Tag Archives: animal research

Clinical trial success for Cystic Fibrosis gene therapy: built on animal research

This morning the Cystic Fibrosis Gene Therapy Consortium (GTC) announced the results of clinical trial in 140 patients with cystic fibrosis, which demonstrate the potential for gene therapy to slow – and potentially halt – the decline of lung function in people with the disorder. It is a success that is built on 25 years of research, in which studies in animals have played a crucial role.

Cystic fibrosis is one of the most commonly inherited diseases, affecting about one in every four thousand children born in the USA, and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene produces a channel that allows the transport of chloride ions across membranes in the body, and the many mutations identified in cystic fibrosis sufferers either reduce the activity of the channel or eliminate it entirely. This defect in chloride ion transport leads to defects in several major organs including the lungs, digestive system, pancreas, and liver. While the severity of the disease and the number of organs affected varies considerably, cystic fibrosis patients often ultimately require lung transplant s, and too many still die early in their 20’s and 30’s as the disease progresses.

In a paper published in Lancet Respiratory Medicine today (1), the GTG members led by Professor Eric Alton of Imperial College London compared monthly delivery to the airway of a non-viral plasmid vector containing the CFTR gene in the liposome complex pGM160/GL67A using a nebuliser with a placebo group who received saline solution via the nebuliser. They reported stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group after a year. This is the first time that gene therapy has been shown to safely stabilise the disease, and while the difference between the treated and control group was modest, and the therapy is not yet ready to go into clinical use, it provides a sound bases for further development and improvement.

Blausen_0286_CysticFibrosis

The Chief Executive of the Cystic Fibrosis Trust, which is one of the main funders of the GTC, has welcomed the results, saying:

Further clinical studies are needed before we can say that gene therapy is a viable clinical treatment. But this is an encouraging development which demonstrates proof of concept.

“We continue to support the GTC’s ground-breaking work as well as research in other areas of transformational activity as part of our mission to fight for a life unlimited by cystic fibrosis.”

So how did animal research pave the way for this trial?

Following the identification of the CFTR gene in 1989 scientists sought to create animal models of cystic fibrosis with which to study the disease, and since the early 1990’s more than a dozen mouse models of cystic fibrosis have been created. In some of these the CFTR gene has been “knocked out”, in other words completely removed, but in others the mutations found in human cystic fibrosis that result in a defective channel have been introduced. These mouse models show many of the defects seen in human cystic fibrosis patients and over the past few years have yielded important new information about cystic fibrosis, and in 1993 Professor Alton and colleagues demonstrated that it is possible to deliver a working copy of the CFTR gene using liposomes to the lungs of CFTR knockout mice and correct some of the deficiencies observed.

To get a working copy of the CFTR gene to the lungs of cystic fibrosis patients Professor Alton and colleagues needed three things:

• A DNA vector containing the working CFTR gene that is safe and  can express sufficient amounts of the CFTR channel protein in the lungs to correct the disease

• A lipid-like carrier that can form a fatty sphere around the DNA vector to so that it can cross the lipid membrane of cells in the lung, as “naked” DNA will not do this efficiently.

• A nebuliser device that produces an aerosol of the gene transfer agent so that it can be inhaled into the lungs of the patient.

Several early attempts to use gene therapy using viral vectors to deliver the working copy of the CFTR gene to patients failed because the immune response rapidly neutralised the adenoviral vector (see this post for more information on challenges using adenoviral vectors), and while attempts to use non-viral vectors were more promising, it was found that they caused a mild inflammation in most patients, which would make then unsuitable for long term use. As reported in a paper published in 2008 the GTC members developed and assessed in mice a series of non-viral DNA vectors, repeatedly modifying them and testing their ability to both drive CFTR gene expression in the lungs and avoid inducing inflammation. They finally hit on a vector – named pGM169 – which fulfilled both key criteria.

Earlier the consortium had undertaken a study to determine which carrier molecule to use in their non-viral gene transfer agent (GTA). To do this they assessed 3 GTA’s, each consisting of a lipid like molecule that could form a sphere around the non-viral DNA vector; either the 25 kDa-branched polyethyleneimine (PEI), the cationic liposome GL67A, or as a compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. Because there are significant differences in airway physiology between mouse and human they carried out this study in sheep, whose lung physiology more closely matches that of humans. The study identified the cationic liposome GL67A as the most promising candidate, resulting in robust expression of the CFTR transgene in the sheep lungs.

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

It now remained to bring the DNA vector and carrier together. In a 2013 publication the consortium reported that repeated aerosol doses of pGM169/GL67A to sheep over a 32 week period were safe and induced expression of the CFTR transgene in the sheep lungs, although the level of expression varied between individuals (this variation was also observed in human CF patients in the clinical trial reported today). A final study, this time in mice, assessed the suitability of the Trudell AeroEclipse II nebuliser as a device to create stable pGM169/GL67A aerosols, finding that it did so in a reproducible fashion. When aerosolized to the mouse lung, the new pGM169/GL67A formulation was capable of directing persistent CFTR transgene expression for at least 2 months, with minimal inflammation. These studies provided the evidence to support the gene delivery system and dosage strategy used in the clinical trial reported today.

