Author Archives: Blue Sky Science

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

Setting the record straight: Environmental enrichment in animal research.

One of the most important goals of Speaking of Research is to counter the misinformation and mistaken beliefs about animal research that are so prevalent in society, even among those who ought to know better, and a recent series of articles in Ampersand –  the blog of the organization PRIM&R (Public Responsibility in Medicine & Research) – illustrates the value of taking the time to correct these errors when they occur.

PRIM&R is an organization whose goals are to create a “a strong and vibrant community of ethics-minded research administration and oversight personnel, and providing educational and professional development opportunities that give that community the ongoing knowledge, support, and interaction it needs to raise the bar of research administration and oversight above regulatory compliance“. It’s membership includes over 4,000 individual members, and its educational and professional development programs address a range of issues surrounding research involving human subjects and animals.

SBER11

So it was rather disappointing to read a blog post entitled 40 years of Research Ethics: Environmental Enrichment which presented the development of environmental enrichment guidelines and practice in a rather superficial manner, and in particular presented PeTA propaganda concerning the 1981 Silver springs case as fact:

The regulatory mandate for environmental enrichment has a long history. In 1970, as a result of amendments to the Animal Welfare Act (AWA), enclosure standards for all warm-blooded animals were developed.  The need for additional regulations became apparent in 1981 when Alex Pacheco, an animal rights activist and cofounder of the then-newly formed organization People for the Ethical Treatment of Animals, discovered and documented violations of the AWA as a volunteer at the Institute for Biological Research in Silver Spring, MD. Pacheco’s work drew public attention to the care of laboratory animals.

In the years following the Silver Spring Monkey case, a number of bills advancing standards for the care of laboratory animals were introduced in the US House and Senate. In 1985, the Food Security Act amended the AWA to mandate exercise for dogs and a “physical environment adequate to promote the psychological well-being of primates.” While initially the research community responded to the mandate for environmental enrichment with hesitation, today such programs are considered fundamental to a comprehensive animal care and use program

In response to this Allyson J. Bennett, PhD, former chair of the Committee on Animal Research and Ethics at the American Psychological Association (APA) – and  Speaking of Research member – , and Sangeeta Panicker, PhD, director of research ethics at the APA, contacted PRIM&R to express their concerns, and to their credit PRIM&R published their email in new post. In their email Allyson Bennett and Sangeeta Panicker pointed out (among other points) that:

Contrary to well established facts, the post implicitly maligned a distinguished member of the psychological science community (involved in the ‘Silver Spring Monkey case’), and lauded the less than honorable tactics of the individual associated with a group, People for the Ethical Treatment of Animals (PETA), that is publicly opposed to research with nonhuman animals. Furthermore, based on scant, if any, credible evidence, the blog post credited PETA for almost singlehandedly achieving changes to the Animal Welfare Act (AWA) that led to environmental enrichment requirements for research animals.

and that:

Finally, circling back to the events described in the PRIM&R blog post, we note that not only was the researcher in question exonerated on all but one count of AWA violation by USDA, as well as the US judicial system, but three highly respected scientific organizations—the American Association for the Advancement of Science, the Society for Neuroscience, and the American Psychological Association—independently investigated the so-called ‘animal abuse’ and found his conduct to be beyond reproach. Furthermore, in light of the baseless accusations against the researcher, we believe it is incumbent upon PRIM&R, the premier organization in the continuing education of institutional animal care and use committees, to acknowledge the impact of this ethically and scientifically sound research with nonhuman primates on the rehabilitation of individuals recovering from strokes and spinal cord injuries.

Mice in a research laboratory. Image courtesy of Understanding Animal Research.

Mice in a research laboratory. Image courtesy of Understanding Animal Research.

So far, so good, but what is really interesting is that it didn’t stop there. A few months later PRIM&R published another article entitled The Evolution of Environmental Enrichment which gave a far more balanced account which acknowledged that the history of environmental enrichment reaches back far longer than 40 years, and how animal researchers, in particular behavioral researchers, have played a key role in shaping its development. In the introduction to their post the writers acknowledge that the impetus for writing this second post came from the email sent by Drs Bennett and Panicker:

In the spirit of transparency and respectful dialog, PRIM&R has written this second post, which we believe is a more considered treatment of an important and complex issue. We thank Drs. Bennett and Panicker for their feedback and for prompting us to take this second look.

What can we learn from this series of posts?

Firstly, the first post shows  how the myths and misrepresentations spread by animal rights organizations have become so pervasive that even many people who should know better take some of them for granted. This is something we have  encountered time and again, and even many scientists who support the use of animals in research don’t appreciate just how high a proportion of the claims made by animal rights groups are pseudoscience.

