Use of gene altering technology

Of the 3.4 million regulated procedures carried out on animals in 2019 nearly half, 1.67 million, were for the creation and breeding of genetically altered (GA) animals. This number has been steadily increasing over the past 20 years and is the main reason why UK statistics do not show a fall in laboratory animal use over this time.

So what are GA animals, how and why are they created, and are they bringing us closer to understanding and curing human diseases?

Genetically modified (GM) is a term used to describe animals that have had their DNA altered for example by adding, removing, or modifying specific genes. The resulting changes in DNA can have a positive, negative, or neutral effect on the physical characteristics of an organism. In scientific research this is normally a deliberate change to model a disease or investigate the function of a biological process. These animals are referred to as genetically altered (GA), but are also known as genetically modified (GM) or genetically engineered (GE). An organism which has had the genes of another species introduced into its genome is referred to as transgenic.

In the 1970s the first GA mouse was created by inserting a viral DNA into an early-stage mouse embryo. The inserted viral DNA was then found to be present in various organs of the mouse as it matured.  In 1981 the stem cells from two mouse embryos were mixed to produce mice with a combination of both sets of genes which they then passed on to offspring. Later in the same decade the first ‘knockout’ mice were produced. A knockout animal is a genetically altered animal that has had one or more genes removed or rendered inactive. This has proven a very useful technique for helping to understand the role of specific genes, both in normal body function but also in the development and cause of disease. In 2007 the Nobel prize was awarded to the scientists who developed this pioneering technique.¹ Scientists have been fascinated by the ability to alter the genetics of animals and the possible applications of this technology which has unsurprisingly progressed significantly over the past 30 years.

Genetic modification can be used to remove or replace faulty genes in animals. This has potential health and economic benefits, particularly in farming. Whilst meat and milk from genetically modified animals are not currently found on supermarket shelves in the UK that may soon be the case². Genetic modification allows the identification and removal of faulty genes in embryos, and can be used to speed up growth rates, increase milk production, improve meat quality, or increase disease resistance. The wider implications of this are not yet known, which is why genetic engineering has not yet been approved as the way forward for animal production in agriculture.

This technology has opened the door to the potential uses of genetically altered animals in the study of human disease. GA strains of mice in particular have become very popular in medical research. Alongside humans, mice were one of the first animals to have their whole genome sequenced. Researchers quote that of the 10% of our DNA which contains protein-coding genes, around 85% is found to be identical between humans and mice. Of the other 90% of our non-coding or ‘junk’ DNA the similarity is much less at 50% or below.³ For mostly practical reasons the mouse was already a well-characterised and widely used experimental model. Mice are small, easy to handle, house and breed, and their gestation time is short. In addition, as wild animals they show a lot of variation which is helpful for creating different research strains, and as mammals they have similar, although by no means identical, body systems to humans.

With advances in genetic engineering came the rise of the transgenic mouse – an organism that contains genetic material from another species. Specifically, ‘humanised’ mouse strains where mouse genes are replaced with human genes in order to recreate or study human diseases, or drug/pharmaceutical targets, have become commonly used in research. This has undoubtedly provided new knowledge in some areas of research where there are similarities between naturally occurring conditions in mice and humans. These include for example cataracts and muscular dystrophy, the latter of which is an example of a relatively rare genetic disease caused by a naturally occurring faulty gene in both mice and humans.

The advent of modern gene editing technologies such as CRISPR/Cas 9, which became more widely available from 2013 onwards, has further increased the ability to carry out specific genome edits in animals, and create strains of mice for many different disorders on demand. CRISPR systems are faster, more precise, significantly cheaper, and more efficient than the genome editing methods commonly used before this.⁴ GA mice are now widely used in many areas of medical research including cancer, immunology, neurobiology, endocrinology, human genetic diseases such as sickle cell disease and cystic fibrosis, and disorders where genes can partially contribute such as diabetes, rheumatoid arthritis, and Alzheimer’s.

It is not a surprise then that the number of GA animals used in scientific procedures has steadily increased over the past 20 years. This is the primary reason why UK statistics do not show a fall in laboratory animal use over this time.⁵

The use of animals in science in the UK is governed by the Animals (Scientific Procedures) Act 1986 (ASPA). This regulates what is and isn’t allowed to be done to animals and for what purpose, in the name of science. ASPA defines a “regulated” procedure as one performed on a protected animal that has the potential to cause pain, suffering, distress, or lasting harm. Regulated procedures must be recorded, and these figures are published in the UK annual statistics.  Procedures for the creation and breeding of GA animals are classed as a regulated procedure because the resulting altered phenotypes of these animals have the potential to negatively affect them even if these animals are not used in for further experimental procedures after birth.