The trial results announced today are an important accomplishment, but they mark a beginning rather than the end for Cystic Fibrosis gene therapy. It will be necessary to improve the efficiency of the therapy before it can enter widespread clinical use. Animal research will certainly play an important part in this work, notably the observation that the efficiency of CFTR gene delivery using this strategy was varied between individuals in both sheep and humans indicates that sheep are a good model in which to assess changes to improve the consistency and effectiveness of the gene therapy.

If you would like to know more about this cystic fibrosis gene therapy clinical trial you can watch two videos recorded at a meeting for cystic fibrosis patients at ICL on the  Cystic Fibrosis Trust website.

Paul Browne

1) Alton E.W.F.W. et al. “Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial” Lancet Respiratory Medicine Published online July 3, 2015

European Commission rejects Stop Vivisection Initiative

Today the European Commission rejected the Stop Vivisection Initiative that sought to repeal European Directive 2010/63/EU on the protection of animals used for scientific purposes and ban animal research in the EU.

Today, there are effective treatments for many infectious diseases, some forms of cancer, and several chronic diseases such as diabetes. These advancements would have been impossible without the insights gained in animal studies.
[…]
However, the Commission does not share the view that scientific principles invalidate the ‘animal model’. Indeed, despite differences with humans, animal models have been the key scientific drivers to develop almost all existing effective and safe medical treatments and prevention measures for human and animal diseases
[…]
The Commission therefore does not intend to submit a proposal to repeal Directive 2010/63/EU and is not intending to propose the adoption of a new legislative framework.

Read the full EU report here.

Dr Paul Browne, Research Editor at Speaking of Research, said:

We welcome the decision by the European Commission to reject the Stop Vivisection Initiative. EU Directive 2010/63 which governs animal experiments has been a step forward for both animal welfare and better science. They put the 3Rs – Replacement, Refinement and Reduction of animals in research – at the heart of the rules governing animal experiments.

Animal research continues to play a key part in medical advances. Only last week we learned about a new lung cancer therapy that performed very well in clinical trials, allowing patients with the disease to live longer; this treatment was only possible thanks to studies in transgenic mice. “

The Commission’s decision is not, however, unexpected. Directive 2010/63/EU was adopted by the EU Council and Parliament in September 2010 after more than 5 years of discussion and debate, including consultation exercises in which scientists, patient organizations, animal welfare experts, animal rights organizations and members of the public were given the opportunity to submit evidence. At a time when the EU is facing some of the greatest political and economic challenges of its history it was always very unlikely that the EU commission would repeal Directive 2010/63/EU and start the negotiation process again from scratch.

EU_Commission

If the organizers and supporters of the Stop Vivisection Initiative were going to have any chance of persuading the Commission to repeal directive 2010/63/EU, they needed to make a very strong case to the MEPs who gathered to hear what they had to say at the European Parliament session held on Monday 11 May 2015.

They didn’t. The hearing was something of a flop, with reports noting that the majority of MEPs present were unconvinced by the arguments put forward by the proponents of the Stop Vivisection Initiative. It’s not difficult to see why this was the case. The Stop Vivisection Organizers and their witnesses failed to put forward any significant new evidence that had not been examined back when the Directive 2010/63/EU was originally negotiated, and at one point in the hearing descended into outright conspiracy theory thinking.

By contrast supporters of Directive 2010/63/EU made a stronger case, especially Nobel laureate Professor Francoise Barré – Sinoussi, who put forward a very strong case for the value of animal research in advancing medicine.

While this was happening scientists and supporters of medical progress in the EU were not taking any chances, and let the European Commission know in no uncertain terms how important animal research is to medical science. More than 170 organizations (Speaking of Research among them) representing scientists, major funders of medical research  and many millions of patients across the EU have signed up to a statement in support of Directive 2010/63/EU and sixteen European Nobel laureates published an open letter in UK and German newspapers to rebut the Stop Vivisection campaign. Several excellent letters on the importance of animal research were published in the national press, including a letter in the Times by Steve Ford, Chief executive of Parkinson’s UK, as well as articles such as that written by Oxford University Duchenne muscular dystrophy researcher Professor Kay Davies. In addition research funders have added information explaining their position on animal research to their websites, for example the Wellcome Trust, one of the world’s top medical research charities, have published a briefing on “Why we support research involving animals”, and a Q&A on European Directive 2010/63/EU.

We congratulate the European Commission on this good decision for science and patients in Europe, and the EU scientific community for speaking up for science with one voice.

Speaking of Research

Lung cancer immunotherapy, from PD-1 knockout mice to clinical trials

This morning many news outlets, including the BBC, covered a very promising development in lung cancer therapy; the successful clinical trial of the cancer immunotherapy Nivolumab in 582 patients with advanced lung cancer. While the extension of survival was modest in most patients, it is to be remembered that these were patients with advanced lung cancer, which is notoriously difficult to treat, so to see the survival time doubling in some patients was quite dramatic. Future trials will examine whether greater benefits are seen when Nivolumab is given earlier in the course of the disease.