Secondly, the next two posts show how some (regrettably not all) people are willing to listen when presented with the facts, and how this can spur them to become better informed and reconsider their prior assumptions. This is why it is so important that scientists and supporters of scientific research who know the facts take the opportunity to engage with both specialist and general media to correct misapprehensions and misrepresentations.

You can make a difference!

Speaking of Research

Pro-Test Deutschland: Standing up for science in Germany!

Today we welcome the launch a brand new science advocacy organization, and a new member of the Speaking of Research Family, Pro-Test Deutschland!

Pro-Test_Deutschland_image

Pro-Test Deutschland is a grassroots science organization founded by 18 young scientists and supporters of medical progress in the German university town of Tübingen.

The need for such a grassroots campaign in Germany has never been greater, as over the past few years the rhetoric of animal rights activists in Germany has been getting steadily more extreme. This culminated last month with the announcement by Professor Nikos Logothetis, a leading neuroscientist at the Max Planck Institute for Biological Cybernetics in Tübingen, that he would be ending his research with non-human primates. His decision followed a series of false allegations by animal rights activists, and a campaign of vilification and intimidation against him, his family and his colleagues.

A lot can change in a month. Within days of Prof. Logothetis announcement over 4,000 scientists in Germany and beyond had signed a motion in solidarity with him and his colleagues, and the Max Planck Society issued a strong statement of support. The events in Tübingen spurred the wider European scientific community to take a strong public stance on the necessity of animal research, and its intervention played an important role in yesterday’s decision by the European Commission to reject the Stop Vivisection Initiative.

The launch of Pro-Test Deutschland comes at a critical time for science in Germany, and indeed in the EU as a whole, and we look forward to working with our new friends to support animal research that is so crucial to advancing science and medicine.

Below is the text of a press release that Pro-Test Deutschland issued yesterday to announce their launch. They have also issued an invitation letter to anyone who would like to get involved with details on how to get in touch.

Press Release June 3rd, 2015

Pro-Test Germany, a supplier of reliable information and advocate for animal testing in research

Tübingen, June 3, 2015

Pro-Test Germany is an initiative intended to lend a voice to science. Its primary goal is to educate the public on scientific, ethical, legal, social and psychological aspects of animal research. In addition, Pro-Test Germany will provide reliable information to help those better understand the role of animals in research and the benefits to society.

Today the European Commission decided that the 2 010/63/EU directive for the protection of research animals will not be affected by zealous antivivisectionists. This is good news for animal welfare. And it is good news for our society as a whole, as this decision issues a clear vote for science and research in the EU.

The often one sided campaign led by animal research opponents has recently left a huge impression on Tübingen, Germany. For one instance, the renowned neuroscientist Nikos Logothetis had decided to withdraw from his primate research to escape ongoing threats and harassment. Until now there has been very little public support for this research, especially from the scientific community, even Logothetis lamented a lack of support in his decision letter.

A powerful voice in the public debate is largely absent. Where have the scientists been during these one sided discussions? Scientists, whom are the most familiar with this research, are largely afraid to speak out because of the potential hostility or because they may not be understood or able to convey a message that the public understands. Not all scientists are adept at speaking out about their research; however, Pro-Test Deutschland aims to educate and provide a secure platform for scientists to speak and the community to get involved.

The view that animal testing in research is not only ethical but also necessary may be widespread, but it is rarely openly professed. For many people outside of science, it is also often difficult to obtain reliable information, such as reports on the outcomes of animal research and their public benefit. This fundamental problem has been acknowledged by young scientists in Tübingen. So by now, it is time to release Pro-Test Germany, an advocacy group for animal research and a voice lent to science. The founders of Pro-Test Germany believe that animal testing in research is ethically and scientifically necessary. All the while supporting a broad societal discussion based on information and literature that ranges through all sides of the story. Thus, to promote an informed and fair debate, Pro-Test Germany will provide a point of contact for all those who want to learn about the role of animals in science.

Pro-Test Germany is initially aimed at building a website that collects data, facts and personal testimonies concerning animal research and its final outcomes. The homepage at http://www.protestgermany.org is going live tonight on June 3, 2015. Additionally, a social media campaign has already begun on Facebook and Twitter. In due course, further activities will also be tackled, such as informational events, lecture series, open letters, rallies etc. The objective is to push Pro-Test Deutschland as far past the Swabian university city limits as possible.

Website: http://www.protestgermany.org
FaceBook: https://www.facebook.com/protestdeutschland
Twitter: @ProTestDE

Pro-Test Deutschland_logo

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

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