards a more systems based approach to biology, one where knowledge of how networks of genes interact to produce a particular physiological or clinical outcome is applied to areas such as toxicology, the information that studies of GM animals can yield will become increasingly important. This importance has not gone unrecognized by the wider scientific community, the 2007 Nobel Prize in Medicine was awarded to Mario Capecchi, Sir Martin  Evans, and Oliver Smithies for their discoveries of  “ principles for introducing specific gene modifications in mice by the use of embryonic stem cells”.

With this in mind let’s turn briefly to another GM animal that’s been in the news lately – the rat.  While GM mice have become a mainstay of modern scientific research the rat has lagged behind, which is a shame since the larger size, longer lifespan, and more complex behavior of rats make them more effective animal models than mice for studying many human diseases, particularly neurological conditions. The lack of GM rats was due to the difficulty in growing rat embryonic stem cells (ESCs) in culture, a necessary first step in the most common methods of producing GM animals. Last year Matthew Evans wrote an article for the Pro-Test blog discussing how scientists at the University of Cambridge and the University of Southern California had developed a method for growing rat ESCs in culture, and how this achievement paved the way for the production of transgenic rats. Last week the same group of scientists announced that they had employed this method to produce GM rats whose p53 gene, a key tumor suppressor that is defective in several cancers, was deleted.

The humble lab rat, now available in GM. Image courtesy of Understanding Animal Research.

This is not the first time GM rats have been produced, as for the past few years scientists have been able to use zinc finger nucleases to knock-out rat genes. Zinc fingers, so called because one or more zinc ions stabilizes the finger like structure, are found in many proteins, allowing them to bind specifically to a structure within a cell, such as a particular DNA sequence. Scientists found that they could produce artificial zinc fingers that recognize particular genes, and then join a nuclease to that zinc finger so that it cuts out the target gene. This method, discussed in more detail in this excellent article by Elie Dolgin, allows scientists to knock-out genes in rat embryos. The downside of the zinc finger nuclease technique can only be used to knock-out genes, whereas the ESC method is more flexible – it can also be used to add extra copies of a gene, or to delete genes in specific tissues or stages of development.

It is now clear that the rat is joining the mouse at the forefront of the GM revolution in medicine, and that has to be great news for medical science and the patients that depend on it.

Paul Browne

1)      Teslovich T.M. et al. “Biological, clinical and population relevance of 95 loci for blood lipids” Nature Volume 466, Pages 707-713 (2010) DOI:10.1038/nature09270

2)      Musunuru K. et al. “From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus” Nature Volume 466, Pages 714–719 (2010) DOI:10.1038/nature09266

Septic shock: Mice show way to a new treatment

When we think of the immune system we usually think of the adaptive immune system – the B-cells and T-cells that recognize and destroy specific pathogens – which isn’t surprising since this is the arm of the immune system that vaccines are designed to stimulate.  However working alongside the adaptive immune system is the innate immune system which protects us form infection in a non-specific fashion. Key to this system are phagocytes, a diverse set of cells whose primary characteristic is their ability to consume and digest invading microorganisms and secrete a range of chemical messengers known as the proinflammatory cytokines which stimulate other components of the immune system. This usually a useful part of the immune response, but sometimes there is an excessive release of cytokines which causes the patient to enter a condition known as septic shock where the immune system over-reacts and causes serious tissue damage, eventually leading multiple organ failure.  As a consequence of the increase in complicated surgery, implantable medical devices, elderly patients and patients with weakened immune systems, there has been an increase in the incidence of septic shock in recent years, and with around half of septic shock cases proving fatal it is now the number one cause of death in intensive care units.

Overreaction by the immune system to a bacterial infection can cause septic shock

This week a multinational team of scientists based in Bern, Frankfurt, Glasgow and Singapore, and led by  University of Glasgow physician Professor Alirio Melendeza, have published a paper in Science (1) announcing an important development in the struggle to reduce the death toll from septic shock.

They had previously used in vitro cell culture techniques to identify an enzyme called sphingosine kinase 1 (SphK1) in human phagocytes and demonstrated using both RNAi and a specific inhibitor of SphK1 called 5c that SphK1 was involved in stimulating the cellular signaling pathways that promote release of proinflammatory cytokines.  In this study they began by examining phagocytes isolated from 30 septic shock patients, finding that SphK1 levels were higher in these patients than in a control group. They next found that treating the phagocytes from septic shock patients with the inhibitor 5c blocked the production of proinflammatory cytokines by these cells in response to exposure to bacterial lipopolysaccharide, a molecule found on the exterior of some bacteria that usually provokes a strong inflammatory response.

The ability of 5c to block SphK1 dependent inflammation in-vitro was impressive but would the same happen in a whole organism where other pathways might promote inflammation? The team led by Professor Melendez next examined if 5c or RNAi could protect mice which were injected with an otherwise lethal dose of lipopolysaccharide, and they found that both methods of blocking the action of SphK1 did indeed provide complete protection against septic shock.

This was a very exciting result but acute, one-off exposure to lipopolysaccharide in vitro or in vivo is not the same as bacterial infection, where bacteria are multiplying and constantly interacting with the immune system to induce inflammation. Of course it is also vital that when turning down the inflammatory response the treatment doesn’t also compromise the immune system’s ability to fight the infection.  The team therefore assessed whether pre-treatment with 5c or RNAi could prevent systemic inflammation and mortality from septic shock in a mouse model that simulates microbial infection in humans following surgery or injury. Both treatments prevented death from septic shock,  and not only was the immune system’s ability to combat the infection not compromised but the infection was actually cleared more quickly.

Pre-treatment is all very well but in the clinic treatment almost always starts after sepsis develops, so it was cheering to note that  the inhibitor 5c reduced mortality when given up to 12 hours after infection it reduced mortality from septic shock, though it was most effective when given within 6 hours. This was as effective as the broad-spectrum the antibiotic co-amoxiclav, a standard treatment for sepsis, and when co-amoxiclav was administered along with 5c the combination was observed to be considerably more effective than either treatment used alone.

Professor Melendeza and his colleagues have identified an exciting new approach to reducing the toll from septic shock, hopefully work is already underway to translate this promising study from the bench to the bedside.

In other news the 2010 Kavli Prize in Neuroscience has gone to three scientists, Richard H. Scheller, Thomas C. Südhof, and James E. Rothman, who haveused a creative multidisciplinary set of approaches to elucidate the key molecular events of neurotransmitter release”.  Their work, which involved the study of tissues from a variety of species including rats and marine rays and studies of knockout mice, has made a huge contribution to our understanding of how the release of the molecules that carry messages between the cells of the immune system work.  This research may sit squarely in the realm of basic science, but the understanding of nerve cell communication that these three scientists have provided is now informing the development of new therapies for a wide range of psychiatric and neurological disorders.

Both these news items may at first seem unrelated, but what they have in common is animal research at the heart of a multidisclipinary approach that is increasingly typical of how biomedical science is done in the 21st century.

Paul Browne

1)      Puneet P. et al. “SphK1 Regulates Proinflammatory Responses Associated with Endotoxin and Polymicrobial Sepsis” Science Volume 328(5983), pages 1290 – 1294 (2010) DOI: 10.1126/science.1188635

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