Dr Alan Worsley, Cancer Research UK’s senior science information officer, told the BBC that harnessing the immune system would be an “essential part” of cancer treatment, and adding:

This trial shows that blocking lung cancer’s ability to hide from immune cells may be better than current chemotherapy treatments. “Advances like these are giving real hope for lung cancer patients, who have until now had very few options.”

Nivolumab works by blocking the activation of the PD-1 receptor protein found on the surface of many of the immune cells that infiltrate tumours. Another protein named PD-L1 binds to PD-1 and initiates a regulatory pathway that leads to the immune response being dampened down. Usually this is a good thing as it maintains immune tolerance to self-antigens and prevents auto-immune damage to healthy tissue, but unfortunately many solid tumour cells, such as lung cancer cells, also secrete PD-L1, and by activating PD-1 can evade destruction by the immune system. By blocking PD-1 Nivolumab turns off this protective mechanism and allows the immune cells to detect and destroy the tumour cells.

X-ray of a lung cancer patient. Image credit: "LungCACXR" by James Heilman, MD - Own work.

X-ray of a lung cancer patient. Image credit: “LungCACXR” by James Heilman, MD – Own work.

So how was this discovered? This is where the knockout mice come in. Scientists had observed in the 1990’s that PD-1 was highly expressed on the surface of circulating T- and B- immune cells in mice, but didn’t know what role PD-1 played, suspecting that it may be involved in increasing the magnitude of the immune response. To examine the role of PD-1 researchers at Kyoto University in Japan creates a knock-out mouse line where the PD-1 gene was absent, and observed that this lead to some immune responses being augmented. In a paper published in 1998 they reported than rather than being an activator of the immune response PD-1 was actually involved in dampening down the immune response (1).

Subsequent studies in a range of PD-1 knockout mouse strains over the next decade explored the role of PD-1 in regulating the immune system, and also demonstrated that its ligand, PD-L1, could block immune-mediated tissue damage (2).  At the same time as these studies were taking place other research was demonstrating that PD-L1 was produced at high levels by tumour cells, first in   renal cell carcinoma in 2004 (3), but later in many other solid tumours including in lung cancer (4), and that this expression was associated with a decrease in the immune response to the tumour and a poorer prognosis.

This raised an obvious question: would blocking PD-1 improve the immune response against these tumours?

Work was already underway to find out. A paper published in 2007 by scientists from Nara Medical University in Japan demonstrated that blocking PD-L1 binding to PD-1 with monoclonal antibodies enhanced the immune response against established tumours in a mouse model of pancreatic cancer and acted synergistically with chemotherapy to clear the tumours without obvious toxicity (5). Subsequent studies with other monoclonal antibodies in a range of mouse and in vitro models of cancer showed similar results, including the humanized monoclonal antibody MDX-1106, now called Nivolumab, which was obtained by immunizing mice which had been genetically modified to produce human antibodies with human PD-1 (6).

Laboratory Mice are the most common species used in research

Cancer Immunotherapy – adding another accomplishment to an already impressive CV!

MDX-1106/Nivolumab showed promising results in a phase 1 trial against metastatic melanoma, colorectal cancer, castrate-resistant prostate cancer, non-small-cell lung cancer, and renal cell carcinoma, and following larger clinical trials (7) it was approved by the FDA for the treatment of melanoma that cannot be removed by surgery or is metastatic and no longer responding to other drugs, and more recently for metastatic squamous non-small cell lung cancer.

The story of the development of anti-PD-1 cancer immunotherapy is an illustration of how basic or fundamental biological research in animals informs medical science, and drives the discovery of new therapies. As cancer immunotherapy begins to transform the treatment of many previously untreatable cancers, it is well worth remembering that this revolution has its origin in the hard work of countless scientists working around the world, many of whom could only have guessed at the time where their efforts would eventually lead.

Breaking news, 1 June 2015: In another exciting report from the American Society of Clinical Oncology meeting in Chicago, researchers have reported that in a clinical trial of 945 patients with advanced metastatic melanoma a combination of Nivolumab with  Ipilimumab (another cancer immunotherapy that works through a different mechanism) stopped cancer advancing for nearly a year in 58% of cases, with the cancer still stopped in its tracks in many patients when the study period had ended. This is substantially greater effect than is seen with existing therapies, including Ipilimumab when administered alone, and shows how powerful cancer immunotherapies may be when two or more are combined.

