Clean pigs for clear results Newsletter 36 GÖTTINGEN MINIPIGS A/S

Autumn 2011

Göttingen Minipig genome sequenced ! Microarray for mRNA profiling in development

Refinement: Take blood samples from minipigs in a sling !

Transgenic pig models PEG Catheter for biomedical placement in the research Göttingen Minipig

Stem Cell Research in Minipigs: a Potential Model for Mimicking Human Disease

Posters: • Survey of minipig clinical biochemistry, coagulation, and urinalysis parameters • Use of pig, dog and NHP in biomedical research www. minipigs.dk

Soroe Landevej 302 · DK-4261 Dalmose · Denmark · Tel. +45 5818 5818 · Fax +45 5818 5880 · [email protected] · www.minipigs.dk For Göttingen Minipigs in North America please contact Marshall BioResources – [email protected] For Göttingen Minipigs in Japan please contact Oriental Yeast Co., Ltd. - [email protected] ➤ dear reader

I sincerely hope you have enjoyed your summer, despite the book about the minipig, and we are convinced that many will find changeable summer weather we have seen in Northern Europe. it interesting and useful. New information about minipigs is needed Over the summer, we have experienced greater interest than nor- and this new book provides minipig information that has been mal in this period from existing and potential customers, and we lacking but which now will enable readers to get an overview of are pleased to note that this year’s demand for minipigs currently the key minipig strengths and to compare minipigs to other non- exceeds last year’s demand. rodent species. This growth in minipig sales shows that an increasing part of In this newsletter you will find an interesting article from F. the pharmaceutical industry has acknowledged the minipig as an Hoffmann-La Roche AG that describes how the Göttingen Minipig established and suitable animal model. genome has been investigated and sequenced. The availability of One of our more recent customers – a large pharmaceutical genomes for the Göttingen Minipig makes it possible to perform company – has shared with us that the process of implementing a thorough and justified species selection. Again, our well defined the Göttingen Minipig has been very successful. This particular genetics shows its importance. This newsletter also provides you customer has benefited from our advice on optimal design of a with informative articles about stem cell research in minipigs and minipig facility and from participating in our Handling & Dosing transgenic pig models. Course and our Surgery Course. Generally, we have closely We are very pleased that researchers from many different phar- collaborated with them during the process of implementing the maceutical companies, CROs and universities are willing to share Göttingen Minipig as an animal model. We value our customers their expertise and knowledge with us and other minipig users. highly and give high priority to closely cooperating with them, espe- Hopefully, you will value the interesting articles in this newsletter cially when they are preparing for and establishing the Göttingen as much as we do. Minipig as a new animal research model. If you need information about Göttingen Minipigs, you are wel- We hope that more of our customers will make use of our come to visit our website. Please contact us if you cannot find the expertise and experience and, at the same time, we are also very information you are looking for or if you have any other questions interested in hearing about your work with minipigs. relating to minipigs. Although we have many years of experience with minipigs, we realise that we can learn even more about minipigs by working Sincerely, closely with our customers and minipig users around the world. Jens Ellegaard, CEO, Later this year the book “The Minipig in Biomedical Research” Ellegaard Göttingen Minipigs A/S will be published. We look forward to the release of the updated ➤ Content

3 Socialising 10 Stem Cell Research in Minipigs: a Potential 3 Scand-LAS 2011 Minipig Handling Workshop Model for Mimicking Human Disease 4  Poster: Percutaneous Endoscopic 11 Meeting calendar Gastrostomy 12 Poster: Use of pigs, dogs and NHPs in 6 PEG catheter placement in the Göttingen Minipig biomedica 6 Auxiliary equipment 14  Poster: Survey of minipig clinical biochemistry 7 Refinement: Taking Blood Samples from Minipigs in a Sling 16 Reformulating Agricultural Organophosphorus Pesticides to Reduce Global Suicide Rates: 8 Pig models of early-stage cardiovascular disease a Göttingen Minipig Model 9 Minipig Genomics at Roche: State of the Art 17 Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research

If you prefer to receive this newsletter by e-mail, please send an e-mail to elle- If you receive [email protected] or write to us at the address provided below. To correct mailing list problems, please send an e-mail to: [email protected] Ellegaard Göttingen or write to us at this address: Ellegaard Göttingen Minipigs A/S Minipigs A/S Soroe Landevej 302, DK-4261 Dalmose Newsletter by mail ... Thank you PLEASE NOTE: Ellegaard Göttingen Minipigs A/S will not release or give away a sub- scriber’s e-mail address, name or any other information provided.

