Author Archives: Blue Sky Science

Behind the Scenes of Zebrafish Research

Today we have the 2nd in a series of articles by Jan Botthof, a PhD Student at the Cambridge University Department of Haematology and the world renowned Wellcome Trust Sanger Institute. Following his first article “Zebrafish: the rising star of animal models”, Jan discusses here how Zebrafish used in scientific research are housed, cared for and bred.

Today I am going to look at some of the things that have to happen in the background to allow scientists to carry out their research. These things include the rules and regulations covering zebrafish use in research, general care and daily work in the fish facility.

Regulations
Zebrafish research in the UK is covered by the same laws that govern research on all other vertebrates, as outlined in the Animals (Scientific Procedures) Act (ASPA), originally instated in 1986 and recently revised to implement the provisions of the new EU directive. This means that the standard of care is just as high for fish as for mammals. All institutes housing fish need a licence ensuring that standards are met, every research project is evaluated for possible harm to the animals and all of the people involved in research or care for the fish receive mandatory training in order to ensure that the fish are treated correctly. Everyone takes utmost care to ensure that the fish lead a comfortable life in the zebrafish facility.

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish housing
Now that we have covered the basic legislation, let’s talk about essential zebrafish care. Nowadays, fish are usually housed in special rooms, unlike the beginning of their use in research back in the 1970’s, when they were commonly just kept in a few tanks on a shelf in the lab. These rooms are designed to keep a constant temperature (between 24 and 28°C depending on the institution) and the lights are programmed to give a constant light-dark cycle to simulate the sun (usually around 16 hours of light and 8 hours of darkness).

Various commercial fish housing systems exist, but most of their features are very similar. The basic components of such a system are the fish tanks, racks to hold them, an integrated water supply, as well as water filtration and monitoring components.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

The tanks are designed to allow a constant inflow of fresh water, easy removal from the rack and convenient access for feeding. These tanks used to be made of glass, but currently different kinds of plastics are much more popular due to the lower weight, making it much easier to handle them. Unless a procedure requires identification or separation of a specific fish, they are always kept in groups, not only for practical reasons, but also because zebrafish are very social animals and need interaction with other fish.

The water filtration and monitoring system ensures that the water is free of contaminants, has the right pH, salinity, hardness, enough dissolved oxygen and does not contain too much nitrite and nitrate stemming from waste products (i.e. fish excretions, excess food). Apart from the constant flow of fresh water, tanks are cleaned regularly to prevent the accumulation of waste products, as well as microbial and algal growth.

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

A separate quarantine room is also very important. This is where incoming fish from outside facilities are kept on a separate water system to prevent the introduction of parasites and diseases into the main facility. These fish are preferably received as early embryos, which are disinfected before shipping to kill any germs.

Diet
Just like there are different housing systems, there are different possible food sources, ranging from commercially available dry fish flakes to adult or larval brine shrimp. This diet is often supplemented with paramecia (small single celled organisms) to achieve optimal growth and survival rates when the fish are raised to adulthood. Exactly which diet is chosen depends on the individual facility. At the Sanger Institute, fish are fed adult brine shrimp, which are very rich in protein, soft and easily digestible (especially compared to brine shrimp cysts) and they are able to survive and swim even in fresh water. This is much closer to the natural diet that the fish would obtain in the wild than most commercially available diets.

Brine Shrimp. Image: Hans Hillewaert

Brine Shrimp. Image: Hans Hillewaert

Disease prevention
During the daily cleaning and feeding tasks, all tanks are monitored for diseased or injured fish, which are then humanely euthanized to minimize suffering. Euthanasia is usually carried out using an overdose of a common fish anaesthetic followed by destruction of the brain to ensure the death of the animal. Detailed records are kept to identify recurrent problems, such as potential parasitic infections. Dead fish are also removed from the tanks and their data recorded. This monitoring is especially important for fish that have been treated with drugs or that carry mutations likely to cause disease.

Breeding
One essential component of working with fish is setting up matings between them. This is essential if you want to obtain embryos for studying them, or when crossing different genetically modified lines and many other procedures. Fish are placed in small mating tanks in the late afternoon before the actual mating, as zebrafish begin to mate right after sunrise in the wild. These mating tanks have a removable insert between the fish and the floor of the tank, so the fish cannot consume their own eggs, which they would otherwise do.