Paul Browne

References:

  1. Nishimura H1, Minato N, Nakano T, Honjo T. “Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses.” Int Immunol. 1998 Oct;10(10):1563-72. PubMed: 9796923
  2. Grabie N, Gotsman I, DaCosta R, Pang H, Stavrakis G, Butte MJ, Keir ME, Freeman GJ, Sharpe AH, Lichtman AH. “Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart.” Circulation. 2007 Oct 30;116(18):2062-71. PubMed 17938288
  3. Thompson RH1, Gillett MD, Cheville JC, Lohse CM, Dong H, Webster WS, Krejci KG, Lobo JR, Sengupta S, Chen L, Zincke H, Blute ML, Strome SE, Leibovich BC, Kwon ED. “Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target.” Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17174-9. PubMed:15569934
  4. Zhang Y1, Huang S, Gong D, Qin Y, Shen Q. “Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer.” Cell Mol Immunol. 2010 Sep;7(5):389-95. doi: 10.1038/cmi.2010.28. PubMed: 20514052
  5. Nomi T1, Sho M, Akahori T, Hamada K, Kubo A, Kanehiro H, Nakamura S, Enomoto K, Yagita H, Azuma M, Nakajima Y. “Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer.” Clin Cancer Res. 2007 Apr 1;13(7):2151-7. PubMed:17404099
  6. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman WH, Stankevich E, Pons A, Salay TM, McMiller TL, Gilson MM, Wang C, Selby M, Taube JM, Anders R, Chen L, Korman AJ, Pardoll DM, Lowy I, Topalian SL. “Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.” J Clin Oncol. 2010 Jul 1;28(19):3167-75. doi:10.1200/JCO.2009.26.7609. PubMed: 20516446
  7. Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, Sharfman WH, Brahmer JR, Lawrence DP, Atkins MB, Powderly JD, Leming PD, Lipson EJ, Puzanov I, Smith DC, Taube JM, Wigginton JM, Kollia GD, Gupta A, Pardoll DM, Sosman JA, Hodi FS. “Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab.” J Clin Oncol. 2014 Apr 1;32(10):1020-30. doi: 10.1200/JCO.2013.53.0105. PubMed:24590637

A Conversation About Beagle Testing

I received an email one morning from James, a Grade 6 student who wanted to know more about beagles used in research and testing for a school project about his passion. He has a pet beagle named Bagel and had recently watched some videos from the Beagle Freedom Project (BFP written about here and here). James was very curious and quite concerned about the beagles that participated in studies in Canada. He requested some information and to visit the Central Animal Facility at the University Of Guelph. James was invited for a tour and the answers to his questions are as follows:

Job Related

  • What is your job and what do you teach at the University?

I am a research animal technician and my job is to advocate for the animals that are under my care. I instruct those who have not worked with animals how to do so in a compassionate, respectful and ethical manner.

  • Why did you become a technician?

I became a technician because I love animals and people. I also love science and love being a part of making discoveries that improve the lives of millions of people and animals

  • My project is on a passion and I am wondering what your passion is?

I’m passionate about a lot! I am passionate about animals that I have the privilege to care for with compassion and respect. I am passionate about the science that continually makes strides towards new therapeutic advancements. I am passionate about alleviating the suffering of our fellow animals and people who agonize with debilitating and painful diseases. I choose this profession in research because it is my passion.

  • What research do you do in your Lab?

The majority of the work that is done in the facility where I work is basic or fundamental science in a wide variety of areas including oncology, neuroscience, animal behaviour and welfare, molecular biology, physiology, immunology, among others.

Michael Brunt and James during the laboratory visit

Michael Brunt and James during the laboratory visit

Animal Research/Testing

  • Why is it important to use animals/ beagles?

Various non-animal research methods are used together with animal studies to reduce the number of animals needed. These methods include antibodies, stem cells, tissue cultures (all in-directly use animals) and computer models. Non-animal methods account for the majority of biomedical research. Nevertheless, there are important research questions that still require animals. For example, in drug development, a large initial group of chemical candidates may be screened using non-animal methods, and only the most promising ones are taken through animal testing and human clinical trials. Before animal studies can go forward, investigators must detail how they have considered non-animal methods, and why they are not appropriate for answering their research question.

  • What kinds of tests are done?

The Canadian Council on Animal Care has 5 classifications for the purposes of animal use (PAU):
PAU1 – Studies of a fundamental nature in science relating to essential structure or function
PAU2 – Studies for medical purposes, including veterinary medicine, that relate to human or animal disease or disorders
PAU3- Studies for regulatory testing of products for the protection of humans, animals, or the environment
PAU4 – Studies for the development of products or appliances for human or veterinary medicine
PAU5 – Education and training of individuals in post-secondary institutions or facilities

  • What happens with your research findings once you are finished a project?

The findings are published in scientific journals that are available on the internet for everybody to access. The knowledge gained could be used to answer other scientific questions or be applied in translational science to develop new therapies or cures for those that are suffering.

  • What do you do with the animals after you have used them for research/testing?

Ultimately, most of the animals involved in animal research are euthanized. This is because the researchers will often need to further study the body – taking tissue samples and other such tests to make sure they get as much data from any animal they use. To euthanize the animals, researchers use a variety of methods such as an overdose of anesthesia (pain killers) or using CO2 so that the animal slowly drifts into a sleep it never awakes from.

Beagle Research/Testing

  • What is your opinion about beagle research?