2 Newsletter 36 Autumn 2011 Socialising

Animal welfare is our main priority and we continuously adjust our procedures in order to provide the minipigs with the optimal conditions. In the early weaning section we socialise with the pigs every day to accustom them to human contact. At first, minipigs can be shy and do not want to be patted but once they feel safe and comfortable around humans, they love to be scratched behind the ear or on the back. They will also appreci- ate a gentle belly rub. Socialising will improve the welfare for the minipigs and the working conditions for the staff. This picture shows Martin who is socialising with the minipigs in the early weaning section. He sits on the floor and makes no abrupt movements that might scare the minipigs. Most minipigs are curious and want to investigate new things or people in the pen. Martin picks up one minipig at a time and his calm manner shows that they can feel safe with him. The female minipig on his lap obviously feels safe, is very relaxed and enjoys every moment. Please contact us if you would like to know more about how to benefit from socialising with the minipigs.

Scand-LAS 2011 Minipig Handling Workshop

The annual Scand-LAS meeting was held at the University of the opportunity to get practical experience in handling and dosing Copenhagen on 25–28 May 2011 minipigs. During the workshop, participants showed a strong interest in On Thursday 26 May, Ellegaard Göttingen Minipigs conducted a the handling and dosing of minipigs, and feedback from the work- minipig handling workshop attended by participants from several shop has been very positive. pharmaceutical companies and universities. Over the years, we have provided many of our customers with To maximise the benefits for the participants, they were divided training courses in basic handling of minipigs, and you are welcome into four teams of 3–4 people. to contact us if you would like to attend a course or if you are Adrian Zeltner, our Laboratory Technician, has many years of interested in receiving further information. experience in handling minipigs which he shared with the partici- We can run the course at your best convenience at our facility or pants during the workshop. your own facility. Please contact us for further information. Anne, Lise and Mette are staff members working within our We also exhibited at the Scand-LAS symposium and had good barriers and they assisted Adrian with their knowledge and practi- conversations with many people who showed an interest in the cal experience. Four minipigs were used to give the participants Göttingen Minipig.

Newsletter 36 Autumn 2011 3

Percutaneous Endoscopic Gastrostomy (PEG) catheter placement for intragastric dosing studies in the Göttingen minipig

Peter Bollen', Henrik Saxtorph', Helle Lorentsen" & Adrian Zeltner" ') Biomedical Laboratory, University of Southern Denmark, Odense, Denmark ") Ellegaard Göttingen Minipigs, Dalmose, Denmark

Oral dosing is widely used to test compounds on Göttingen minipigs. The procedure of oral dosing by gavage is stressful for the animals and may require up to 3‐4 technicians to perform. Some of these studies are chronic dosing studies with multiple daily dosing. In humans, PEG (Percutaneous Endoscopic Gastrostomy) catheters are commonly used to feed patients who are not able to ingest food, and where the placement of a nasal intragastric catheter is not possible. We suggested that oral dosing studies in minipigs would be easier to perform, and would cause less distress for the animals, when using PEG catheters, due to the absence of the need for restraint. For this reason, we investigated if PEG catheters could be placed and maintained in Göttingen minipigs, and we examined the degree of tissue reaction three weeks after placement.