It is also possible to tilt the separation between the floor of the tank and the fish to further stimulate the fish, as they prefer shallow water for egg laying. If you need the embryos at a specific stage you can use tanks with a separator between the male and female, so you can control the time of the mating. An occasion when you would need to do this is when using the gene editing technique CRISPR to modify zebrafish genes, a process which requires injections of the Cas9 enzyme and appropriate guide RNAs during the first stage of development. Matings can be done in small groups or in pairs. It is very important to be able to correctly identify the sex of the animals – not only do you obviously need a male and a female to have a successful mating, but you also need to know this when you combine different transgenic lines. Here you would take a male from one line and a female from another, so you can put them back in the correct tank after the mating, as it is otherwise nearly impossible to identify individual fish.

Zebrafish mating tank with removable separation before and after assembly.

Zebrafish mating tank with removable separation before and after assembly.

Once the eggs have been laid and fertilized, you can collect them in a sieve, and place them in petri dishes containing water with some salts and minerals essential for development. Embryos are then raised at 28.5°C. Here at the Sanger, the zebrafish larvae are placed in nursery tanks when they are five days old and the yolk that feeds them during early development has run out. These fry reach sexual maturity within three months, from which point on they are considered adults and housed in the main facility. Zebrafish in the lab can live about two to three years, but usually we use younger fish for breeding, as they  lay more eggs.

In summary, a lot of work needs to be done before any actual research can be carried out. Moreover, a lot of effort is put into ensuring the health and welfare of all laboratory animals. The next time you read about some exciting new discovery made using animal research, try to picture how much effort was needed before any actual science was done!

Jan Botthof

Cotton Rats, Calves and Clinical Trials: New RSV vaccine shows great promise.

Respiratory syncytial virus (RSV) affects almost two-thirds of babies in their first year of life, and is a leading cause of bronchiolitis and severe respiratory disease in infants, young children, immunocompromised individuals, and the elderly throughout the world. It is a major cause of hospital admission for infants, and results in up to 200,000 deaths per year in children under the age of 5 years worldwide. Development of an effective vaccine is a public health priority, but has proven difficult, in part due to fact that RSV infection cannot be easily studied in the standard mouse and rat species that are most commonly used in laboratory research.

Oxford University researchers have announced the successful completion of the first trial of a vaccine against RSV in adult humans, which indicated that the vaccine was safe and could induce a robust immune response (though this Phase 1 study did not evaluate its ability to protect against RSV).

In winter RSV accounts for more than 10% of UK infant hospitan admissions.

In winter RSV accounts for more than 10% of UK infant hospital admissions.

In two papers published back-to-back in the journal Science Translational Medicine this week, the University of Oxford team and their colleagues at The Pirbright Institute and the Italian biotechnology firm Okairos (now Reithera Srl) report on the successful Phase 1 clinical trial in adult human volunteers, and the animal studies that led to the trial (1,2).

The basis for their vaccine was a vector derived from the chimpanzee adenovirus PanAd3, which was modified to express several highly conserved human RSV (HRSV) proteins, which they had shown could provide a good level of protection against RSV infection in cotton rats, which are one of the less commonly used laboratory animals, but a very useful model of viral infection of the respiratory system. The cotton rat immune system more closely resembles that of humans at a genetic level than that of more commonly used laboratory mouse and rat species, and because of this similarity cotton rats have been successfully used to evaluate novel therapies for RSV prior to clinical trials, including predicting the efficacy of these drugs in children. As we discussed in an earlier post on the development of gene therapy for Hemophilia B, choosing the right adenoviral vector for the task is critical, and the chimpanzee adenovirus was chosen because there is no preexisting immunity to it in the human population that would compromise its effectiveness as a vaccine vector.

Because studies with other virus-vectored vaccines had shown that heterologous prime/boost with Chimpanzee adenovirus based vector followed several weeks later by a modified vaccinia Ankara (MVA) – a vector used in several experimental vaccines, including HIV vaccines – generates a stronger  immune response than with chimpanzee adenoviral vector alone, the researchers next examined this strategy in cotton rats, demonstrating that intranasal prime/boost immunization was effective in protecting against infection, and did not lead to adverse effects.