Animal research plays a vital role in the development of modern medical and veterinary treatments. Much of our understanding about the biological processes in the body, and the diseases that affect them, comes from studies in animals. I believe that animal research should be conducted with the utmost care, responsibility and respect towards the animals. All personnel involved in animal research should strictly follow the pertinent guidelines, regulations and laws.

  • When did beagle testing begin?

Hundreds of years ago to begin to understand blood movement and the interactions of organs.

  • Why are beagles used for testing?

Health Canada requires that all new drugs, medical devices, and procedures first be evaluated in animals for safety before clinical trials involving human volunteers can begin. The most common “product” that is tested using animal models is new medications. Animals are used to determine that the drug shows a reasonable likelihood of working as conceived and to determine unforeseen side effects. For instance, a researcher may find that a new drug to control high blood pressure does so, but there is a possibility of a side effect such as liver damage. That information needs to be known before it is used in clinical trials with humans.

  • How many beagles are used a year?

0.3% of the animals used in Canada in 2011 were dogs. Mice, rats and fish accounted for 78.5% of the animals.

  • Where do you get your dogs?

Our beagles are provided by companies who breed dogs for research, teaching or testing purposes.

  • How are the dogs treated?

With love, compassion and respect.

  • Why don’t some companies let beagles see sunlight play or even touch grass during their testing time?

At our institution our animals go outside for walks every day with their dedicated paid dog walker and our volunteer dog walkers.

  • How many beagles die each year from testing?

I don’t have an answer to that question. In Canada in 2011, 10,199 dogs were utilized in science. However, that isn’t how many were humanely euthanized at the end of the projects. Our institution has adopted 100s of beagles into our community.

James and Bagel

James and Bagel (Photos reproduced with permissions from copyright owner)

Feedback

James and his parents met a number of our animals, including our beagles, during their tour and I asked him to provide some feedback on his experience.

At first I thought beagle research and testing was inhumane, unbeneficial and cruel. But when I went to the University of Guelph my perspective changed and I learned that research and testing is very important and it helps 1000s of humans and animals because of the research on animals. The people treat all the animals to a good life like every other animal in the world. They play with all the animals mice/rats/dogs and turkeys. One of the reasons that they euthanize the animals is to further discover the effects of a drug to make it safer for humans and other animals. All the animals there are well cared for, like the animals are their family. If we didn’t have research and testing we would never have a treatment to help the people suffering with cancer. 1000s of products have helped humans and other animals because of the work done with beagles. How many people would have died without animal research and testing on the drugs to know if they are safe. What I thought about beagle testing at first was nothing compared to what it is now. I now know that it very helpful. Most of the websites that say all the bad things are not aware of all the things the beagles and animals have done for advancing medicines. Another part of my visit included seeing Dr Woods and he told me about the research he did on mice for prostate cancer. They use mice cells because they react to the cancer like the humans cells do. Dogs are closer to humans than mice in DNA and they need to see how much of the drug they can give without it being toxic. All chemotherapy has been tested through rats, mice and beagles before humans. In my opinion all the beagles and animals who are involved in research are Heroes.”

My interview and tour with James demonstrates that everyone must seize opportunities to engage with members of the public. It is a chance to present accurate information about the ethical use of animals in science and allow people to make informed opinions. These instances foster a culture of understanding, acceptance, value and recognition for the contributions animal research plays in improving the lives of millions of animals and people every day. They are opportunities that should not be squandered.

Michael Brunt

Stop vivisection Initiative fails to impress at EU hearing

In March we discussed a new attempt by animal rights supporters to ban animal research in Europe, The Stop Vivisection European Citizens’ Initiative, which was signed by  1.2 million people (half of them in Italy). The initiative calls for “the European Commission to abrogate directive 2010/63/EU on the protection of animals used for scientific purposes and to present a new proposal that does away with animal experimentation”. On Monday 11th May the organizers of the initiative had an opportunity to present it to a joint session of  several European Parliament committees, in a hearing that was also addressed by scientists who spoke in favor of keeping directive 2010/63/EU.

So how did it go?

Well, an editorial in last week’s edition of Nature gave a fair assessment of it when they described the session as “a pretty grey affair” in which the duo who presented the initiative – Gianni Tamino and Claude Reiss – “spoke calmly but unconvincingly” to a half-filled auditorium. A transcript and summary of the key points made by the European Animal Research Association and put together the key points that were said during the meeting (download here) indicates that the initiative is almost certain to fail in its objective of  persuading the EU Commission to repeal Directive 2010/63/EU.