Methods Two male Göttingen minipigs with a body weight of 6 kg were brought into general aneasthesia by administration of 0.04 mg/kg medetomidine, 0.2 mg/kg midazolam and 0.05 mg/kg atropine IM, followed by IV administration of 3‐5 mg/kg Figure 6: Schematic placement of a Flowcare PEG catheter. propofol IV. Anaesthesia was maintained by 3.5% sevoflurane. Via a gastric endoscope, the Discussion ventricle was insufflated with CO2, and the The study demonstrated that PEG catheter Figure 3: An endoscopic view of the inserted cannula (white) and mucosa of the ventricle was inspected for pulling snare (blue). placement in the Göttingen minipig is possible optimal placement of the PEG catheter. The without major complications. Apart from mild catheter (Nutricia Flowcare) was placed through Results tissue reactions to the foreign body, no major the mouth, by retracting a transabdominal snare. PEG placement was performed without inflammation was observed. The animals The security rings were attached, and the minipig complications, and the animals recovered tolerated the PEG catheter well, and had a was recovered from anaesthesia. The animals uneventful from anaesthesia. Appetite was fine, normal body weight gain. Correct placement of were housed post‐operatively for three weeks, and a normal weight gain was observed. In one the PEG catheter is essential. The adhesion to after which they were euthanized for animal, no visible signs of inflammation the spleen was possibly due to damage of the pathological examination. (erythema, exudation) were observed. In one splenic capsule. Therefore, extra attention animal a slight formation of clear exudate was should be paid to the correct placement of the observed in combination of light reddening of catheter, and the catheter should be placed as the skin. Pathological inspection revealed a sheet far as possible from palpable and visible organs, of connective tissue around the catheter, with like spleen and liver. This study investigated only slight inflammation in both animals. In the acute tissue reaction to PEG catheter placement, animal with exterior exudation, a small (1x1 cm) and a follow‐up study over a longer period adhesion to the spleen was observed. In both should be performed for investigating chronic animals the ventricle, intestines and liver were tissue reactions, and the effect of growth on PEG mobile. catheter placement.

A B

Figure 1: Placement of the endoscope through the mouth and oesophagus into the ventricle.

Figure 4: Skin reaction to catheter placement in two Göttingen minipigs. Minipig A had no erythema and exudation. Minipig B had slight erythema and exudation.

Figure 7: The loose end of the catheter is kept in place by a net stocking. This animal started eating within 15 minutes after recovery from anaesthesia.

Figure 2: The site of PEG catheter placement is clearly visible by Acknowledgments translumination. Associate professor Lars Rasmussen MD, Odense University Hospital, is kindly acknowledged for instructing the PEG placement procedure. Associate professor John Chemnitz MD, conservator Annette Møller Dall PhD and conservator assistant Figure 5: The catheter encapsulated in connective tissue in the Brian Kenneth Hansen, Dept. of Anatomy, University of Southern abdominal cavity, with vascularisation and slight inflammation. Denmark, are kindly acknowledged for preparing the plastinates.

Percutaneous Endoscopic Gastrostomy (PEG) catheter placement for intragastric dosing studies in the Göttingen minipig

Peter Bollen', Henrik Saxtorph', Helle Lorentsen" & Adrian Zeltner" ') Biomedical Laboratory, University of Southern Denmark, Odense, Denmark ") Ellegaard Göttingen Minipigs, Dalmose, Denmark

Oral dosing is widely used to test compounds on Göttingen minipigs. The procedure of oral dosing by gavage is stressful for the animals and may require up to 3‐4 technicians to perform. Some of these studies are chronic dosing studies with multiple daily dosing. In humans, PEG (Percutaneous Endoscopic Gastrostomy) catheters are commonly used to feed patients who are not able to ingest food, and where the placement of a nasal intragastric catheter is not possible. We suggested that oral dosing studies in minipigs would be easier to perform, and would cause less distress for the animals, when using PEG catheters, due to the absence of the need for restraint. For this reason, we investigated if PEG catheters could be placed and maintained in Göttingen minipigs, and we examined the degree of tissue reaction three weeks after placement.