The cotton rat - a valuable model for studying lung disease. Image: J.N. Stuart

The cotton rat – a valuable model for studying lung disease. Image: J.N. Stuart

While the cotton rat is a valuable model for the study of respiratory infections, it does not demonstrate all the clinical features of RSV infection, the infection tends to be less severe and to be cleared more quickly in cotton rats than in human infants, and  the extent to which vaccine efficacy in the cotton rat model of HRSV can predict efficacy in humans  is unclear. The scientists therefor evaluated the prime/boost vaccine in a more stringent model, calves, which are the natural hosts of the bovine form of RSV – called bovine RSV (BRSV). The disease course and epidemiology of BRSV infection in calves is very similar to that of HRSV in children, and the very high degree of similarity in sequences of the BRSV proteins to their HRSV equivalents used to develop the vaccine  suggested that the calf would be a valuable preclinical animal model to evaluate the safety and efficacy of their prime/boost vaccine strategies (2).

The results of this evaluation indicated that the different vaccination strategies they assessed were safe and did not cause any adverse immune responses, and showed that heterologous vaccination strategies that used an intranasal administration of the  PanAd3-based vector followed by intramuscular administration of the MVA-vased vector provided superior protection against BRSV infection compared to the PanAd3-based vector alone, or repeated doses of the PanAD3-based vector. As the authors described in the article reporting on the successful phase 1 trial (1) noted in their introduction, these studies paved the way for the evaluation of this vaccine strategy in 42 human volunteers:

In developing this approach toward an RSV vaccine in humans, homologous and heterologous combinations of PanAd3-RSV, including IN vaccination route, and MVA-RSV were tested in preclinical models. The genetic vaccines elicited RSV-specific neutralizing antibodies and T cell immunity in nonhuman primates and protective efficacy in challenge experiments in rodents with human RSV and in young seronegative calves with bovine RSV (32, 33). Of critical importance in both rodent and bovine challenge models was the absence of immunopathology associated with ERD after vaccination, with the calf model acting as a translational model for the development of a vaccine for the pediatric population. All regimens fully protected the lower respiratory tract from bovine RSV infection in the calf, and heterologous combinations resulted in sterilizing immunity in both upper and lower respiratory tracts (33).

The demonstration that the prime/boost vaccine strategy developed by the Oxford University team can safely induce a strong immune response in adult humans, and protect against RSV infection in both the cotton rat and calf models, is very promising, and pave the way for further clinical trials.  Professor Andrew Pollard of the Oxford Vaccine Group, who lead the clinical trial, is keen to now move the development of this much needed vaccine forward:

Both components of the vaccine were found to be safe and to create an immune response.

‘While I am delighted with these results, this was just a first trial. We need this vaccine for children and the elderly and that is where the efforts in vaccine development will now focus.’

Paul Browne

  1. Christopher A. Green et al. “Chimpanzee adenovirus– and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults” Science Translational Medicine, Vol 7, Issue 300, 300ra126, 12 August 2015 Link
  2. Taylor G. et al.”Efficacy of a virus-vectored vaccine against human and bovine respiratory syncytial virus infections” Science Translational Medicine, Vol 7, Issue 300, 300ra127, 12 August 2015 Link

Animal models are essential to biological research: issues and perspectives

The following article by Françoise Barré-Sinoussi and Xavier Montagutelli was published on 31 July 2015 in the journal Future Science OA, and is reproduced here under a Creative Commons Attribution 4.0 License

Françoise Barré-Sinoussi leads the Regulation of Retroviral Infections Division at the Institut Pasteur in Paris, and was awarded the Nobel Prize in Physiology or Medicine in 2008 for her role in the discovery of HIV, and Xavier Montagutelli is head of the Central Animal Facility of the Institut Pasteur. This article follows the recent decision by the European Commission to reject 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.

Animal models are essential to biological research: issues and perspectives

Françoise Barré-Sinoussi (1) & Xavier Montagutelli*,(2)

The use of animals for scientific purposes is both a longstanding practice in biological research and medicine, and a frequent matter of debate in our societies. The remarkable anatomical and physiological similarities between humans and animals, particularly mammals, have prompted researchers to investigate a large range of mechanisms and assess novel therapies in animal models before applying their discoveries to humans. However, not all results obtained on animals can be directly translated to humans, and this observation is emphasized by those who refute any value to animal research. At the same time, the place of the animals in our modern societies is often debated, particularly the right to use animals to benefit human purposes, with the possibility that animals are harmed. These two aspects are often mixed in confusing arguments, which does not help citizens and politicians to get a clear picture of the issues. This has been the case in particular during the evaluation of the European Citizen Initiative (ECI) ‘Stop Vivisection’ recently presented to the European Commission [1].