European-Parliament

A look through the EARA report  shows why. Any MEPs (Members of the European Parliament) hoping to hear new evidence from Dr Ray Greek and Dr Andre Menache, the scientific advisors who the Stop Vivisection Initiative organizers had brought along, were in for a  disappointment, as instead they presented a veritable greatest hits of anti-vivisection claims. Their testimony included Dr Ray Greek’s trademark  misrepresentation of what “prediction” means in biomedical research, while Dr Menache reheated the old 0.0004% myth. Surprisingly, these were far from being the worst claims made by supporters of the Stop vivisection initiative. Particularly low points came when MEP, and initiative supporter,  Anja Hazekamp stated that there has been massive increase in animal testing (The EU’s own statistics show the opposite) and when Claude Reiss, one of the organizers of the Stop Vivisection petition, ventured deep into conspiracy theory territory with a claim that there is a patent on HIV treatment that completely cleans the virus from the body, but has not been developed because it is not profitable.

In contrast the voice of science was very ably represented. Professor Francoise Barré – Sinoussi, 2008 Nobel Laureate in Physiology or Medicine for her role proving that HIV causes AIDS, put forward a very strong case for the importance of animal research in advancing medicine, and repeatedly demolished false claims made by anti-vivisectionists, particularly claims that animal research had not made a useful contribution to HIV research and the development of a vaccine against HIV infection. On this she is on safe ground as there is no doubt that animal research has made very important contributions to HIV research and development of therapies (for examples see here, here and here), and while development of an effective vaccine has been slow – because it’s very, very difficult – there has been real progress in recent years, and most HIV experts is that studies in  non-human primate models of the infection have a critical role to play in evaluating potential vaccination strategies.

Francoise Barré - Sinoussi, undoubted star of the EU parliament hearing.

Francoise Barré – Sinoussi, undoubted star of the EU parliament hearing.

Throughout the hearing one very important voice was conspicuous by its absence, that of the patients who rely on medical research. MEP Françoise Grossetête, who spoke in favor of retaining Directive 2010/63/EU, noted in particular that EURORDIS, the organization that represents rare disease patients in Europe, had not been invited to present evidence at the hearing. We hope that the EU commission will now actively seek the advice of EURORDIS and other European patient organizations before making their final decision.

What happens now?

At the hearing the Vice-President of the European Commission confirmed that the Commission will provide a formal response to the initiative by 3 June 2015. On the basis of what we saw at the hearing, and the fact that the majority of MEPS present were in favor of retaining Directive 2010/63/EU, it is a near certainty that the EU commission will reject the Stop Vivisection initiative and retain the Directive.

In 2017 the Directive will undergo it’s first 5 year review, which is likely to focus on its implementation across the EU, but the commission have also promised to organize a scientific conference that year to discuss the validity of animal research. With that in mind it’s good to see that last week’s Nature editorial noted that scientists across the EU are becoming increasingly – and refreshingly – vocal on the need to support animal research as a pillar of scientific and medical progress. In recent weeks we’ve seen thousands of scientists sign a motion of solidarity with a neuroscientist targeted by animal rights extremists in Germany, more than 140 research organizations, patient organizations, medical research funders and scientific associations sign up to a statement in support of Directive 2010/63/EU, Sixteen European Nobel laureates publish an open letter in UK and German newspapers to rebut the Stop Vivisection campaign. We’ve also seen several excellent letters appear in the national press, including a letter in the Times by Steve Ford, Chief executive of Parkinson’s UK, on the importance of animal research, and articles such as that written by Oxford University Duchenne muscular dystrophy researcher Professor Kay Davies.

The Stop Vivisection Initiative may have almost run its course, but the threat to the future of biomedical science in the EU is sadly never very far away. We hope that the current re-invigoration of the European scientific community continues, and that scientists strengthen and expand their engagement with politicians, journalists and citizens in the run-up to 2017 and beyond.

Speaking of Research

Zebrafish: the rising star of animal models

Today we have a guest article by Jan Botthof, a PhD Student at the Cambridge University Department of Haematology and the world renowned Wellcome Trust Sanger Institute. Together with the EMBL-European Bioinformatics Institute – with which it shares the Genome Campus a few miles south of Cambridge – the Sanger Institutes is one of the World’s top centres of expertise for genome research. As EMBL-EBI’s associate director Ewan Birney highlighted in a recent article for the MRC Insight blog, by studying the biology of a wide variety of model organisms – including humans and zebrafish among many others – the more than one thousand scientists working on the Genome Campus a gaining critical insights into biology that are advancing 21st century medical science.

When most people think of animal research, they imagine mice, rats or maybe fruit flies. However, other models are increasingly being used in addition to the more traditional organisms. The number of zebrafish (Danio rerio) in particular is steadily increasing in biomedical research each year. You might be wondering why scientists are using fish instead of animals more closely related to humans for their studies. Let’s have a look at some of the advantages of the zebrafish to explain this matter. This list is obviously not going to be comprehensive, because many advantages are field-specific and quite technical, but it should give you an idea why researchers might want to choose fish over other animals.

The zebrafish, a rising star star of medical research.

The zebrafish, a rising star star of medical research.

First of all is something that makes zebrafish more attractive to scientists who pressed for time, such as PhD students wanting to graduate punctually (like me!): zebrafish reproduce at a rapid rate. Each female can lay several hundred eggs each week, which will develop into mature adults in about three months. This is especially useful if you need to breed a large number of animals very quickly, or when you want to cross several lines with modified genes. Rapid breeding also greatly reduces the time it takes to introduce novel genetic modifications into the animals, as several generations are required before a stable modification of the gene in question is achieved. This makes zebrafish a very efficient species for research.

