Monthly Archives: March 2010

SR at UCLA – April 6th 2010

Two days before the upcoming Pro-Test for Science rally, Tom Holder will address members of the UCLA community about the importance of standing together in support of lifesaving medical research.

The presentation will be held in the Gonda 1st Floor Conference Room on the UCLA Campus starting at noon on Tuesday April 6th 2010. I encourage you to tell your friends and colleagues – this is a perfect opportunity to discover ways in which you can help improve the public understanding about the role of animals in research.

Standing up for Science

Animal research has been a divisive issue for many years, however much of the problem lies with the public’s general mistrust of science. This mistrust is a reflection of the average person’s lack of understanding about how science works and the animal research issue is no exception. Many people are unable to see the connection between the animal experiments and the huge array of medical drugs that they take for granted. If we are to convince people to support scientific activities such as animal research then we need to be more active in explaining how it affects the lives and welfare of the public.

The scientific community in California and beyond must be ready to meet the challenge of a growing animal rights movement. Despite isolated incidents of violent activity, researchers must realise that the only way to reverse this trend is to put their head above the parapet and provide the public with the scientific argument for biomedical research. The UK provides a clear example of how the scientific community can bring the public onside and combat the rise in animal rights extremism – and there are signs of a similar movement within the US. From the scientists doing the research to the animal care technicians whose sole priority is the welfare of the animals, we need people in the industry to become advocates for science.

Regards

Speaking of Research

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

Magic Bullets and Monoclonals: A Breakthrough in Bioscience

The Federation of American Societies for Experimental Biology (FASEB) is one of the world’s largest and most influential scientific organizations, representing as it does 23 independent scientific societies and over 90,000 individual scientists.  As a coalition that represents tens of thousands of US medical researchers FASEB has policies and positions on all kinds of issues which affect scientific research, from federal funding of research to the legal status of embryonic stem cells and human cloning, and you will probably not be altogether surprised to learn that FASEB has taken a very strong position in support of animal research and the scientists who undertake it.

FASEB also takes its responsibility to educate and inform members of the public about the role of biomedical research very seriously and has produced the excellent Breakthroughs in Bioscience, a series of essays written with the help of leading scientists on the research that led to important advances in medicine. While these essays do not of course focus solely on the role of animals in research, key discoveries have after all been made through approaches as disparate as clinical observations and X-ray crystallography,  they do illustrate how important animal research has been as an integral and frequently vital part of the research process.

The most recent essay entitled Magic Bullets and Monoclonals: An Antibody Tale is a great example of this;  I would encourage anyone who is interested in finding out how the role of antibodies in the immune system was first uncovered and how this eventually lead to the development of these “magic bullets” to read it.

A couple of years ago I wrote on the Pro-Test blog about the role of animal research in the development of the monoclonal antibody drug Lucentis that is used to treat the wet form of age-related macular degeneration, a common form of blindness , but it is only one example out of many.  The Breakthroughs in Bioscience essay focuses on the development other monoclonal antibody drugs including Rituximab, a treatment for cancers of the immune system such as non-Hodgkin lymphoma, infliximab, a treatment for autoimmune diseases such as rheumatoid arthritis, and trastuzumab, better known as Herceptin and used to treat breast cancer. While the essay discusses how animals were vital to the production of these monoclonal antibody drugs, the contribution of animal research to the development of these treatments went far beyond just that, as the following two examples illustrate.

Herceptin (1) targets the HER2/neu receptor, a protein whose normal function is to regulate the growth of cells but which is produced in excess in some breast cancers where it promotes tumor growth. HER2 was first discovered to have a role in cancer through studies of cancer in rats and mice, and scientists following up on this discovery then found that it was over-produced in some breast cancers.  Subsequently research in transgenic mice enabled scientists to understand how HER2 promoted tumor growth, while xenograft models where  immunodeficient mice wre injected with  of HER2 positive human breast cancer cells were used to screen candidate monoclonal antibodies, eventually identifying the antibody that was taken into successful human trials as trastuzumab.

