Research Review 2019

Pioneering Better Science About the NC3Rs Contents

1 Introduction 04

2 Case studies 08

Ferrets and flu transmission studies Professor Wendy Barclay: Imperial College London 10 The National Centre for the Replacement, We support the commitment of the Refinement and Reduction of Animals scientific community to the 3Rs by Fly infectivity assay for prion disease Dr Raymond Bujdoso: University of Cambridge 12 in Research (NC3Rs) is a scientific funding research and early career organisation that leads the discovery development, facilitating open innovation Bioreactor for Cryptosporidium oocysts and application of new technologies and the commercialisation of 3Rs Professor Joanne Cable: Cardiff University 14 and approaches that minimise the use technologies, and stimulating changes Blood flow in mouse stroke models of animals in research and improve in policy, regulations, and practice. Professor Claire Gibson: University of Nottingham 16 animal welfare (the 3Rs). Self-structuring bone in vitro Further information can be found Professor Liam Grover: University of Birmingham 18 at www.nc3rs.org.uk We collaborate with scientists Zebrafish and skin cancer research and organisations from across Dr David Hill: Newcastle University 20 the life sciences sector, nationally Pathways for cardiotoxicity and internationally, including Dr Luigi Margiotta-Casaluci: Brunel University London 22 universities, the pharmaceutical, Virtual heart for drug screening chemical and consumer products Professor Blanca Rodriguez: University of Oxford 24 industries, other research funders, MRI and pancreatic cancer and regulatory authorities. Dr Jane Sosabowski: Queen Mary University of London 26 TB challenge in vitro Dr Rachel Tanner: University of Oxford 28 Dancing parasites and mice Dr Joseph Turner: Liverpool School of Tropical Medicine 30 Flies and neuronal ageing research Dr Alessio Vagnoni: King’s College London 32

3 Annexes 34

References 36 2019NC3Rs researcher-led funding schemes 38 4 | 7 Research Review 2019

Funding research is a key part of the NC3Rs strategy to provide robust and reliable 3Rs models, technologies and tools that enhance scientific discovery.

We have demonstrated that research focused With strategic grants and highlight notices, on the 3Rs not only leads to reductions in we have continued to target funding in animal use and improvements in animal welfare, areas where there are specific concerns but also to wider impacts that benefit human about the numbers of animals used or their and animal health, protect the environment, welfare, or where there are opportunities and generate commercial opportunities. to build capacity or take advantage of new technologies with 3Rs potential. Over the last Since the NC3Rs was launched in 2004, we five years, we have made awards totalling have committed £62.3M in grants and early £25M, covering a range of strategically career awards to advance the 3Rs. Of this, important areas from replacing animal models £39.4M (63%) was for research focusing on of bovine TB research through to encouraging Introduction replacement, £10.1M (16%) for refinement, the use of human tissue. and £12.8M (21%) for reduction. This balance across the ‘Rs’ primarily reflects the focus of the applications that are submitted. Since the NC3Rs Applications are reviewed by expert independent panels using rigorous criteria to assess both the quality of the scientific was launched in proposal and potential 3Rs impacts. 2004, we have Based on our experience and feedback from the community, we have recently committed £62.3M evolved the researcher-led schemes that we 1 offer. We have introduced two new funding in grants and early schemes; training fellowships to promote the development of early career scientists career awards to with less than three years’ post-doctoral experience, and skills and knowledge transfer advance the 3Rs. awards to help bridge the ‘valley of death’ that exists between the development and application of established 3Rs tools into routine practice. 6 | 7 Research Review 2019

in partnership with F1000Research to provide Working in partnership with other research funders To date, we have a platform for our grant holders to report their and organisations has been critical to our success and 3Rs model development, detail its performance made 332 awards characteristics and how it was validated. The we have secured co-funding from other public sector gateway is open access with open peer review at 80 research allowing immediate access and transparency. and charitable organisations to support our remit. There are already eight papers on the gateway, institutions, funding which in total have been viewed 5,500 times, more than 560 with 840 downloads. This has increased the number of awards we leaders, nurturing early career researchers is In this review, we provide case studies from are able to make and extended our reach across particularly important. We invest considerable principal and across our portfolio showing the breadth of the a range of disciplines. Since 2014 we have resource in additional opportunities to engage in science we fund through our various schemes received £2.8M for co-funding opportunities, the 3Rs, for example through dedicated summer co-investigators and the 3Rs and scientific impacts we deliver. working in partnership with the BBSRC, EPSRC schools and training events, that also cater In some cases, the grant holders we showcase and the British Heart Foundation. This year, for the development of transferable skills that and supporting have had more than one NC3Rs award, and we we have new funding collaborations with underpin a successful career in science. describe the value this has had in widening the Cancer Research UK for grants to facilitate the development impact and inspiring other advances. the sharing of 3Rs approaches across the It is essential that the 3Rs impacts of the cancer research community, and with Unilever science we support are widely disseminated of 144 PhD students Finally, we would like to thank the MRC and for PhD studentships focusing on non-animal and implemented. We work closely with our BBSRC, our core funders, for their sustained approaches for safety testing. researchers to ensure the 3Rs advances they support which has allowed us to invest in deliver not only benefit their own laboratories and fellows. amazing science and talented individuals. but are shared across their institute and 63% of the awards field through publications, workshops and Dr Vicky Robinson CBE presentations. In some instances, maximising Discovery Catapult at Alderley Park, who will Chief Executive have been for these benefits requires further research, scale- match the funding we commit with in-kind Professor Stephen Holgate CBE up or commercialisation, and access to industrial contributions such as access to specialist Chairman replacement, 16% collaborators is key. We facilitate this through our equipment and facilities. The first awards will CRACK IT Solutions partnering hub and seed- be made in the Spring. for refinement and funding scheme to enable grant holders to work with potential end-users in the pharmaceutical, Many of the publications that describe research 21% for reduction. biotechnology and (agro)chemical industry funded by the NC3Rs report primary findings sectors nationally and internationally. only. While this is important in showing how We provide scientists across the life Recently, we have introduced the Technologies 3Rs models, tools and technologies can be sciences with an opportunity to engage in to Tools (T2T) scheme, with awards of up used, helping to build confidence in new 3Rs research regardless of their career stage. to £50k, to support the translation of in approaches, it can mean that the detailed To date, we have made 332 awards at 80 vitro models and non-animal technologies, methodology is not published. This limits the research institutions, funding more than 560 developed with NC3Rs grant funding, into ability of others to understand or use the 3Rs principal and co-investigators and supporting research tools that can be applied effectively model, tool or technology and can prevent the the development of 144 PhD students and in the pharmaceutical industry. The T2T work from being reproduced. To address this, fellows. As the next generation of research scheme is in partnership with the Medicines in May 2018 we launched the NC3Rs Gateway 8 | 7 Research Review 2019