European-Parliament

Humans and other mammals are very complex organisms in which organs achieve distinct physiological functions in a highly integrated and regulated fashion. Relationships involve a complex network of hormones, circulating factors and cells and cross-talk between cells in all the compartments. Biologists interrogate organisms at multiple levels: molecules, cells, organs and physiological functions, in healthy or diseased conditions. All levels of investigations are required to get a full description and understanding of the mechanisms. The first two, and in some instances three, levels of organization can be studied using in vitro approaches (e.g., cell culture). These techniques have become very sophisticated to mimic the 3D and complex structures of tissues. They represent major scientific advances and they have replaced the use of animals. On the other hand, the exploration of physiological functions and systemic interactions between organs requires a whole organism. It is, for example, the case for most hormonal regulations, for the dissemination of microorganisms during infectious diseases or for the influence of the intestinal microorganisms on immune defense or on the development of brain functions. In these many cases, no in vitro model is currently available to fully recapitulate these interactions, and investigations on humans and animals are still necessary. Hypotheses and models can emerge from in vitro studies but they must be tested and validated in a whole organism, otherwise they remain speculative. Scientists are very far from being able to predict the functioning of a complex organism from the study of separate cells, tissues and organs. Therefore, despite arguments put forward by the promotors of the ECI, studies on animals cannot be fully replaced by in vitro methods, and it is still a long way before they can.

Animal models have been used to address a variety of scientific questions, from basic science to the development and assessment of novel vaccines, or therapies. The use of animals is not only based on the vast commonalities in the biology of most mammals, but also on the fact that human diseases often affect other animal species. It is particularly the case for most infectious diseases but also for very common conditions such as Type I diabetes, hypertension, allergies, cancer, epilepsy, myopathies and so on. Not only are these diseases shared but the mechanisms are often also so similar that 90% of the veterinary drugs used to treat animals are identical or very similar to those used to treat humans. A number of major breakthroughs in basic science and medical research have been possible because of observations and testing on animal models. Most vaccines, which save millions of human and animal lives every year, have been successfully developed using animal models. The treatment of Type I diabetes by insulin was first established in the dog by Banting and McLeod who received the Nobel Prize in 1921 [2]. Cellular therapies for tissue regeneration using stem cells have been engineered and tested in animals [3]. Many surgical techniques have been designed and improved in various animal species before being applied to humans. The discoveries in which animal models played a critical role are indeed numerous and led to many Nobel Prizes.

It is, however, noticeable that the results obtained on animals are not necessarily confirmed in further human studies. Various reasons can be evoked. First, despite large similarities, there are differences between a given animal species and humans. For example, over 95% of the genes are homologous between mice and humans but there are also differences for example in the members of genes families, in gene redundancies and in the fine regulation of gene-expression level. These genetic differences translate into physiological differences which are increasingly better described and understood. While some people like the ECI promotors use these differences to refute the value of animal models, many including ourselves strongly advocate for further improving our knowledge and understanding of these differences and for taking them into account in experimental designs and interpretation of observations [4]. Moreover, these differences may provide opportunities to unravel novel mechanisms and imagine innovative therapies.

Research in mice has led to many medical advances - most recently the development of PD-1 inhibitors for treating cancers http://speakingofresearch.com/2015/05/30/immunotherapy-lung-cancer-pd-1-knockout-mice/

Research in mice has led to many medical advances – most recently the development of PD-1 inhibitors for treating cancers http://speakingofresearch.com/2015/05/30/immunotherapy-lung-cancer-pd-1-knockout-mice/

The second reason is due to genetic and physiological variations within each species or between closely related species. Laboratory mice have been developed as inbred strains which have highly homogeneous genetic composition to increase the reproducibility of results and the statistical power of experiments. Reports on animal models of human conditions often speak of ‘the mouse model of…’, referring in fact to observations made in a given genetic background. However, the clinical presentation often varies if another mouse strain is considered. A striking example is provided by a study published in November 2014 in Science by a team who reported that some mouse strains are fully resistant to Ebola virus, others die without specific symptoms and others develop fatal hemorrhagic fever [5]. Another example is the difference of responses to SIV, the monkey homolog to human HIV, between Rhesus macaques which develop simian AIDS and sooty mangabeys which do not develop symptoms despite high levels of circulating virus [6]. This range of responses reflects in fact the variety of clinical observations among human patients. These examples illustrate how animal models must be considered: no single animal model is able to mimic a given human disease which is itself polymorphic between patients, but the differences between strains or species provide unmatched opportunity to understand disease development and differential host response, and to eventually find new cures.