4-day old zebrafish embryo.

4-day old zebrafish embryo.

Another really useful trait of zebrafish is that their embryos are relatively large and initially transparent. This makes it easy to manipulate the embryo, which is very helpful if you are injecting various substances to modify their properties. In my case, I’m using a technique called CRISPR-Cas9 to very precisely switch off certain genes, but there are many other applications.  An added advantage is that you can treat these embryos chemically to stop pigmentation from forming, making it very easy to study early embryonic development (Figure 1). Moreover, the embryos are permeable to many chemicals and drugs – making them ideal for screening large numbers of toxicology samples or drug candidates.

The zebrafish genome has been fully sequenced, which is a must-have for model organisms nowadays.  This effort showed that their genome is remarkably similar to the human one, with at least 70% of human genes having a zebrafish equivalent – a figure that is even higher when only disease-causing genes are considered. There are also efforts underway (by the same group that sequenced the zebrafish genome, which coincidentally happens to be right next to my research group) to mutate every single gene in the zebrafish genome. This can be very helpful if you study a certain gene and wonder what happens to the whole organism when it is lost – and having such large scale resources can save the wider research community huge amounts of time and effort.

Apart from fish with mutations in specific genes, there are also numerous lines containing genes from other organisms (transgenic lines). Usually the proteins encoded by these genes are fluorescent and are used to mark specific cells, as we can control (at least partially) in which tissue a protein is made. One of these is called green fluorescent protein or GFP (originally from a jellyfish). Using techniques such as GFP it is possible to visualize changes in specific cell populations in real time in living animals. Just to give you a personal example: I study blood development, so naturally I want to look at the different types of blood cells. Depending on what cell type I want to look at, I can select an appropriate zebra fish line, where this type is labelled. For an example, have a look at Figure 2, which shows early blood cells during embryonic development labelled with GFP. As these fluorescent proteins come in different colours, it’s possible to look at two or more different cell types at the same time.

A recent advance is the generation of fully transparent adult zebrafish, aptly named “Casper” after the popular cartoon ghost. You can look up the freely available original paper here if you want to see what these fish look like. Of course, scientists are not making transparent fish just because they look cool, but they are very useful tools for research. One application is the easy study of tumour metastases, as the cancer cells are just much easier to spot in transparent fish. Adding fluorescent labelling as described above can make this technique even more powerful.

Usually when we use zebrafish, we take advantage of the fact that many fundamental processes have been evolutionary conserved between fish and humans. Because of these similarities, we can use zebrafish as a model for what happens in humans. Sometimes differences between animals and humans can be more telling though. For example, many animals can regenerate much more efficiently than humans (you might have heard about the ability of salamanders to regrow lost limbs or tails) and this is also true to some extent for zebrafish. One very well studied research area is heart regeneration. Humans are unable to regenerate heart muscle tissue, which is of course problematic when parts of it die off during a heart attack or following injury. In contrast, zebrafish can use stem cells to regenerate the lost tissue – if we could induce a similar process in humans, it might help treating people recovering from cardiac injury. The British Heart Foundation is funding important research in this particular field through its “Mending Broken Hearts” campaign. In the even longer term, it might be possible to adapt similar principles to other tissues and thereby help in treating a variety of injuries.

The regenerative capacity of zebrafish isn’t only interesting for medical research, but it has a very practical advantage: you can cut a tiny part of the tail fin off and use it to extract DNA from the tissue. Then, the mutation status of a specific gene can be determined, which is essential when you want to know whether an animal is a carrier for the mutation you are interested in. The fin then grows back within two weeks, so the animal is not harmed.

Lastly, I just want to mention some financial considerations. Animal research in general is really expensive, which is one of the reasons why alternatives are used whenever possible. These costs are largely determined by how much effort and space is required to house, feed and care for the animals. Of course, this makes large or exotic species especially expensive, so they are used less often. However, even rodent colonies can cost quite a lot of money to maintain. Zebrafish require much less space per individual, are relatively inexpensive to feed , and it’s also relatively straightforward to ship animals (usually as embryos) between labs. This facilitates collaborative research and reduces the number that need to be used, since they don’t have to recreate the same genetically modified line all over again.

In conclusion, zebrafish have a lot of useful characteristics that make them very practical and useful model organisms, which explains their rising  popularity among researchers.

In the next article of this series, I’m going to have a closer look at zebrafish care, as well as daily work in a fish facility and some of the rules and regulations surrounding fish welfare.

Jan Botthof

Guest Post: Animal models in research are necessary and ethical

The following post was originally published in The Daily of the University of Washington on April 26, 2015. It has been reproduced with permission from the newspaper and the original author. Benjamin Cordy is a neurobiology student at UW, he is also the Editor-in-Chief of Grey Matters Journal – an undergraduate neuroscience journal whose mission is to educate the public and develop effective science communicators.