Case 2 Studies 10 | 11 Research Review 2019

A typical experiment designed to test when Added value during the infection transmission occurs uses In 2017, Wendy presented the IVTT at the four donor ferrets and depending on the number Ferrets and flu ‘Transmission of Respiratory Viruses: From of time points up to 64 sentinel animals. The basic knowledge to evidence-based options studies do not completely recapitulate what for control’ conference in Hong Kong. happens in humans where contact is much Subsequently groups in the USA and Japan transmission studies shorter in duration and may be over a greater have adopted a similar device, further reducing distance. Better understanding of how much the use of ferrets. Based on the work supported infectious virus a ferret exhales and its stability by the NC3Rs funding, Wendy has secured a in airborne droplets could improve the relevance £1.2M Wellcome Collaborative Award to study of the studies to humans and help explain why the evolution of the influenza virus, which will Professor Wendy Barclay was awarded funding some viral strains are reported to be more exploit the use of the IVTT. There have been to reduce the number of ferrets used to study transmissible than others. three papers published from the NC3Rs-funded research. Wendy has collaborated with a small influenza transmission. 3Rs benefits (actual and potential) company to use the IVTT to test materials suitable for use in face masks that aim to protect Wendy and her team have designed, against respiratory virus transmission. She is Influenza viruses cause flu-like symptoms in a because they exhibit similar clinical signs and manufactured and tested a novel piece of currently working with a major pharmaceutical range of species, including birds, pigs, horses, receptor distribution in the airways as humans. equipment called the influenza virus transmission company using the IVTT to test the effects of dogs and humans. Each year, seasonal flu Studies in ferrets show good correlation with tunnel (IVTT) which avoids the need to use antiviral drugs on influenza transmission. affects 10% to 15% of the UK population and human transmission patterns. Animals are sentinel ferrets. The IVTT consists of a 100 cm is associated with 12,000 deaths. Children, lightly anaesthetised and infected intranasally long tube along which sentinel cell culture plates the elderly and those with pre-existing with the virus and daily viral titres are measured containing MDCK cells (an immortalised canine medical conditions are especially vulnerable. in nasal flushes or swabs. Post-infection, the cell line that is highly susceptible to influenza Influenza viruses evolve rapidly giving rise ferrets (referred to as donors) are housed next viruses) are positioned. Infected donor ferrets to novel antigenic variants that can evade to naïve sentinel animals for up to 14 days are placed for a maximum of ten minutes in a natural or vaccine-induced immune responses. and subsequent nasal flushes of sentinels chamber attached to the IVTT and viral plaque Occasionally, influenza strains, which would at multiple time points are used to establish counts on the plates are used to determine the ordinarily cause disease in one species, acquire whether transmission has occurred. Exposure titre of infectious virus in the animal’s exhaled the ability to transfer to a new host. This can can be by close contact where donor and breath. The IVTT can be attached to a nebuliser enable rapid viral spread in the absence of host sentinel animals are housed in the same allowing fully in vitro experiments. It is also immunity, leading to influenza pandemics. cage, akin to a household setting, or to model amenable to use in human infection studies. respiratory droplet and airborne routes, Ferrets are considered the ‘gold standard’ for animals are placed in adjacent cages Scientific and technological benefits modelling influenza airborne transmission separated by perforated sides. Using the IVTT, the Barclay laboratory have demonstrated that the amount of virus in the ferret’s nose is not a reliable indicator of the amount of viable virus in exhaled air. They showed that factors affecting virus survival in airborne droplets, such as stability of the virus particle, can be more readily studied in the IVTT than in the animal-to-animal transmission studies. Virus recovered from the MDCK cells infected by the airborne route can be easily interrogated using molecular techniques, for example, to PI: Organisation: Award type: Start date: Duration: Amount: define the genetic determinants that enhance Professor Imperial Project grant 2013 3 years £400k transmission – studies which are challenging Wendy College to do in the ferret as the site from which virus is Barclay London transmitted is unknown or inaccessible without culling the animals. 12 | 13 Research Review 2019

Prion infectivity is typically measured by than the mouse. This increased sensitivity has intracerebral or peripheral inoculation of a important practical applications. For example, Fly infectivity assay test material such as brain homogenate into Raymond has shown that PrP transgenic an experimental host. There is often a long Drosophila can detect prion-infected blood incubation period and clinical signs such as from asymptomatic scrapie-infected sheep, weight loss, ataxia and paralysis can take opening the possibility for a confirmatory for prion disease months or years to appear depending on route blood test for prion diseased individuals, and dose of inocula, or prion strain used. The including humans. Importantly, assessing prion studies require close monitoring of the animals infectivity in the fly takes about six weeks to and the judicious use of humane endpoints complete while testing the same inoculum in mice can take months or years. Dr Raymond Bujdoso was awarded funding given the potential for animal suffering. to test whether PrP transgenic Drosophila 3Rs benefits (actual and potential) Added value are a suitable invertebrate host to measure Raymond and colleagues generated Drosophila Raymond has published seven papers arising transgenic for ovine PrP to establish an from the project grant, including in Brain, the mammalian prion infectivity. invertebrate model of scrapie. Un-inoculated PrP Journal of Virology and the Biochemical Journal. transgenic flies show no adverse phenotypic A composite summary of the research was effects. However, by feeding PrP transgenic published on the NC3Rs gateway in 2018 – the Drosophila at the larvae stage with scrapie- paper has been downloaded 58 times to date. Prion diseases or transmissible spongiform Prion diseases can arise spontaneously or infected sheep brain homogenate, Raymond Based on the utility of the model, Raymond has encephalopathies (TSEs) are a family of fatal be transmitted between individuals of the demonstrated that the adult flies subsequently established new collaborations with research neurodegenerative disorders that affect both same and different species. This zoonotic had hallmark features of mammalian prion groups in Europe and North America and has humans and animals. They include Creutzfeldt- potential led to the BSE crisis in the UK in the disease, including the accumulation of infectious generated bovine and cervid PrP flies in order Jakob disease (CJD) in humans, bovine 1980s and the emergence of variant CJD in prions, a neurotoxic phenotype as assessed by to support these and replace the use of the spongiform encephalopathy (BSE) in cattle, humans. Much of the early research into prion locomotor assay, and enhanced mortality rate natural hosts of prion diseases. Raymond and scrapie in sheep. The infectious agents are infectivity, including disease transmissibility compared to controls. was awarded an NC3Rs skills and knowledge misfolded versions of the PrP protein, termed and the existence of different strains, has been transfer grant in 2017 for the validation of a fly prions. PrP is a cell surface glycoprotein, found dependent on the use of animals, primarily Raymond investigated other aspects of prion bioassay for classical and atypical BSE prion throughout the brain. It is highly conserved non-human primates, sheep and goats. transmission and infectivity in the fly model. infectivity. Working with collaborators, at the across vertebrate species and, although The demonstration that scrapie could be This included studies in which head homogenate UK’s Animal and Plant Health Agency, a major relatively little is known about its normal experimentally transmissible to rodents has, from scrapie-exposed PrP transgenic flies was reference centre for TSEs, Raymond has shown function, evidence suggests it is involved however, led to the widespread use of wild inoculated into ovine PrP transgenic mice. The that bovine PrP transgenic Drosophila provide in a diverse range of biological processes type and genetically modified mice as the mice subsequently developed prion disease a highly sensitive and rapid bioassay to assess from cellular differentiation through to the ‘gold standard’ model. indicating that the fly can effectively propagate BSE infectivity. Following further validation work, maintenance of myelin. mammalian prions, providing a new model for this new invertebrate prion model could replace studying prion biology. The Bujdoso laboratory the use of 750 mice a year at the agency. have replaced the use of the rodent bioassay with the PrP fly model, saving around 200 mice a year.