Of the 3.4 million regulated procedures carried out on animals in 2019 nearly half, 1.67 million, were for the creation and breeding of GA animals, mainly mice. Whilst this number has been very slowly decreasing since 2013,⁶ it is still worryingly high.  Eighty-eight percent of that 1.67 million were procedures on animals purely for the maintenance of already established GA lines, and the breeding of offspring to keep the lines going for future research/clients. The animals themselves were not used in experimental procedures. That’s almost 1.5million procedures carried out to breed animals with genetic alterations that could cause pain and suffering, that were not actually used in any experimental procedures.

If we consider all 1.73 million regulated experimental procedures carried out on animals in 2019, just under half (42%) used GA animals.⁶ The statistics also show that whilst the overall number of experimental procedures has been slowly falling, the number of procedures carried out on GA animals has risen and is currently holding steady. It must be noted that more than one procedure may be carried out on the same animal, the statistics record the number of procedures themselves rather than the total numbers of animals used. This still reflects the current popularity of GA animal use and is illustrated in this graph taken from the 2019 UK statistics.⁶

Overall, it is clear that GA animal use is the main driving factor responsible for the high number of scientific procedures performed on animals in the UK. Reducing the use of GA animals in research and the number of procedures required for creating and breeding them, would have a major influence on reducing animal use in scientific experimentation.

The EU releases statistical data for all member states every five years. The last of these published reports covering 2015 – 2017 stated that ‘Genetically altered animals are used almost exclusively for research purposes. In 2017, basic research (understanding living systems and disease processes) accounted for 75% of uses of genetically altered animals and translational and applied research (developing potential drug interventions and testing these for efficacy) for 21%.’⁷ This explains where these GA animals are being used, it is not in regulatory testing to meet legislative requirements, but for more open research purposes including drug discovery research prior to regulatory tests. Basic research consists of research to increase knowledge of the structure, function and behaviour of living organisms and the environment. In the 2019 UK statistics the most common areas of basic research that involved regulated animal procedures were on the immune system, the functioning and disease of the nervous system, and cancer (oncology) indicating research for medical and human health purposes.

This high use of animals must also be weighed against the value of GA animal models in different research fields. In cases where they are adding little or no value to human disease research is their continued use ethically warranted? Are the high numbers of animals required to create and maintain these lines justifiable?  The remainder of this article highlights some of the issues with the continued use of GA animals and suggests some alternative ways forward.

The use of GA animals has increased knowledge of the function of various genes, both in mice and in humans, and has helped us understand aspects of some human conditions and disorders. The media report stories of GA animal research successfully identifying the role of a specific gene, but fewer examples of how this has led to significant progress in understanding or treating a human condition. There are undeniably some however, for example the use of ‘knock-out’ mice to discover the role of orexin, a peptide found in the brain, in sleep regulation. Similarities between mice and humans in this case contributed to the development of a drug to help treat narcolepsy in people.⁸

Spurred on by this apparent success scientists are now starting to move on from single ‘knock-out’ mice to study the effect of one gene, to more complex models with multiple modifications to study human conditions that are polygenic (caused by more than one gene). We say ‘apparent success’ as there are many publicly available examples of positive medical research findings obtained from GA mouse models. However, there is very little publicity, or indeed actual data concerning research that proves inconclusive, gives negative results, or fails to translate into human research; the vast majority of such experimental data is never published (a commonly identified phenomenon across scientific fields known as publication bias). Consequently, the available research is not an accurate reflection of the ultimate success or failure of GA animal models in different research areas.

But back to the complex GA models.  These are used in research into diseases such as obesity⁹, diabetes¹⁰ and psoriasis¹¹; human diseases caused not only by complex genetic factors but also aging, lifestyle and environmental factors which are hard to replicate in an animal model. Modelling the genetics of a varied human population, would potentially require multiple GA animal lines with different genetic backgrounds, leading to more animals and yet more expense. The value of producing more complex GA animal models of human diseases must be considered not only ethically and financially, but also weighed against the scientific relevance of the model and the number of animals required to create it. The scientific relevance of replicating age and lifestyle related diseases in animals with short lifespans and no similar naturally occurring conditions, must be questioned. Despite this GA animals are still widely used in research into human age-related conditions and diseases such as ALS¹³ (or MND – Motor Neurone Disease), Parkinson’s and Alzheimer’s¹⁴. These are complex conditions where much research still relies heavily on mice to try to gain new understanding, but some scientists are now arguing that a blinkered animal research approach in some areas may be hindering scientific progress.¹⁵

Research into such complex conditions is difficult and expensive to conduct, and many animals may be required to investigate one human condition. Researchers will often work on one aspect of a condition and the role of a particular gene hoping to ascertain knowledge that will ultimately help understand the disease and treatment options. This approach requires multiple animal models across many projects to gain a full picture of the mechanisms of one disease. A 2014 paper in Respirology¹⁷, suggests that ‘…asthma endotypes and mouse models can be aligned, resulting in a stratified approach to preclinical asthma research…’ whilst acknowledging in the conclusion that the main approaches to treating asthma have not evolved significantly in 20 years ‘indicating a need for change in the approach to asthma research and clinical management.’