The story was similar for infliximab, which works by blocking the action of a chemical messenger called Tumour Necrosis Factor-alpha (TNF-alpha) that promotes inflammation and is a key factor in the development of several autoimmune disorders.  Studies in rodents and dogs played a key role in the isolation and identification of TNF-alpha, and in subsequently animal research that demonstrated its role in both the normal immune system and in inflammatory and autoimmune diseases. This work included studies in transgenic mice which provided the definitive evidence that TNF-alpha plays a crucial role in the development of rheumatoid arthritis , which formed the basis for studies which demonstrated that a chimeric human/mouse monoclonal antibody against TNF-alpha could protect transgenic mice which produced human TNF-alpha from inflammation-induced cachexia (2). Follow up studies in transgenic mice expressing human TNF-alpha provided important pre-clinical information about the safety of infleximab (3).

The examples above show just how important animal research is to both basic research which seeks to understand what is going on in normal physiology and disease, and translational research which seeks to take that knowledge and apply it to developing treatments that can be used effectively in the clinic.  We’re delighted by the work that FASEB is doing to ensure that the public is aware of how all types of research contribute to medical progress, and hope that they continue these efforts for many years to come.

Paul Browne

1)      Pegram M. and Ngo D. “Application and potential limitations of animal models utilized in the development of trastuzumab (HerceptinR): A case study”  Advanced Drug Delivery Reviews Volume 58, Pages 723-734 (2006) DOI:10.1016/j.addr.2006.05.003

2)      Siegel S.A. et al. “The Mouse/Human Chimeric Monoclonal Antibody cA2 Neutralizes TNF In Vitro and Protects Transgenic Mice from Cachexia and TNF Lethality In Vivo” Cytokine Volume 7(1), Pages 15-25 (1995) DOI:10.1006/cyto.1995.1003

3)      European Medicines Agency report http://www.ema.europa.eu/humandocs/PDFs/EPAR/Remicade/190199en6.pdf

Added Download Ability to Videos

We’ve added a download button for all our videos (except YouTube ones). These can make a perfect addition to a presentation (Learn how to add video to PowerPoint), or can be used on their own to show colleagues. Check out the Media/Downloads section to see most of the videos.

Cheers

Tom

p.s. I added a download button to the video in the previous post

So what ARE conditions in labs like?

There is much misinformation about what conditions are like in modern research laboratories. Animal rights activists spread pictures and videos (often decades old) showing some of the few labs which have failed in their duty of care to their animals. However, such footage is the exception not the rule – so here we provide some footage provided by the UK non-profit Understanding Animal Research, from inside labs.

Download (Right click and select “Save target/link as”)

UAR also have a fantastic YouTube channel – animalevidence – which provides viewers with a look at the welfare considerations put in laboratories across the country. Here is an example:

This video shows the standard caging used for mice in animal houses. It is important to note that mice are much smaller than the other mammals used in research, so although their cages appear small, there is plenty of room for their needs. They are social animals, benefiting from being housed together in small groups and their natural behaviour involves grouping together in small spaces. Other natural behaviours including nesting and tunneling, and is why they are provided with fairly deep bedding. Mice and other small rodents also like to hide inside dark spaces, which is the reason for the plastic casing in the cages. It is termed the red mouse house and is beneficial as the mouse cannot see out of the box and so feels secure, but the researcher can see in. This video has no sound.

In the US, Americans for Medical Progress also have a YouTube channel providing views with a look at the Cedars-Sinai Medical Center research labs:

These videos provide excellent tools for spreading a realistic vision of what goes on inside laboratories – we encourage other research labs to provide footage and prevent AR groups spreading misinformation about the conditions in these labs.

Regards

Tom Holder

Pro-Test for Science – April 8th 2010

Dear Friends,

In 2009, Pro-Test for Science held an historic rally on the UCLA campus; this event brought over 700 people onto the streets in support of the scientists and researchers who carry out crucial medical research using laboratory animals. Animal research continues to play a vital part in the development of scientific knowledge and modern treatments for human disease.