Scientific and technological benefits To test the sensitivity of the PrP transgenic fly model compared to the equivalent mouse model, animals were exposed to serial dilutions of scrapie-infected sheep brain homogenate. PI: Organisation: Award type: Start date: Duration: Amount: The flies showed a significant neurotoxic Dr Raymond University of Project grant 2013 2 years £262k response to dilutions from 10-2 to 10-10 of the Bujdoso Cambridge brain homogenate while the mouse bioassay detects a 10-6 dilution – in other words the fly bioassay is 10,000 times more sensitive 14 | 15 Research Review 2019

to severe weight loss and a high risk of death). Added value Importantly unlike Cryptosporidium parasites Jo and Anna organised a workshop, part funded generated through in vivo infection models Bioreactor for by the NC3Rs, at the 2018 British Society for which show batch-to-batch variation in their Parasitology Spring meeting, to promote the transcriptomic and proteomic profiles, in vitro use of the bioreactor for the production of production generates genetically identical parasites and other applications to advance Cryptosporidium oocysts parasites helping to ensure findings are Cryptosporidium research. This has led to five reproducible from one experiment to another. new collaborations, including with the NHS Cryptosporidium Reference Unit in Wales, 3Rs benefits (actual and potential) with the Cable laboratory acting as a training hub for researchers and organisations. Wide Following training in the Yarlett laboratory, Dr Anna Professor Joanne Cable was awarded funding to uptake of the bioreactor system could have Paziewska-Harris, the post-doctoral researcher on bring to Europe, via a transatlantic exchange, a high significant cost saving benefits. For example, the grant has established the bioreactor system at more than 20 water companies in the UK each throughput in vitro culture system for the enteric Cardiff University. spend around £60k per annum on purchasing Cryptosporidium parasites for positive controls protozoan parasite Cryptosporidium. Calf-derived oocysts were initially required for their daily drinking water screening which is for primary seeding of the bioreactor, but required by law – use of the bioreactor could cut subsequently oocysts produced in vitro have the cost to around £20k per annum. been used for experiments or for seeding of Cryptosporidium is a waterborne pathogen neonatal mouse infection models, because additional bioreactors. The bioreactor has allowed Data from studies using Cryptosporidium which poses a major threat to farm animals production of in vitro systems was difficult the Cable laboratory to replace the use of calves oocysts produced by the in vitro system and humans because there is no available with poor yields and low infectivity. for parasite production – saving a minimum of have been included in successful funding drug treatment and no immediate prospect of six animals per year. Further savings have been applications totalling £290k to date. This vaccine development. Together, two species In 2016, Professor Nigel Yarlett from Pace made for experimental work. Because parasite includes a Marie Curie COFUND Fellowship of the parasite, C. parvum and C. hominis, are University, New York, published methodology viability decreases when stored outside of the to Anna, and a Global Challenges Research responsible for more than 200,000 infant deaths for the continuous culture of C. parvum using a host, each experiment requires a fresh batch Fund project and a BBSRC-funded PhD attributable to diarrhoea in South Asia and sub- hollow fibre bioreactor seeded with human colon of oocysts – without the bioreactor, a recent studentship to Jo. Saharan Africa each year, but even in developed tumour-derived HCT-8 cells. The bioreactor series of experiments by the Cable laboratory on countries outbreaks can occur. provides an environment that mimics the gut environmental factors affecting sporozoite The project has been showcased at various by delivering nutrients and oxygen through the viability would have required four calves. public engagement events including the There is an urgent need to understand the lumen of the fibres to the basal layer of host Welsh Eisteddfod festival in August 2018, host immunological response and how the cells growing on the outside of the fibres, while with a ‘soapbox’ presentation on the 3Rs. parasite interacts with the host microbiome, in allowing separate redox and nutrient control Scientific and technological benefits Further-a-field, Jo has talked about the order to develop effective therapeutics. Until outside the fibres for parasite development. The The bioreactor produces 106 oocysts per bioreactor in Namibia during a launch event recently the generation of parasites (specifically system produces a high oocyst yield, replacing week for over six months, however, through for Phoenix Waters, a collaboration between transmissive stages, termed oocysts) for the use of animals and avoiding the suffering manipulation of nutrient content in both the Universities of Cardiff and Namibia to experimental purposes was dependent on the associated with infection (such as diarrhoea, compartments, the Cable laboratory has shown understand issues relating to water security use of neonatal calf and immunocompromised dehydration, inappetence and lethargy leading that it can be scaled-up to produce 108 oocysts and quality in sub-Saharan Africa. per ml/week. This is similar to the commercial production in calves where the typical quantity is 108 to 109 oocysts per batch. Anna has also optimised the viability assay for oocysts and the gliding motility assay for sporozoites providing an estimation of the parasites’ condition following the in vitro culture. Together PI: Organisation: Award type: Start date: Duration: Amount: with a newly developed 3D infectivity assay for Professor Cardiff Skills and 2017 2 years £75k sporozoites using HCT-8 cell spheroids, the Joanne University knowledge assays constitute a quality control system for Cable transfer assessing the phenotype of parasites produced grant by the in vitro platform. 16 | 17 Research Review 2019

appropriately. Many studies are under- suggests this reduces body weight loss when powered as a result – because of factors compared with animals where the external Blood flow in mouse such as cost, ethical considerations, and carotid artery is temporarily ligated. practical issues involved with the complex surgery and subsequent intensive care of Scientific and technological benefits the animals required. stroke models The improved surgical methods for the MCAO model were published in Disease Models 3Rs benefits (actual and potential) and Mechanisms in 2017, with the detailed During the MCAO procedure, it is typical to methodology reported in the Journal of permanently ligate the common carotid artery Visualized Experiments. Claire was awarded an Professor Claire Gibson was awarded funding to and reperfusion of the MCA occurs via vessels NC3Rs skills and knowledge transfer grant in that form the Circle of Willis. This structure 2017 to provide hands-on training to four major improve a mouse model of ischaemic stroke to reduce is anatomically highly variable, particularly in stroke research groups in Europe and North inter-animal variability. the commonly used C57Bl/6 mouse strain, America enabling them to adopt the model and which might account for the heterogeneity subsequently act as training hubs for others. seen in lesion volume. To address this, Claire The improved surgical approach may also have applicability in rodent models of embolic stroke. Approximately 85% of strokes in humans and associated with significant animal welfare investigated whether it was possible to enhance are ischaemic in nature. This is modelled concerns in the immediate days following reperfusion by repairing the common carotid experimentally, usually in rodents, by the induction of experimental stroke, including artery rather than relying on the Circle of Willis. Added value temporarily or permanently occluding the death, weight loss, sensorimotor deficits Claire used a small tissue pad coated in Claire was awarded NC3Rs funding for a PhD middle cerebral artery (MCA). Occlusion can and seizures. student in 2015. Michaela Bayliss, the PhD fibrinogen and thrombin to seal the incision be achieved by various methods including student, has investigated further refinements following removal of the filament, rather than electrocoagulation and pharmacological The primary outcome measure used in in the MCAO model in rats and mice, including permanently ligating the common carotid artery. intervention but mechanical obstruction by in vivo stroke research is the infarct or lesion whether improved environmental enrichment Using histological assessment and magnetic intraluminal insertion of a filament is often the volume. There is, however, considerable inter- benefits animal welfare following stroke resonance imaging techniques to monitor method of choice, being used by almost 80% animal, experiment and laboratory variation surgery and whether it has any impact on cerebral blood flow, Claire demonstrated of UK stroke researchers. in the size of the infarct in the MCA occlusion ischaemic damage. To date Michaela has one the repair of the vessel wall was stable and (MCAO) model even when following the same first author paper, published in PLOS ONE, reperfusion was enhanced compared with the The filament is inserted into the intracranial defined protocol. In some instances, despite which shows that in normotensive rats (unlike standard ligation model. Importantly, repairing internal carotid artery through an incision the animal undergoing surgery, there is no hypertensive rats) a pre-stroke craniotomy the common carotid artery reduces lesion usually made in the common carotid artery. lesion. Evidence suggests that this variability does not improve animal welfare in terms of volume variability between animals. Based on The filament prevents blood flow into the MCA can be caused by differences in the strain decreased body weight loss, improved survival this, Claire has shown that a study to determine which, after a period of ischemia typically up and age of the rodents used, cerebrovascular and fewer neurological deficits. to 90 minutes, is restored by removal of the anatomy, and the size and type of the filament. treatment effect would use 40% fewer mice (35 filament and ligation of the common carotid The variability reduces the statistical power of rather than 58 per group), assuming a power of artery to prevent blood loss through the the experiment necessitating the use of large 0.8, significance level of 0.05 and a predicted incision. The surgery is technically demanding numbers of animals to power the experiment 30% reduction in lesion volume between control and test animals.