One of the perceived scientific benefits of using a GA animal line is the increased validity that comes from reducing variation in a sample. Using genetically similar animals should reduce variation in the data collected leading to more conclusive results. Scientists use inbreeding and genotyping (studying DNA to identify differences) to help maintain this genetic similarity and reduce genetic drift (variations of a specific gene in a population). Whilst, from a scientific perspective this is the best approach to increase reproducibility¹⁸, it requires a high number of animals to maintain the integrity of a GA colony and as stated previously many genetically similar, inbred colonies to represent a varied human population.

In contrast to this there are studies that suggest that in preclinical research ‘outbred’ mice may more realistically reflect the human body, for example in how the immune system functions.^18,19 Limiting genetic and environmental variation in scientific studies is key to obtaining reproducible outcomes. Inbreeding GA mice helps maintain the desired DNA alteration whilst limiting natural variation caused by genetic drift. The aim of this is to reduce variation in the data, unfortunately at the same time this inbreeding is potentially producing data even less relevant to humans. Gene editing technology today can be used directly on human tissue to establish the role of certain genes directly in human cells without the need to use animals.²³ A number of scientists and organisations hold the view that this is where funding can be used more effectively to break new ground in medical research than focusing on more advanced GA animals.²⁴

Another issue is that gene manipulating technology is not perfect and often leads to common ‘off-target’ effects where other genes are mutated as well as the target gene. This can result in unplanned or unwanted phenotypes that were not desired and in extreme cases these cause unintended suffering in the animal bred. An example of GA lines with a potentially severe phenotype are SOD-1 transgenic mice (Superoxide dismutase 1) which are commonly used in motor neurone disease research (a neurodegenerative disorder for which there is no treatment). They are born seemingly normal but show progressive weight loss, the inability to right themselves by 21days, develop onset of paralysis at around 88 days and eventual death. ²¹ The severity classification of the protocol being used depends on the planned humane endpoint (point at which the animal is killed in the research to prevent further suffering). SCID (severe combined immunodeficiency disease) mice and other GA mice have the potential to experience severe suffering as a result of their genetic modification. These mice are modified to be immunodeficient and lack white blood cells. ²²

Established GA animal lines have desired phenotypes (physical characteristics caused by genes) which are classified as harmful or not harmful depending on the potential of the gene modification to impact welfare. The procedure being carried out on the animal is also classified for severity as sub-threshold, mild, moderate, or severe based on the suffering the animal will experience because of the procedure, and the phenotype of the animal being used. When considering the ethics of research involving GA animals, the severity of the model being used is also therefore relevant. Yet there is currently no system or requirement to publish all licensed animal research, or to publicly share retrospective project reviews that license holders are required to submit to the Home office. The same GA lines, including those with harmful phenotypes, can therefore be repeatedly bred, adapted, and used to answer research questions where the use of the GA model has led to little or no scientific progress, the lack of a requirement to publish meaning scientists are not aware of previous negative or null findings. This can lead to more breeding and research happening with harmful phenotypes than is necessary, increasing the numbers of animals that may potentially suffer as a result.

The creation and maintenance of GA lines requires many regulated procedures for example for the administration of drugs to cause super ovulation in females, to collect eggs, and to implant genetically modified eggs into surrogate mothers. In 2019 there were 1.67 million procedures carried out for this purpose, nearly half of all regulated procedures that year.⁶ The UK Government guidelines on severity classification of GA animals under the Animals (Scientific Procedures Act) 1986 (ASPA), provides more detailed classification of the severity of breeding procedures and harmful phenotypes relating to creating and using GA animals.²¹

So what can be done to stem the tide of GA animal use?  We would suggest the following:

• Registration of all animal studies. Requesting that all animal research studies that are approved under ASPA project licenses be registered on a chosen, publicly available register for animal/preclinical research to ensure that all completed animal research is visible whether published or not. This is already a legal requirement for clinical studies^{. 25} If this register included simple project outcomes this would increase transparency around GA animal use, allow researchers to make more informed decisions around the use of GA animals and help regulators assess the scientific relevance and likely value of proposed projects. Some registers already exist such as the Preclinical Trials Register^{, 26} the German Animal Study Registry. ²⁷
• Funding and supporting non-animal methods. Funding bodies and journals help drive what research happens and what research is published. They must be not just open to funding and publishing papers where non-animal technologies have been trialed/used, but actively encourage submissions in areas where GA animals may previously have been popular. Journals should not be asking authors to validate modern, non-animal approaches with animal model work, or actively prioritising publication of projects that have done this. This flawed thinking suggests that animal models are the ‘gold standard’, which we know is false, and actively goes against the fundamental principles of the 3Rs. Funders can actively prioritise the funding and promotion of human-relevant, non-animal methods, particularly those that use gene editing technology to help progress medical research in these areas. They can also help to support the training and education of researchers to understand the availability of all research tools – animal and non-animal – and facilitate discussions to identify barriers that may be hindering the uptake of alternatives in areas where animals are still heavily used.
• Ensuring published research using GA animals is of value. We must ensure published papers that use GA animals are scientifically relevant considering historic and current work in the field, and available alternative, non-animal methods. This rigour should start during the early project planning stages, be evaluated when a project licence application is submitted to the Home Office and continue through the review process when papers are submitted for publication in journals.
• Training and knowledge enhancement for decision makers. The Home office ASRU inspectors, AWERBS (Animal Welfare Ethical Review Body), NACWOs (Named Animal Care and Welfare Officer) and other named persons at research establishments all play a role in reviewing potential research projects. They are in key positions to help question and reduce research using GA animals. These vital roles require time, commitment, and knowledge, and must be supported in light of the changing face of animal research in the UK.

GA animal use is a fascinating area of science that has captured the imagination of a generation of scientists, with its exciting potential to better understand and treat disease.  However, there are many flaws in the ways in which the technology is being used resulting in a lack of concrete outcomes of direct relevance to human disease, as well as avoidable animal suffering.  Once again both the ethics and the science lean towards the prioritisation of human relevant methods to enable researchers to produce valid outcomes from their work.

The considerable time and funding being invested in developing new and better ways to create GA mice that display human ailments is a waste of scarce resources. We would get further, faster, by prioritising human-relevant models of enquiry.

Further reading

• Of mice and men – are mice relevant models for human disease (europa.eu)
• The use of genetically modified animals (royalsociety.org)
• The yin and yang of genome editing (FRAME)

References

1. Biologists claim Nobel prize with a knock-out: Nature News
2. Genetically modified farm animals and fish in agriculture: A review – ScienceDirect
3. Why Mouse Matters (genome.gov)
4. CRISPR | Broad Institute
5. Biotechnology | Genetically altered & cloned animals | Research (rspca.org.uk)
6. Annual Statistics of Scientific Procedures on Living Animals Great Britain 2019 (publishing.service.gov.uk)
7. EUR-Lex – 52020DC0016 – EN – EUR-Lex (europa.eu)
8. The use of genetically modified animals (royalsociety.org)
9. Biology of Obesity: Lessons from Animal Models of Obesity (hindawi.com)
10. Comparison of Two New Mouse Models of Polygenic Type 2 Diabetes at the Jackson Laboratory, NONcNZO10Lt/J and TALLYHO/JngJ (nih.gov)
11. A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice – PubMed (nih.gov)
12. ALIGNING MOUSE MODELS OF ASTHMA TO HUMAN ENDOTYPES OF DISEASE (nih.gov)
13. Mouse models of ALS: Past, present and future – ScienceDirect
14. Alzheimer’s research: a ‘mighty mouse’ is still a mouse | FRAME
15. Animal experiments are slowing down progress for ALS | Cruelty Free International
16. FRAME praises animal research facility closures | FRAME
17. ALIGNING MOUSE MODELS OF ASTHMA TO HUMAN ENDOTYPES OF DISEASE (nih.gov)
18. Laboratory mice born to wild mice have natural microbiota and model human immune responses | Science (sciencemag.org)
19. The unexpected advantages of outbred mice in research | Diverse mouse strains at The Jackson Laboratory (jax.org)
20. Guidelines on severity assessment and classification of genetically altered mouse and rat lines – PubMed (nih.gov)
21. asp-severity-classification-of-genetically-altered-animals.pdf (publishing.service.gov.uk)
22. SCID Mouse – an overview | ScienceDirect Topics
23. New Screening Approach Reveals Novel Regulators of Microcephaly | The Scientist Magazine® (the-scientist.com)
24. Why Nobel Prize-winning CRISPR should be the game-changer for animals – Animal Free Research UK
25. Clinical Trials Register
26. https://preclinicaltrials.eu/
27. https://www.animalstudyregistry.org/asr_web/index.action