Pro-Test for Science is once again taking to the streets for this all-important cause; with that in mind, we are calling upon you to stand together with other members of the community in supporting the crucial efforts of researchers on this campus and to condemn those who try to block medical progress with threats, harassment and intimidation tactics.
This rally, on the UCLA campus seeks to:

  • Communicate a better understanding about animal research to the public, its importance, and the what we all stand to lose if such work were to stop.
  • Celebrate the successes of animal research in the development of treatments for disease, new diagnostic procedures/instruments, and surgical techniques.
  • Defend the rights of researchers to pursue their work free from harassment and intimidation.

The rally will begin on Thursday April 8th at 11:30 AM, on the north-east corner of Westwood Blvd. and Le Conte Ave., which will be followed by a march to Wilson Plaza, where members of the UCLA family and representatives from the National Institutes of Health will speak to the incredible advances in human health and welfare that have come from animal research.

Please check the news section and www.pro-test-for-science.org regularly for updates and further communications.

Join us to make this second rally at UCLA an unprecedented event in scientific activism!

Pro-Test for Science Committee

David Jentsch, Dario Ringach, Tom Holder, Lynn Fairbanks, Kathy Wadsworth, Megan Wyeth, Jennifer Perkins

The 2009 Pro-Test for Science rally was a huge success

In defense of “The scientific basis for the support of biomedical science”

During our panel discussion, Dr. Greek criticized a classic study that appeared in the pages of Science by Comroe and Dripps, entitled “The scientific basis for the support of biomedical science”, which set out to analyze the time sequence of discoveries that had led to major medical advances.

Comroe and Dripps analyzed the top ten clinical advances in cardiovascular and pulmonary medicine in the last 30 years (prior to the study which was done in 1976).   Their goal was to identify the key scientific discoveries had led to these advances.  With the help of consultants and physicians, they read and carefully examined 4,000 individual articles, identifying 2,500 of them as being essential for the development of the body of knowledge that lead to these breakthroughs.

The main result of the study was 41% of all articles considered to be essential for later clinical advances were not clinically oriented at the time of the study, and that 62% of key articles were in fact the result of basic research exploring fundamental questions of biology.  This figure could in fact be considered lower bounds (underestimating the value of basic research), as any given study was categorized as “clinically oriented” even if it was done entirely on animals with a basic question in mind but merely mentioned in passing an interest or relation to a particular disease.

Another interesting outcome of this study was a very rather detailed chronological list of the key elements involved in the development of electrocardiography.  From the early manifestation of electricity in ancient times, to Galvani and the discovery of bio-electricity, Volta, Purkinje, the first ECG recording in frogs and humans, and the development of ECG devices (see their Table 3).  Such a clear sequence of causal events leading to major breakthroughs is what the opposition usually demands as proof for the contribution of animals in medical advances.

The methodology of the study was criticized by Richard Smith eleven years after the publication of the original study.  Here are his central complaints:

“Comroe and Dripps [asked] 40 physicians to list the advances that they thought most important.  They do not, however, say in their paper whether they asked 40 and fewer replied or whether they asked more and only 40 replied.  Nor do they say how they selected these 40, and nor do they say in their methods why they chose only physicians, although they are defensive in their discussion about having done so

From the replies Comroe and Dripps produces a list of the top cardiovascular and pulmonary advances and sent them to ’40 to 50 specialists in each field’ and asked them to vote on the list.  ‘Their votes selected the top t10 advances’.  Again this is very imprecise for a paper published in Science.  Exactly how many specialists were contacted?  How many responded?  How were they asked to vote?  How were the votes put together?”

Richard Smith concluded the study was therefore “unscientific”.