To make surgical access easier, it is common practice in the MCAO model to temporarily ligate the external carotid artery prior to making an incision in the common carotid artery. However, reducing vascular supply to the external carotid artery, even temporarily, can affect the muscles PI: Organisation: Award type: Start date: Duration: Amount: involved in chewing and swallowing. Claire was Professor University of Pilot study 2014 1 year £75k also able to demonstrate with the pilot grant Claire Nottingham grant that it is possible to conduct the surgery without Gibson ligating the external carotid artery with no effect on lesion volume. Preliminary evidence 18 | 19 Research Review 2019

3Rs benefits (actual and potential) that block ossification. The model also has other benefits as it is possible to harvest Self-structuring Alexandra Iordachescu, the PhD student, has matrix vesicles from it. These exosomes are developed a self-structuring in vitro model for the thought to have a central role in controlling development of mature bone, which has replaced bone mineralisation but are poorly understood the use of animals for screening potential due to difficulties extracting them. Alexandra bone in vitro demineralisation agents and studies of early has developed a new protocol for harvesting bone formation in the Grover laboratory. the vesicles using immunoprecipitation, which should allow more detailed study of the process The model consists of a fibrin gel cast between and regulation of mineralisation. The protocol two ceramic anchors into which osteoblastic was published in RSC Advances in 2018. Professor Liam Grover was awarded funding to develop cells, from the rat femoral periosteum, are seeded. The culture is maintained with a an in vitro model for studying osteogenesis. continuous source of calcium phosphate, Added value supplemented with osteogenic factors. Using Alexandra has presented the in vitro model at various imaging modalities, Alexandra has shown international conferences including the World Osseous tissue forms in various physiological The animal models are often associated with that the periosteal cells deposit an ordered Biomaterials Congress (WBC) in Montreal, where circumstances ranging from normal bone severe suffering and while they have been useful matrix that closely resembles mature bone in she was the recipient of a WBC Trainee Award. In development and fracture repair, to pathological in understanding some aspects of ossification, terms of chemistry (e.g. the collagen/mineral 2019, Alexandra was awarded an NC3Rs training heterotopic bone formation in extraskeletal they are not always representative of pathological ratio) and cellular composition (e.g. osteoblasts fellowship to further develop the in vitro model tissues following muscle trauma, traumatic brain states and often are too complex to investigate and osteocytes are present). The model remains by including mechanical unloading. This will or spinal cord injury, or surgical procedures early phase bone formation. There are few viable in culture for over a year, recapitulating allow the model to be used to study the loss of of the hip and knee. Animals such as rodents, alternative models available due to difficulties the successive phases of ossification from bone in ageing and during osteoporosis, helping rabbits and sheep are used in research on normal differentiating and maintaining osteocytes initiation of bone formation through to the to replace procedures such as the hindlimb and heterotopic ossification to understand the in vitro and although immortalised cell lines exist, differentiation of osteocytes from osteoblasts. unloading model in which the rat is suspended dynamic role of osteoclasts, osteoblasts and these show reduced levels of key markers such Importantly, the model includes the presence of from its tail for two to three weeks so that only osteocytes. Many studies focus on manipulating as sclerostin, the secreted protein which inhibits canalicular networks, essential for intracellular the forelimbs touch the cage floor. the expression of bone morphogenetic proteins osteoblast differentiation. communication, which have not been previously in transgenic mice or involve subcutaneous or observed in in vitro culture. Details of the model Liam also secured further funding from intramuscular implantation of bone marrow- development and validation were published in the NC3Rs in 2018 for a PhD studentship derived mesenchymal stem cells. However, for Advanced Biosystems in 2017. in collaboration with Dr Amy Naylor at the trauma models it can be necessary to cause University of Birmingham. This also aims to physical damage by crushing a muscle or Alexandra and Liam are working with groups further advance the model developed by fracturing a bone. at the University of Oxford and Imperial Alexandra, by including human osteoblasts and College London to transfer the model into their osteoclasts (primary cells and mesenchymal laboratories, helping to further replace the use stem cells) so that the process of bone of animals. re-modelling can be studied.

Scientific and technological benefits Alexandra demonstrated in a pilot study that it is possible to test compounds that inhibit ossification in the in vitro model. The study was conducted with two compounds, one which is used to treat acquired and congenital heterotopic ossification and the other which has been shown to reduce heterotopic ossification PI: Organisation: Award type: Start date: Duration: Amount: in transgenic murine models of Fibrodysplasia Professor University of PhD 2014 3 years £90k ossificans progressiva. Both compounds led Liam Grover Birmingham studentship to a decrease in matrix and mineral formation. Since then, the model has been used by the Grover laboratory to identify new compounds 20 | 21 Research Review 2019

3Rs benefits (actual and potential) re-growth of the tumour. Using the zebrafish embryo model, David and his colleagues have During his Fellowship, David focused on two demonstrated that combined treatment with Zebrafish and skin approaches that have replaced the use of drugs that specifically block both MEK and mice in his melanoma research, saving around autophagy reduced the invasion of trametinib- 200 animals a year. David has optimised resistant melanoma cells – this is the first time and validated a 3D in vitro model of human cancer research that inhibition of autophagy has been shown to skin that recapitulates the early melanoma overcome resistance to targeted MEK inhibitors. microenvironment. The model contains both The work was published in the British Journal a dermal and epidermal equivalent into which of Dermatology in 2019 and could have clinical cancer cells can be introduced. These form relevance. David is now using the in vitro human groups or nests at the dermal/epidermal interface Dr David Hill was awarded funding to develop a 3D skin model to determine the effect of autophagy before breaching the basement membrane and modulation on the invasion of melanoma cells as in vitro human melanoma skin equivalent and a invading the dermis. This mirrors early tumour a potential preventative therapy for metastasis invasion with, for example, the characteristic in high risk patients. zebrafish xenograft model to investigate melanoma breakdown of type IV and VII collagens as is observed in human cutaneous tumours in vivo. invasion, migration and metastasis. Details of the model were published in Molecular Added value Cancer Therapeutics in 2015. The 3D skin model is now routinely used by Alcyomics, a Newcastle-based company which David has also established a xenograft model conducts preclinical skin tests on chemicals and Malignant melanoma is the most lethal form of immunodeficient mice. In some cases, transgenic for investigating cancer cell invasion and pharmaceutical compounds to assess immune skin cancer and the fifth most common cancer in mice that develop spontaneous tumours are used. metastasis in zebrafish embryos which can be hypersensitive reactions. This has replaced the the UK. It is a growing world health concern, with completed prior to five days post-fertilisation use of up to 200 rodents a year in skin contact an incidence that has risen more than any other For the study of metastatic disease, melanoma (the point when they become regulated under hypersensitivity experiments. malignancy in the last 40 years. Although curable cells are implanted subcutaneously and allowed to the Animals (Scientific Procedures) Act 1986). by surgical resection at early disease stages, late spread to distant and multiple sites. The primary Fluorescently-labelled human melanoma cells David has received funding for two PhD stage metastatic melanoma is highly invasive and engrafted tumour may be surgically removed injected into the yolk sac can be tracked in real studentships from the BBSRC and EPSRC to currently incurable. to prevent humane endpoints based on tumour time by time-lapse imaging. Using transgenic fish, further develop the in vitro skin model. The first, size being breached (and thus requiring animals for example with GFP-labelled endothelial blood in collaboration with Professor Stefan Przyborski Melanoma research, like that for most other to be killed) while the study of the metastasis is vessels, David has been able to track local non- at Durham University, is to apply the model to cancers, has a heavy reliance on mouse ongoing. Alternatively, melanoma cells may be vascular spread, intravasation of metastatic cells study skin ageing to replace the use of mice, xenograft models for investigating tumour injected directly into the circulation via the tail vein and subsequent haematological and lymphatic and the second in collaboration with Alcyomics development and progression, and responses or the metastatic site of interest (e.g. intracranially). metastatic dissemination. The latter requires focuses on bioprinting to increase the throughput to therapeutics. Studies of melanoma initiation Depending on the cell line or patient source, interaction with the endothelium during entry and of the assay. In 2015, David was awarded the and invasion into the skin typically involve the metastasis to the lungs, liver, kidney or brain are exit, and the metastatic melanoma cells show British Society of Investigative Dermatology engraftment of human skin, or skin reconstructs common and careful monitoring of the mice is characteristic sticking and rolling behaviours on Young Investigator of the Year award. containing human melanoma cell lines or patient- essential as the potential for pain and suffering the surface of vascular endothelium indicative derived melanoma cells, onto the backs of is high. of specific interactions between human melanoma and zebrafish endothelial cells. Details of the zebrafish model were published on the NC3Rs gateway in 2018 – the paper has been downloaded 100 times to date.