Methods Two male Göttingen minipigs with a body weight of 6 kg were brought into general aneasthesia by administration of 0.04 mg/kg medetomidine, 0.2 mg/kg midazolam and 0.05 mg/kg atropine IM, followed by IV administration of 3‐5 mg/kg Figure 6: Schematic placement of a Flowcare PEG catheter. propofol IV. Anaesthesia was maintained by 3.5% sevoflurane. Via a gastric endoscope, the Discussion ventricle was insufflated with CO2, and the The study demonstrated that PEG catheter Figure 3: An endoscopic view of the inserted cannula (white) and mucosa of the ventricle was inspected for pulling snare (blue). placement in the Göttingen minipig is possible optimal placement of the PEG catheter. The without major complications. Apart from mild catheter (Nutricia Flowcare) was placed through Results tissue reactions to the foreign body, no major the mouth, by retracting a transabdominal snare. PEG placement was performed without inflammation was observed. The animals The security rings were attached, and the minipig complications, and the animals recovered tolerated the PEG catheter well, and had a was recovered from anaesthesia. The animals uneventful from anaesthesia. Appetite was fine, normal body weight gain. Correct placement of were housed post‐operatively for three weeks, and a normal weight gain was observed. In one the PEG catheter is essential. The adhesion to after which they were euthanized for animal, no visible signs of inflammation the spleen was possibly due to damage of the pathological examination. (erythema, exudation) were observed. In one splenic capsule. Therefore, extra attention animal a slight formation of clear exudate was should be paid to the correct placement of the observed in combination of light reddening of catheter, and the catheter should be placed as the skin. Pathological inspection revealed a sheet far as possible from palpable and visible organs, of connective tissue around the catheter, with like spleen and liver. This study investigated only slight inflammation in both animals. In the acute tissue reaction to PEG catheter placement, animal with exterior exudation, a small (1x1 cm) and a follow‐up study over a longer period adhesion to the spleen was observed. In both should be performed for investigating chronic animals the ventricle, intestines and liver were tissue reactions, and the effect of growth on PEG mobile. catheter placement.

A B

Figure 1: Placement of the endoscope through the mouth and oesophagus into the ventricle.

Figure 4: Skin reaction to catheter placement in two Göttingen minipigs. Minipig A had no erythema and exudation. Minipig B had slight erythema and exudation.

Figure 7: The loose end of the catheter is kept in place by a net stocking. This animal started eating within 15 minutes after recovery from anaesthesia.

Figure 2: The site of PEG catheter placement is clearly visible by Acknowledgments translumination. Associate professor Lars Rasmussen MD, Odense University Hospital, is kindly acknowledged for instructing the PEG placement procedure. Associate professor John Chemnitz MD, conservator Annette Møller Dall PhD and conservator assistant Figure 5: The catheter encapsulated in connective tissue in the Brian Kenneth Hansen, Dept. of Anatomy, University of Southern abdominal cavity, with vascularisation and slight inflammation. Denmark, are kindly acknowledged for preparing the plastinates.

PEG catheter placement in the Göttingen Minipig

Investigations concerning whether PEG catheters can be placed and would like to share their knowledge with other minipig users. and maintained in Göttingen Minipigs have been carried out in They possess the equipment required for placing PEG catheters close cooperation with Peter Bollen and Henrik Saxtorph of the and make this equipment available for hire. Furthermore you are Biomedical Laboratory, University of Southern Denmark. welcome to contact Peter Bollen and Henrik Saxtorp to have them At Ellegaard, we find this procedure beneficial because placing assist you in placing the catheter. PEG catheters can eliminate oral dosing, thus improving animal Peter Bollen can be contacted by email: [email protected] welfare. or by phone: +45 6550 3798. Peter Bollen and Henrik Saxtorph are experienced in this field

Auxiliary equipment

We offer various types of If you would like a demonstration of the equipment, you can sign up equipment that can make your for one of our Handling and dosing courses work with minipigs easier.

You are welcome to contact Minipig scale Bite bar us if you need any auxiliary equipment.

Restraint chair for gavaging

Restraint bench for blood sampling

Sling frame

6 Newsletter 36 Autumn 2011 Refinement: Taking Blood Samples from Minipigs in a Sling

Did you know that it is possible to take blood samples from unanaesthetised minipigs almost without restraint ?