There is, in fact, some degree of validity in the criticism that there is a lack of detail in the original Comroe and Dripps.  However, I would submit these are minor flaws and it should be possible to fill in the missing pieces in a reasonable way  to allow a replication of the study.   Calling the results of the study “unscientific” is not warranted.

In any case, in an effort to take a second look at these issues Grant et al in 2003 decided to address a similar question to that of Comroe and Dripps but using different methods (this was the study cited by Dr. Greek in our panel discussion).  First, Grant and colleagues opted to look at the leading advances in neonatal intensive care.  The first part of the study was performed in a similar way as that of Comroe and Dripps: coming up with a list of clinical advances in one specific area (neonatal intensive care) using a Delphi survey.  The top three advances they identified by voting of experts in this area were mechanical ventilation, replacement surfactant and antenatal steroids (their Fig 2.1).

Second, instead of reading and reviewing articles from the literature to identify the key elements of knowledge that contributed to these advances, this team opted for an automated bibliographic analysis of the literature based on a genealogy tree of articles.   Basically the method works as follows.  First, start by searching for articles within the last 5 years that deal with one of the clinical advances of interest (such as lung surfactants using a keyword search).  Keep the top 5% of the most cited papers.  Presumably, this set is of some importance and it will represent the first generation of papers.  Next, generate the next set of papers by looking at the full set cited by the first set.  Rank this new set according to the number of times they each have been cited and, again, keep the top 5% (this set represents the 2nd generation of papers).  And so on.

The method seems automatic and bias-free.  But is it generating meaningful results?  Where is the 5% threshold coming from?  Normally, in science, only a handful of studies in any one period of time provides the key elements that are necessary to drive a breakthrough.  In Table 3 from Comroe and Dripps, for example, they identified only 21 key studies between 1900 to 1967 — about 1 key discovery every 3.2 years.    Thus, I would suggest that 5% is too high a threshold, which only helps to add a tremendous amount of noise in the literature under study at each generation.   Further, the authors never consider citation rates.  So, a paper that received 50 citations in 5 years might rank higher than a paper that received 49 citations in 2 months.

Even if we assume the analysis is yielding a reasonable collection of papers, the authors split the papers in each generation into five categories or levels: level 1 (clinical observation), level 2 (clinical mix), level 3 (clinical investigation) and level 4 (basic research) and finally level 5 (unknown).  Yet, such classification was arrived at depending on the journal the research was published, which seems a rather crude method.

As a matter of fact, in a recent email communication with Dr. Jonathan Grant about these issues, he wrote back saying that:

“The issue I have with my analysis is […] the metric (research levels) for classifying basic and other research. Although I know of no other way of doing this bibliometrically I have come to believe it is too crude.”

In addition to this issue, one should note that by splitting all the papers into more categories (not just basic and clinical as Comroe and Dripps did) the absolute percentage numbers one would expect for each is category is automatically reduced.  In fact, the highest percentage of studies in any one generation falls in the “unknown” category (~40% of them).  Comparing absolute percentage levels from this study versus Comroe and Dripps is not possible.

Grant and colleagues were careful enough to express reservations about their results when they wrote:

“In reaching this conclusion we are acutely aware of the significant limitations to the revised methodology and, therefore, we caution against the over-interpretation of our results.”

A caveat Dr. Greek should consider mentioning when referring to this study.

In closing, I’d like to offer the readers a challenge.  Consider the two medical advances in neonatal intensive care Grant et al identified with the help of experts: ventilation and surfactants.   It seems to me that anyone with knowledge of medical history in the field will immediately recognize the role animals played in their development.  Don’t take my word for it.  Instead, read the story as told by one investigator that was directly involved in these discoveries.   It is a wonderful tale that will take you from the basic physics of capillarity and surface tension (yes, basic science again), to the elucidation of the composition of lung surfactants in animals, to the treatments that save the lives of thousands of premature babies each year which would have otherwise died 50 years ago.

The inescapable conclusion is that lives of countless premature babies are saved today thanks basic research with animals.

It is as simple as that.

Regards

Dario Ringach