Scientific and technological benefits In patients with malignant melanoma, a sub- PI: Organisation: Award type: Start date: Duration: Amount: set of tumour cells often become resistant Dr David Hill Newcastle David 2014 3 years £195k to mitogen-activated protein kinase kinase University Sainsbury enzymes (MEK) inhibiting drugs, such as fellowship trametinib. This drug resistance correlates with an increase in autophagy and leads to 22 | 23 Research Review 2019

3Rs benefits (actual and potential) Added value During this nine-month project, Luigi developed Luigi has presented the AOPs at five Pathways for the first AOPs for cardiotoxicity. These form conferences and workshops, including a mini-network of AOPs that describe three the Safety Pharmacology Society 2018 different mechanisms by which the blockade Annual Meeting where he was awarded a cardiotoxicity of L-type calcium channels can lead to adverse Junior Investigator Award. As a result of the effects, including heart failure. The AOPs were expertise developed with the NC3Rs award, developed by identifying, critiquing and collating Luigi is a member of a €6.7M Horizon 2020 information from over 150 primary publications GOLIATH consortium where he will guide the on the effects of calcium channel blockers on development of an AOP network for chemical- different parts of the cardiovascular system, and induced metabolic disruption. Dr Luigi Margiotta-Casaluci was awarded funding to publications examining the effects of genetic curate published information on cardiotoxicity, caused manipulations on different components of the L-type calcium channel. by blocking L-type calcium channels, to underpin the The AOPs have been published on the AOPWiki development and use of non-animal approaches. to allow peer review as part of the process towards OECD endorsement.

Scientific and technological benefits Over the last decade there has been considerable In the short-term AOPs can be used to help design interest from industry and regulatory bodies in the bespoke mechanistic in vitro and in silico models Luigi curated over 1,100 in vitro, ex vivo and wider use of non-animal approaches to investigate for specific toxicological endpoints and to identify in vivo data points relevant to blockade of how chemicals, including pharmaceuticals, knowledge gaps which need to be addressed L-type calcium channels into a database. interact with biological processes and biochemical – together supporting the long-term goal of The database includes quantitative pathways to help predict whether they will be replacing animal use in safety testing. Because of pharmacokinetic and pharmacodynamic harmful to humans and/or the environment. their potential benefits, the OECD has provided data that can be used to inform and interpret The concept of the adverse outcome pathway guidance and infrastructure to encourage the in silico and in vitro models, for example, (AOP) has been developed to support this by development and use of AOPs – standardising dose response data for each drug/endpoint, providing a framework to organise information into the language used and outlining a process for exposure duration and quantification method. a description of the critical steps that occur from review and endorsement. To date, nine AOPs have the initial chemical interaction (referred to as the been published by the OECD, including AOPs for An innovative approach to graphically molecular initiating event) through to molecular, neurological and reproductive adverse events. represent the strength of evidence supporting cellular and tissue events that ultimately lead to each key event relationship in the AOP was also adverse effects at the organ and organism-level. developed by Luigi. This could be applied to other AOPs to help highlight knowledge gaps and assess the completeness of the AOP as part of the endorsement process.

PI: Organisation: Award type: Start date: Duration: Amount: Dr Luigi Brunel Strategic 2017 9 months £30k Margiotta- University grant Casaluci London 24 | 25 Research Review 2019

3Rs benefits (actual and potential) simulated human cardiac cells with unique profiles, allowing better modelling of disease The Computational Cardiovascular team at state, drug responses and genetic variation. Virtual heart for Oxford initially generated the Virtual Assay The population featured in the Frontiers in software, with EPSRC funding, to simulate the Physiology publication consisted of 1,213 effect of drugs on the electrophysiology, and unique cardiac cellular simulations, which were calcium dynamics linked with contractility, of drug screening then used to run an in silico drug trial with populations of human cardiomyocytes. reference compounds. The software has been enhanced by the availability of a comprehensive database of To improve Virtual Assay’s physiological human in silico models, funded by the NC3Rs, relevance the team are also adapting it to for specific disease conditions such as heart Professor Blanca Rodriguez was awarded funding account for other cardiac comorbidities. failure, myocardial ischaemia, genetic For example, cardiotoxic adverse events to accelerate the uptake of human-based in silico disorders and cardiomyopathies. are more likely to occur in patients where electromechanical function of the heart is methodologies for the evaluation of cardiac drug With NC3Rs funding, the team have gone on impaired. Such comorbidities are, however, safety and efficacy in industry, regulatory and to compare predictions using simulations from rarely reproduced in in vivo studies which use Virtual Assay with data from clinical trials, animal healthy animals. A computer simulation for in clinical settings. models, and in vitro methods, helping to build silico drug trials in single cell electromechanical confidence in in silico approaches. A publication models is currently being validated, with plans to in Frontiers in Physiology in 2017 describes how extend the model to simulate multiple cells and Drug-induced cardiotoxicity is one of and heart rate) are assessed as part of a Virtual Assay simulations predict clinical risk of eventually the whole heart. the leading causes for attrition during core battery of regulatory pharmacology tests drug-induced Torsade de Pointes with higher pharmaceutical development and can usually performed in dogs or non-human accuracy than animal experiments for more also result in drugs being withdrawn after primates. The number of animals used per than 60 reference compounds. The simulated Added value market approval. International guidelines drug at this stage is four or eight animals repolarisation abnormalities were shown to be a In 2017, Dr Elisa Passini, a senior post- (e.g. ICH S7B) require compounds to be depending on whether the study is conducted more accurate, specific and sensitive biomarker doctoral scientist in the team, was awarded evaluated for their effects on ventricular in Europe or the USA respectively. In some for arrhythmia risk than action potential duration the inaugural Safety Pharmacology Society repolarisation and proarrhythmic risk using cases, it may be necessary to conduct further measured in animals – with the Virtual Assay Technological Innovation Award as well as the in vitro and in vivo tests. Assessment is animal studies to understand potency and simulations having an 89% accuracy compared NC3Rs International Prize which is sponsored carried out during the early stages of drug mechanisms of action. to 75% in the rabbit isolated heart model. The by GSK. Blanca was awarded the MPLS Impact development in in vitro assays using heart paper has been viewed more than 10,000 times. Award for Commercial Impact in 2018 by the tissue and cells, typically from guinea pigs, It is estimated that 20% to 50% of University of Oxford. rabbits, dogs or non-human primates. drug candidates are abandoned due to Virtual Assay is being evaluated by four major The primary function of these assays is to cardiotoxicity despite in vitro and in vivo pharmaceutical companies for use in early drug Blanca and the team have given presentations identify changes to electrophysiological testing. Worldwide there are a number of development to assess arrhythmic risk, with on Virtual Assay at a range of national and properties (e.g. action potential duration efforts to provide more predictive tools initial estimates of a 30% to 33% reduction international scientific meetings. In 2018, and/or cardiac ionic currents). Later in that exploit the potential of human-induced in animal use per year across the sector. with funding from the NC3Rs, they hosted a development, cardiovascular safety including pluripotent stem cell-derived cardiomyocytes The software is also freely available with symposium titled ‘In Silico Drug Safety and electrophysiology and mechanical read-outs and in silico models in order to avoid the an academic licence and is currently being Efficacy’ in Oxford with 100 delegates from of cardiac function (e.g. ECG, blood pressure current reliance on animal models. used by six research groups. academia, industry and regulatory authorities. There have also been extensive outreach Scientific and technological benefits activities which include Elisa presenting at the Hay Festival and organising an event through Typically, computer models of heart function The Royal Institution, where school pupils are based on one generic model representative used the Virtual Assay in a masterclass in of a generic cell and therefore may not be computational biology. An article published in PI: Organisation: Award type: Start date: Duration: Amount: representative of the wider population where The Conversation ‘Why computer simulations Professor University Infrastructure 2016 5 years £512k cardiac response to drugs may differ. An should replace animal testing for heart drugs’ Blanca of Oxford for Impact advantage of the Virtual Assay is that rather has been viewed more than 20,000 times. Rodriguez grant than this ‘one model fits all’ approach, the software generates a large population of 26 | 27 Research Review 2019