The second issue regarding the use of animals for scientific purposes is animal protection and welfare. This is the scope of the European Directive 2010/63/EU, which has set the regulatory framework for all animal research. Scientists have recognized for decades the importance of giving full consideration to three fundamental principles [7], which have become the backbone of the European Directive. First, animals must not be used whenever other, non-animal-based, experimental approaches are available, with similar relevance and reliability. Second, the number of animals used must be adjusted to the minimum needed to reach a conclusion. Third, all provisions must be taken throughout the procedures to minimize any harm inflicted to the animals. These principles, known as ‘the three Rs rules’, for replacement, reduction and refinement, have become the standard to which every project involving the use of animals is evaluated.

Animal research is conducted in compliance with regulatory provisions which cover the inspection and licensing of animal premises, the training and competence of all personal designing projects, performing animal procedures and taking care of animals and the mandatory authorization of every project by a competent authority upon ethical evaluation by an Animal Ethics Committee. The criteria for evaluation are based on the 3Rs rules and a cost–benefit analysis to evaluate if the potential harm to the animals, which must be reduced to the lowest possible level, is outweighed by significant progress in terms of knowledge on human or animal health. Regulation imposes that ethics committees include members concerned by animal protection and not involved in animal research. In response to the ECI, the European Commission has underlined, in a statement issued on 3 June 2015 [8], that animal experimentation remains important for improving human and animal health. At the same time, it is committed to promoting the development and validation of non-animal-based approaches, and to enforcing the application of the 3Rs rules by all players, including the research community. Europe has therefore implemented one of the strictest regulatory frameworks for the protection of animals used in research.

21st century medical research is highly interdisciplinary, a fact that is reflected in the design of new research institutions such as the Francis Crick Institute in London

21st century medical research is highly interdisciplinary, a fact that is reflected in the design of new research institutions such as the Francis Crick Institute in London

The greatest challenges faced by modern biomedical research concern complex, multifactorial, diseases such as cancer, cardiovascular diseases, infectious diseases, neurodegenerative disorders, pathological consequences of aging among others, for which all experimental approaches are indispensable because of their complementarity: biochemistry, genomics, cell culture, computer modeling, animal model and clinical studies. Research on relevant, carefully designed, well-characterized and controlled animal models will remain for a long time an essential step for fundamental discoveries, for testing hypotheses at the organism level and for the validation of human data. Animal models must be constantly improved to be more reliable and informative. Likewise, animal protection requires permanent consideration. These two objectives, far from being antagonistic, must be anchored in high-quality science.

References:

1. The European Citizens ‘Initiative – Stop vivisection. http://ec.europa.eu
2. Nobelprize.Org – The discovery of insulin. www.nobelprize.org
3. Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin. Invest. 98(1), 216–224 (1996). [CrossRef] [Medline] [CAS]
4. Ergorul C, Levin LA. An example on glaucoma research: solving the lost in translation problem: improving the effectiveness of translational research. Curr. Opin. Pharmacol. 13(1), 108–114 (2013). [CrossRef] [Medline] [CAS]
5. Rasmussen AL, Okumura A, Ferris MT et al. Host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance. Science 346(6212), 987–991 (2014). [CrossRef] [Medline] [CAS]
6. Liovat AS, Jacquelin B, Ploquin MJ, Barre-Sinoussi F, Muller-Trutwin MC. African non human primates infected by SIV – why don’t they get sick? Lessons from studies on the early phase of non-pathogenic siv infection. Curr. HIV Res. 7(1), 39–50 (2009). [CrossRef] [Medline] [CAS]
7. Russell WMS, Burch RL. The Principles of Human Experimental Technique. Methuen, London, UK (1959).
8. European Commission – Annex to the communication from the commission on the European Citizen’s Initiative, ‘Stop Vivisection’. http://ec.europa.eu

Affiliations:

Françoise Barré-Sinoussi
1. INSERM & Unité de Régulation des Infections Rétrovirales, Institut Pasteur, 75724 Paris, France
Xavier Montagutelli
2. Animalerie Centrale, Institut Pasteur, 75724 Paris, France

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