Helle Lorentsen

At our handling and dosing courses, we teach participants how to If you are considering this handle the minipig while taking blood samples from its jugular vein. blood sampling method, Many minipig users are there are few things to be probably familiar with the aware of: method of using a blood • If you have a sling, you bench and rolling the mini- need to cut an extra hole pig on its back. This proce- to expose the insertion dure requires 2 to 4 per- site. This can be done sons, depending on the size easily with scissors. and temperament of the • Before placing the mini- minipig. pig in the sling, you can Some minipigs struggle mark the insertion site as during fixation – most likely the skin tends to curl up a Smaller animals are usually because being manipulated little due to pressure from easy to handle in the blood on the back is an unnatural the sling. bench posture for an animal. For large blood volumes, you can use an extension which • After taking the blood Therefore, taking blood samples while the minipig rests in a sling makes the sampling easier sample, remember to is an alternative method worth considering. apply pressure to the One of the advantages site of the vein puncture. of taking blood samples • Also consider the ergonomic aspects. The best solution is if the while a minipig is in a sling height of the sling can be adjusted to make the insertion site is that the procedure is readily accessible. less stressful for minipig (refinement) and person- Apart from training the minipigs to nel alike. Also fewer peo- feel comfortable in the sling, atten- ple need to be involved in tion should also be paid to training the procedure. personnel for the procedure. At Ellegaard we have You are always welcome to attend tried this method on one of our handling and dosing Taking blood samples from a untrained minipigs at dif- courses at our facility in Dalmose, minipig in a sling ferent ages. Even when Denmark. You can cut an extra minipigs were placed in We can also arrange a visit to your hole in the sling for the sling for the first time, facility where the method can be taking blood samples it was possible to perform demonstrated. Please do not hesitate stress-free blood sampling to contact us if you have any ques- on each animal. Some of tions or need further information regarding this blood sampling the minipigs were even so procedure. relaxed that one person We appreciate having good contact with you in any matter con- could do the procedure cerning a successful experience with the minipig. alone. If you already have taken blood samples from minipigs in a sling, we would like to hear your experiences and comments.

Helle Lorentsen, Head of Veterinary Services, Ellegaard Göttingen Minipigs A/S [email protected]