Magnetic resonance imaging (MRI) is already ensure the advantages of MRI imaging, such used in mouse studies on pancreatic cancer. as the ability to do longitudinal imaging on the MRI and It is readily applicable, even for inexperienced same animals, can be applied more readily to operators, with the fast-growing localised genetically altered models of pancreatic cancer. tumours that occur with orthotopic transplants. However, MRI image analysis is challenging Scientific and technological benefits pancreatic cancer with genetically altered models, such as the KPC mouse, where tumour growth is typically The low field MRI instrument used by Joseph slower (three to six months) and where there was funded through an NC3Rs Infrastructure for is a gradual transformation, sometimes across Impact grant in 2013 to Professor John Marshall the whole pancreas. Although there are at QMUL, with Jane as a co-investigator. The Dr Jane Sosabowski was awarded funding to commercially available tools to aid with image instrument has been applied to a wide range of interpretation, these largely only work for well- animal studies at QMUL including cancer (e.g. improve non-invasive imaging of mouse models defined organs such as the liver and stomach pancreatic, brain and lung) and trauma injury and not the pancreas. models, demonstrating the utility of low field of pancreatic cancer. MRI. Impacts include developing new methods to assess lung tumours in mice to improve the 3Rs benefits (actual and potential) humane endpoints used, and demonstrating that There are almost 10,000 new cases of pancreatic development in the appropriate pancreatic MRI can be applied to monitor tumours of the cancer in the UK each year. While survival rates microenvironment. Joseph Brook, the PhD student, has used a omentum (a fold of the peritoneum connecting for most cancers have increased over the last 40 low field, small animal MRI instrument to build the stomach and the abdominal viscera), which is years, the prognosis for patients with pancreatic The mouse pancreas lies in the upper abdomen a library of images of the healthy pancreas adjacent to the pancreas. cancer is poor, with few surviving more than a year behind the stomach and is soft and diffuse in and pancreatic tumours in the KPC mouse. after diagnosis. Research on pancreatic cancer comparison with the human pancreas which By calculating various features for each 3D John and Jane have tracked the reduction in and the development of treatments predominantly makes it difficult to define and distinguish from pixel in the MRI images (e.g. intensity, gradient, animal use from the instrument with their ‘Mouse uses mice. This includes genetically altered the surrounding tissues. These factors mean that spatial and probability features), Joseph Lives Saved’ Counter, with the number currently animals and xenograft models either with cell lines sizing orthotopic tumours and monitoring their has used machine learning to develop an standing at 2,600 mice. or patient-derived tissue. growth over time, for example to assess treatment autosegmentation tool that can automatically efficacy or for the purposes of humane endpoints, identify a tumour-positive pancreas with 95% The KPC mouse model is commonly used. This is challenging. Palpation is difficult and does accuracy compared to veterinary radiologists Added value has a conditional point mutation in both the p53 not provide accurate quantitative information. and image analysis experts. Where the model Joseph has presented his machine learning and KRAS genes. It develops pancreatic ductal Ultrasound can be used but animals have to be and the image analysis experts disagreed, in the tool at a number of international conferences, adenocarcinoma with similarities to human shaved, image acquisition is highly operator vast majority of the cases, this was because the including the World Molecular Imaging Congress tumours as well as associated comorbidities dependent, and analysis of volumetric images is model had correctly identified a tumour earlier in Seattle in 2018 where he won a poster prize. He of cachexia, jaundice and ascites. For xenograft time consuming. Longitudinal studies typically than the experts. This early identification can has participated in more than 50 hours of public models, cells or patient samples are either require mice to be culled and the pancreas inform the use of humane endpoints and the engagement activities, including as a STEM transplanted heterotopically or directly into the removed. Consequently, studies require large monitoring of mice. Ambassador, to champion the 3Rs. In 2017, Jane pancreas. Orthotopic tumours have the advantage numbers of mice for each time point and gave a presentation on the 3Rs and imaging at of providing the opportunity to study tumour individual animals cannot be tracked. Joseph and Jane are working with the an NC3Rs-hosted event in London as part of the Boston-based company Invicro to incorporate ‘Pint of Science’ festival. Working with veterinary the pancreatic segmentation tool into their colleagues at QMUL, Jane has published papers existing 3D mouse atlas software. It is also on anaesthesia and monitoring animals during being applied to other projects at the Barts MRI and other welfare considerations. Cancer Institute, Queen Mary University of London (QMUL), where there are more than 90 researchers working on pancreatic cancer, and PI: Organisation: Award type: Start date: Duration: Amount: through new collaborations with researchers in Dr Jane Queen Mary PhD 2015 3 years £90k Glasgow, Cambridge and London. Research at Sosabowski University studentship the Institute has already shown that the use of of London MRI for orthotopic tumours reduces the number of mice used per study from 12 to eight animals. Wider uptake of the autosegmentation tool will 28 | 29 Research Review 2019