Guest editorial: Animal models in research are necessary and ethical

On Saturday hundreds gathered in Red Square to voice their opposition to scientific research. At its core, this is the true message of the animal rights movement, which believes that research should never rely on animal models. The march on UW was about stopping science altogether. Is this really the best move for society?

Debates about animal models in research are emotional, contentious, and unfortunately, often fraught with demonstrably false “facts.” This is a serious problem. It is impossible to have a thoughtful conversation about the role of science and medical research in society if a position is based on misinformation and inaccurate beliefs.

Two of the most frequently repeated claims of the animal rights movement are that animal models are not actually useful in science and that there are more effective, humane ways to engage in research. While appealing, both statements are wrong.

The history of science provides countless examples of the utility of animal research. For example, until as recently as 1940 and the development of the “antibiotic age”, a knee scrape, if it became infected, could be a death sentence.

In 1928 Alexander Fleming discovered that when grown in proximity to one another, the mold Penicillim notatum killed the colonies of the often-fatal bacteria Staphylococcus aureus. Unfortunately, Fleming’s test-tube studies failed to show the antimicrobial properties he expected from Penicillin. These results, and the difficulty of isolating Penicillin, ultimately led Fleming to believe that it might only be useful as a topical antiseptic.

Although Fleming’s work showed some promise, Penicillin was not a high priority for antimicrobial researchers. In addition to being very difficult to isolate, its therapeutic properties seemed to be inactivated in blood — making it a poor candidate for treating systemic infections. But by 1940 enough Penicillin was isolated for testing. In a landmark study Ernst Chain and Howard Florey infected eight mice with a deadly dose of Streptococcus pyogenes. One hour later, four of the mice were injected with Penicillin. These mice survived the infection and changed modern medicine forever.

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(Left to Right) Alexander Fleming, Howard Florey and Ernst Chain – Shared the Nobel Prize for Physiology or Medicine in 1945

The amount of Penicillin required to treat a human infection is 3,000 times greater than for a mouse. If animal models were unavailable to Chain and Florey, they would have had to undergo the perfectly unreasonable task of isolating huge quantities of a substance that, as far as they could understand, had no therapeutic value. Simply put, without animal models Penicillin would not have been developed.

Fortunately, the story of Penicillin is not unique. There are literally thousands of medical interventions, drugs, and procedures whose discovery and development required the use of research animals. Modern therapies that require animal models include: vaccines, organ transplants, cancer treatments, HIV/AIDS drug development, and thousands more. The claim that animal models are “bad science” and fail to provide important insights into biological understanding and therapeutic development is dishonest and wrong.

The second position of the animal rights movement is that there are alternatives that are simultaneously more effective and humane. The three most often suggested alternatives are human cell cultures, computer models, and experimentation on human subjects.

Tissue and cell culture experiments are extremely powerful research techniques. Their use provides important insights into the function of individual cells and helps identify potential targets for future therapeutics. However, these studies, by their very nature, can only reveal a fraction of the whole picture. For example, a few cells could never describe the complexity of an entire organ — much less the entire organism. Though important for reducing the number of animals used, these techniques could never replace them.

Computational techniques are another tremendously valuable tool. With mathematical models and data analysis, computers allow researchers to better understand the systems they study. But again, computation is a supplement to animal research, not a replacement. Every computer model has to be validated against data collected from animal research. There is no other way to ensure that a modeling program is accurate.

Furthermore, animal rights activists overestimate the power of computer models. In 2007 researchers were able to simulate a virtual brain of 8,000,000 neurons, roughly the complexity of half a mouse brain. While impressive, this is less than 1/10,000th the number of neurons in a human brain and likely much less complex. The simulation ran on the fastest supercomputer and could only do so for 10 seconds at 1/10th the speed of a real brain. In all, this program required the world’s most powerful supercomputer to model one second of one half a mouse brain. How could a desktop PC possibly predict the behavior of the human brain?

The most troubling alternative proposed by animal rights activists is the use of human volunteers for basic science. In practice, such policies would effectively halt biomedical research. For one, the cost of recruiting and paying human subjects would bankrupt already sparse science funding within months. This of course, assumes that enough people volunteer to participate. Considering that clinical researchers already have difficulty in recruiting people for fairly benign studies, it is highly improbable that eight people would volunteer to receive a deadly dose of Streptococcus pyogenes, for example.

Beyond the practical limitations of using only human subjects, there are serious questions about the morality of doing so. Which population is likely to accept payment for becoming test subjects: the socioeconomically disadvantaged or the wealthy? The argument that humans ought to replace research animals raises real concerns about the exploitation of disadvantaged communities.

It was not long ago that I was sympathetic to some of the positions of the animal rights activists. But, as I learned the science behind biomedical therapeutics, it became clear that because animal models save millions and millions of lives, they are necessary. A powerful research program, which includes the use of animal models, is the responsibility of an ethical society.

Benjamin Cordy, UW neurobiology student