Rachel had previously undertaken optimisation easy to manipulate individual components of the of the MGIA in a number of species, including the assay, it can be used to determine the immune TB challenge in vitro NHP. This involved demonstrating a correlation mechanisms controlling mycobacterial growth between the in vitro response using PBMCs and identify correlates of protection – studies taken from macaques and in vivo protection which are difficult and costly to do in vivo. in BCG and Mtb challenge studies in the same animals, as well as clinical concordance based on Added value comparison with epidemiological data. Despite this, the MGIA has been under-utilised by the TB Rachel has presented the MGIA data at various research community because of concerns about international vaccine and TB conferences. the reproducibility and reliability of mycobacteria- This includes organising a workshop, with 25 Dr Rachel Tanner was awarded funding to transfer based functional assays. participants from 14 different institutions, alongside the TBVAC2020 meeting in the mycobacterial growth inhibition assay (MGIA) With NC3Rs funding, Rachel is working with Switzerland. She was awarded the TB Vaccine to two end-user laboratories to refine the use of scientists at Public Health England (PHE) and Initiative Young Scientist Award in 2017 which the Biomedical Primate Research Centre (BPRC) allowed Rachel to present work arising from monkeys in tuberculosis (TB) vaccine studies by in the Netherlands to build confidence and the NC3Rs grant at the Global Forum for TB facilitate the adoption of the NHP MGIA in their Vaccines in Delhi; and the Collaboration for TB avoiding challenge infection studies. TB vaccine programmes. Vaccine Discovery Junior Investigator Award in 2018 which allowed her to present at its annual meeting in Seattle. There are ten million new cases of TB and As in humans, animals that become infected By using PBMCs from previous in vivo challenge 1.6 million deaths worldwide each year. The experience clinical signs that may include raised studies in the NHP MGIA, Rachel and her Rachel, who was only awarded her PhD in 2015, disease can be cured by targeting the causative temperature, respiratory distress and weight collaborators Dr Sally Sharpe at PHE and Dr has recently secured a two-year Fellowship agent Mycobacterium tuberculosis (Mtb) with loss, and humane endpoints are used to avoid Frank Verreck at BPRC have shown that the assay from VALIDATE (an MRC/BBSRC funded appropriate antibiotics. However, limited access unnecessary suffering. is highly reproducible between tests, operators, network) to continue her research on TB vaccine to drugs in developing countries and the recent labs and institutions. The MGIA is currently being development and antigen discovery. Working emergence of multi-drug resistant Mtb strains used alongside the in vivo challenge study to 3Rs benefits (actual and potential) with Professor Helen McShane at the University makes TB an increasing global health threat. The provide additional validation data. As well as the of Oxford and collaborators, Rachel has only established vaccine, the BCG vaccine, is not The MGIA is an in vitro functional assay that animal welfare benefits of avoiding challenge contributed to a publication describing a refined always protective, particularly against pulmonary can be used to refine the use of animals in studies, in the future the MGIA could lead to non-virulent NHP model of mycobacterial disease and in areas where TB is prevalent. Mtb vaccine research by avoiding the need a combined 30% (= 45 animals) reduction in infection and the development of a MGIA for Consequently, there is considerable international to undertake infection challenge studies. macaque use at the two institutions annually. studying TB infection in cattle, as well as three effort from governmental and philanthropic In the MGIA, peripheral blood mononuclear This is because the PBMCs can be used to publications on the human MGIA. She has also organisations to develop new vaccines to halt cells (PBMCs) from naïve and vaccinated screen multiple clinical isolates and vaccine published a paper in Frontiers for Young Minds, the TB epidemic. Vaccine candidates are typically animals are cultured over four days with Mtb candidates. More broadly the work has a science journal for children and teenagers. evaluated using mice, guinea pigs and non- (or other mycobacteria) and the inhibition of underpinned a general shift in the acceptance The paper titled ‘The 3Rs: What are medical human primates (NHPs), which are immunised bacterial growth is used as an estimate of of the MGIA by the TB research community and scientists doing about animal testing?’ with a test vaccine prior to challenge with Mtb. protective immunity. through the NC3Rs grant Rachel has helped has been viewed more than 3,500 times. to set up the assays for various species at 13 institutions in Europe, North America and Asia, providing protocols and technical support.

Scientific and technological benefits The MGIA may be used for early vaccine evaluation as part of the ‘gating’ strategy PI: Organisation: Award type: Start date: Duration: Amount: that has been established by the global TB Dr Rachel University of Skills and 2017 1 year £75k community to accelerate the development Tanner Oxford knowledge of the most promising vaccine candidates. transfer Since the MGIA measures most aspects of the grant complex host immune response to Mtb and it is 30 | 31 Research Review 2019

The lack of biomarkers also means animals can with AbbVie Inc. and the Drugs for Neglected develop welfare issues either due to chronic Diseases initiative. The work has been accepted Dancing parasites parasitism (e.g. peritoneal adhesions, ascites, for publication in Science Translational Medicine adverse behavioural changes) or ageing. and the drug candidate has now entered phase II clinical development. 3Rs benefits (actual and potential) and mice Amy has also optimised a co-culture system Amy Marriott, the PhD student, has validated of dual human lymphatic endothelial cell the use of immunodeficient inbred mice monolayers where adult female worms can instead of gerbils to provide a more consistent be maintained with full viability for up to three and reproducible filarial parasite burden for weeks – three times the duration of commonly Dr Joseph Turner was awarded funding to reduce the Brugia malayi. This has allowed a reduction in used in vitro systems. The longevity of the animal numbers from 14 gerbils to eight mice system allows for long-term and robust in reliance on animals in filariasis research, particularly per compound tested (including donor animals vitro assessment of preclinical therapeutic and those excluded due to low infection rate). candidates prior to a decision whether in vivo for preclinical testing of therapeutics. studies are justified. It has been adopted by In the clinic, ultrasonography is used for Professor Mark Taylor at the Liverpool School of diagnostic purposes by detecting the Tropical Medicine in collaboration with Professor Filarial diseases are caused by a group of parasitic significant investment from governmental and random thrashing movement of parasites David Sattelle at University College London to worms, known as filariae. Infection occurs when private philanthropic stakeholders to develop in the lymphatic system, referred to as the screen the potency of a new anthelmintic class the parasites are transmitted through the bites of curative drugs (macrofilaricides) that eliminate filarial dance sign (FDS). Amy has shown that of molecule against adult stage filariae. Beyond flying insects, such as mosquitoes. Conditions are filariasis as a public health problem. the FDS can be used in the laboratory to drug screening, the co-culture system can be life-long and a major cause of disability, affecting accurately assess the presence or absence used to study host/parasite interactions. approximately 150 million people worldwide, with a Animals are used for filarial research purposes of Brugia malayi infection in mice and gerbils further 1.3 billion at risk of infection. The most well- both to grow the parasites to the life cycle stage (with 100% sensitivity and specificity when Added value known filarial diseases are elephantiasis (lymphatic of interest (typically the adult stage) and for the more than five worms are present) and also filariasis) and river blindness (onchocerciasis) testing of potential drugs. The gerbil is the most The research has been presented at 12 to predict drug effectiveness in a proof-of- which are caused by Brugia malayi and common model. A major challenge is achieving conferences, including at the British Society for principle study using known macrofilaricidal Onchocerca volvulus respectively. Filarial diseases reproducible levels of adult parasite burden Parasitology where Amy was awarded a best and non-macrofilaricidal drugs. Tracking FDS are also of veterinary importance, affecting both in gerbils because of the high incidence of poster presentation prize. Joseph is funded allows longitudinal studies in the same animal cats and dogs. infection failures and high intra-group variation. as part of the Bill & Melinda Gates Foundation avoiding the need for separate groups for For evaluation of the efficacy of an experimental Macrofilaricide Drug Accelerator Consortium experiments with multiple time points. Mass drug administration community control drug, parasites are harvested from donor and has been able to share the 3Rs advances programmes currently use chemotherapeutic gerbils, counted and surgically implanted into with academic and industrial partners from The use of ultrasonography has led to a further agents, such as ivermectin, to target the the peritoneum of recipient animals. There are Europe and North America. In 2018, Joseph reduction from eight to five mice for drug transmission stage of the parasitic cycle. However, no biomarkers to assess adult parasite infection was awarded an NC3Rs project grant of £288k studies in the Turner laboratory. In addition, these are not effective at directly killing the adult status or infectious load and consequently to validate alternative models to cats and dogs implantation surgery can now be avoided in parasites and treatment has to be continued efficacy studies typically involve culling animals at for heartworm drug testing. He has established both mice and gerbils as it is possible to inject annually for five to ten years (depending on the various time points, often up to eight months post- a new collaboration with Bayer Animal Health reproductive cycle of the parasite). There is treatment, to quantify the number of parasites. microscopic larvae intraperitoneally into the which will provide veterinary compounds for experimental animals and track infection by testing in heartworm drug screening models ultrasonography rather than having to take as part of the project. adult parasites from donors. Amy has a first author paper describing the work published in Scientific Reports in 2018.

PI: Organisation: Award type: Start date: Duration: Amount: Scientific and technological benefits Dr Joseph Liverpool PhD 2015 3 years £90k The ultrasonography technique developed Turner School of studentship by Amy has been adopted in the preclinical Tropical validation testing of the candidate Medicine macrofilaricide ABBV-4083, in partnership 32 | 33 Research Review 2019

3Rs benefits (actual and potential) Evidence suggests that axonal transport of Amyloid Precursor Protein (APP), which plays During his Fellowship, Alessio expanded the a major role in the development of Alzheimer’s Flies and neuronal utility of the fly wing model by developing new disease, requires kinesin-1. Based on the strength assays to assess interactions between the of the fly wing model, Alessio has established a mitochondria and endoplasmic reticulum (which collaboration with Professor Chris Miller at King’s ageing research are often perturbed in neurodegenerative College London generating mutant Drosophila diseases) and to measure intracellular calcium to measure in vivo axonal transport of vesicles levels following neuronal stimulation. To validate containing APP in ageing animals. This has the model as a potential replacement for avoided the use of approximately 160 mice. rodents, comparative mitochondrial trafficking Alessio also has active collaborations to replace Dr Alessio Vagnoni was awarded funding to studies were conducted in the exposed sciatic the use of rodents and zebrafish with flies in nerve of ageing MitoMice – transgenic mice laboratories in the UK and Italy. build confidence in the use of Drosophila to in which mitochondrially-targeted fluorescent proteins are selectively expressed in neurons. study mitochondrial transport in neuronal ageing The findings are being prepared for publication. Added value and neurodegeneration to replace some studies Alessio has published two first author papers As an additional 3Rs advance, working with in Nature Protocols and Current Biology, involving rodents. Professor Giampietro Schiavo at the Institute of highlighting key findings from the project, as Neurology, Alessio also maximised the use of well as being a co-author on a paper reviewing the MitoMice. After the imaging studies, neurons methodological advances in intravital imaging. Deficiencies in the active intracellular transport generation of specific transgenic lines expressing from the dorsal root ganglion of young and He has given two public presentations on of organelles, such as mitochondria, along axons fluorescent proteins to track the movement of old mice were rapidly extracted and cultured, the importance of the 3Rs in research on are associated with neuronal ageing and are a ‘cargo’ along the axon. They are also limited by providing an opportunity to compare the neurodegeneration at ‘Pint of Science’ festivals hallmark of many neurodegenerative diseases. tissue transparency and imaging depth, and the in vivo and in vitro properties of two subsets of held in Cambridge and London in 2017. Understanding the cell biology of neuronal ageing relatively long lifespan of mice makes studying the sensory neurons derived from the same animal. could lead to the identification of new targets for cell biology of neuronal ageing difficult. This correlation between the animal and in vitro Alessio is now building an independent research therapeutic intervention. To date axonal transport data could help to reduce the numbers of mice career in neuronal ageing at King’s College has been extensively studied in cultured primary Working with Dr Simon Bullock at the MRC London, securing £500k to start his own neurons and tissue explants, however, these Laboratory of Molecular Biology, Alessio had used as well as facilitating the use of functional laboratory. The funding includes a van Geest models do not always consistently reflect what previously developed non-invasive procedures assays that cannot be performed in vivo. Fellowship and a BBSRC Collaborative Training happens in vivo because of the lack of other for imaging axonal mitochondrial transport in Partnership PhD award with Ely Lilly to study cell types (e.g. muscle cells), myelination and the sensory neurons of the marginal nerve in the Scientific and technological benefits the neuronal cell biology of human ageing in excitatory/inhibitory input into cell bodies. Drosophila wing. Since the wing is translucent and vitro. Alessio has also received a UK-Israel the fly has a short lifespan, axonal transport can be Previous work by Alessio and colleagues had Science Lectureship grant from the British Recent advances in intravital techniques have studied in the intact nervous system throughout shown that the number of actively transported Council and the UK Science & Innovation allowed the process to be monitored in real time ageing. This combined with the genetic tools mitochondria declines significantly in the wing Network. The grant will support a series of in vivo in a range of species. Studies in mice available to manipulate the Drosophila genome sensory neurons during ageing. In his Fellowship, lectures at Israeli universities and strengthen have focused on axonal transport in the sciatic (including the generation of neurological disease- Alessio demonstrated that the cAMP/protein collaborations between laboratories, allowing nerve and axons of the spinal cord and dorsal related models ) provides a powerful system for kinase A (PKA) pathway promotes mitochondrial Alessio to further disseminate his fly model. roots. The procedures are invasive (involving understanding neuronal ageing, with advantages transport in adult Drosophila wing neurons and surgery), technically demanding and require the over other in vivo approaches. that feeding aged flies with a small molecule agonist of PKA is sufficient to suppress the decline in mitochondrial transport, with upregulation of the motor protein, kinesin-1, an important output for PKA activation.

PI: Organisation: Award type: Start date: Duration: Amount: Dr Alessio King’s College David 2016 3 years £195k Vagnoni London Sainsbury fellowship 34 | 7 Research Review 2019

Annexes 3 36 | 37 Research Review 2019

References

Barclay Grover Rodriguez

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NC3Rs researcher-led funding schemes

Total Research grants awarded to date (£)

Project For research projects, of up to 36 months in duration, which support the development £36M grants of new 3Rs approaches and technologies. Average award value £300k.

Pilot study For proof-of-principle work with awards up to £75k for 12 months. £1.7M grants

Strategic For research projects in areas identified as strategically important by the NC3Rs £6.4M* grants because of 3Rs potential or concerns about animal numbers/welfare.

Non-research grants

Infrastructure For projects of up to 60 months duration which focus on infrastructure, networks and for Impact £2.3M other resources to underpin 3Rs research. grants

Skills and Launched in 2016, to promote the wider adoption and use of models, tools knowledge and technologies with proven 3Rs impacts through the transfer of knowledge, skills £0.5M transfer and expertise. Awards are a maximum of £75k for up to 24 months. Joint awards with grants Cancer Research UK from 2019.

Early career awards

PhD For the training of graduate scientists. Awards are £90k for 36 months. Joint awards £11.1M** studentships with the British Heart Foundation and from 2019, Unilever.

Training Introduced in 2016, to support the development of promising early career fellowships researchers with less than three years’ post-doctoral experience, focusing on developing new skills and gaining a breadth of research relevant to the 3Rs. £1.2M Awards cover salary and up to £15k per annum for other directly incurred research costs and are 24 months in duration.

David For talented, 3Rs-minded scientists with two to six years’ post-doctoral experience to Sainsbury support the transition to an independent career. Awards cover salary and up to £30k £3.1M fellowships per annum for other directly incurred research costs and are 36 months in duration.

Available to NC3Rs grant holders only

Technologies For the translation of non-animal technologies, developed with NC3Rs grant funding, First awards to to Tools into research tools that can be used in the pharmaceutical industry to support be made in improved drug discovery. Applicants can apply for up to £50k for projects up to 24 Spring 2019. months in duration. This is a partnership with the Medicines Discovery Catapult who Call budget will provide in-kind contributions equivalent to the funding provided by the NC3Rs. is £0.2M

Public For outreach activities aimed at encouraging awareness of the 3Rs in the general engagement £12K public. Awards of up to £1,500 are available. grants

*includes £1.3M and £0.5M co-funding from BBSRC and EPSRC respectively **includes £0.9M co-funding from the British Heart Foundation NC3Rs Gibbs Building 215 Euston Road London NW1 2BE

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