Newsletter 36 Autumn 2011 7 Pig models of early-stage cardiovascular disease

Malene Muusfeldt Birck, PhD thesis 2011

According to the World Health Organization, cardiovascular *Division of Laboratory Animal Science and Welfare, disease (CVD) is the leading cause of death worldwide. This PhD Department of Veterinary Pathobiology, Faculty of thesis focuses on atherosclerosis, which is an important contributor Life Sciences; †Section of Pathology, Department of Veterinary to the growing burden of CVD. In order to reduce the number of Pathobiology, Faculty of Life Sciences; people suffering from atherosclerosis, risk factors that may initiate ‡Veterinary Reproduction and Obstetrics, Department of Large and progress atherosclerosis have been identified. Conventional Animals Sciences, Faculty of Life risk factors such as smoking, hypertension, diabetes, obesity and Sciences; §Department of Human Nutrition, Faculty of Life hypercholesterolemia are not the only factors that can contribute Sciences, University of Copenhagen, Denmark to the development of atherosclerosis which can then cause CVD. This article has been published in Laboratory Animals, Vol. 42, Inflammation has been shown to play an important role in the 331-337. development of atherosclerosis and this has increased the focus on infection as a possible risk factor. In early life, infection often The thesis substantiates that the pig is a good animal model for causes inflammation and, hence, infection may have an impact on atherosclerosis research. the initiation of atherosclerosis. The results show that repeated infection is associated with The aim of this PhD project was to develop a pig model with endothelial dysfunction and that the degree of impairment is related endothelial dysfunction and early atherosclerosis lesions based to the pathogen burden. Hypercholesterolemia seems to modulate upon infection-induced inflammation. the infection-induced inflammatory response, reducing inflamma- tion and lessening severe dysfunction. In the morphological study, The results are presented in three articles: the combination of hypercholesterolemia and infection seemed to – Infection-induced coronary dysfunction and systemic speed up atherogenesis as increased numbers of foam cells were inflammation in piglets are dampened in hypercholester- present in the subendothelial space of infected animals, compared olemic milieu to non-infected animals fed a cholesterol diet. Infection was also Malene M. Birck,1 Erkki Pesonen,2 Michal Odermarsky,2 associated with increased endothelial cell death and degenera- Axel K. Hansen,1 Kenneth Persson,3 Henriette Frikke- tive changes. Further studies are needed to take a closer look at Schmidt,1 Peter M. H. Heegaard,4 and Petru Liuba2 (2011) the interaction between infection, inflammation and altered lipid 1Department of Veterinary Disease Biology, Faculty of metabolism and its role in atherogenesis. In addition, the somewhat Life Sciences, University of Copenhagen, Frederiksberg, contradictory findings between the functional and the morphologi- Denmark; 2Division of Paediatric Cardiology, Department of cal studies deserve further investigation. Paediatrics, Skåne University Hospital, Lund and 3Department This thesis has shed light on some new aspects of athero- of Microbiology, Skåne University Hospital, Malmö, Sweden; sclerosis and also stresses the importance of applying preventive and 4Innate Immunology Group, National Veterinary Institute, measures early in life. The results reveal new and interesting Technical University of Denmark, Copenhagen, Denmark. knowledge about the dynamics of infection-induced inflamma- This article has been published in American Journal of tion, hypercholesterolemia and endothelial dysfunction and should Physiology – Heart and Circulatory Physiology, Vol. 300 (5), hopefully encourage additional studies about this topic. H1595-H1601. – Intimal changes in the coronary artery of infected hyper- Two additional articles based on this PhD project have been cholesterolemic minipigs published: Malene M. Birck,1 Antti Saraste,2 Poul Hyttel,3 Michal – Expression studies of the obesity candidate gene FTO in Odermarsky,4 Petru Liuba,4 Pekka Saukko,5 Axel K. pig. Hansen,1 and Erkki Pesonen4 M. B. Madsen, M. M. Birck, M. Fredholm, S. Cirera (2010 1Department of Veterinary Disease Biology, Faculty of Life Biotechnol. Vol. 21 (1), 51-63 Sciences (LIFE), University of Copenhagen, Denmark; – Expression profile of miR-122 and its target CAT-1 in 2Department of Medicine, University of Turku, Finland; high-cholesterol fed minipigs (Sus scrofa) 3Department of Basic Animal and Veterinary Sciences, LIFE, S. Cirera, M. M. Birck, P. K. Busk, M. Fredholm (2010) University of Copenhagen, Denmark; 4Division of Paediatric Comparative Medicine Vol. 60 (2), 136-141. Cardiology, Department of Paediatrics, Skåne University Hospital, Lund, Sweden; and 5Department of Forensic Medicine, University of Turku, Finland This article has been submitted. – A novel method for trans-uterine identification of piglets Malene M. Birck*, T. Iburg†, M. Schmidt‡, P. T. Sangild§ and A. K. Hansen* (2008)

8 Newsletter 36 Autumn 2011 Minipig Genomics at Roche: State of the Art

Prof. Dr Ulrich Certa,1 Dr Martin Ebeling,2 Dr Thomas Weiser1 and Dr Thomas Singer1

Today, minipigs are becoming increasingly appreciated as animal genes that are only expressed by a small subpopulation of white models in drug research and predictive drug safety and toxicology. blood cells have low-expression mRNA levels which are likely to Their physiology and metabolism as well as responses to toxicants escape detection by microarrays. and medicines resemble the human situation in many aspects. By For more standard mRNA profiling applications like toxico- contrast with more established animal models like the mouse, rat genomics, we are currently designing microarrays for mRNA pro- or the cynomolgus monkey, only limited sequence information is filing in collaboration with Roche-NimbleGen (Madison, USA). This available for many gene families of interest, like the cytochromes technology is particularly suited for the design of prototype arrays p450, drug transporters or cytokines. In addition, the choice of since errors are easily corrected. Based on control hybridisations, minipig-specific diagnostic assays and kits for clinical chemistry we will assess performance and eliminate bad performing probes or histopathology is limited. As a result of the Macaca fascicularis if necessary. An initially validated version of this array should be genome project, we realised that knowledge of all mRNA and pro- available in the autumn of this year, with probes for about 18,000 tein sequences is highly beneficial for the prediction of cross-reac- transcripts. Although the sensitivity of deep sequencing is con- tivity of tests and reagents originally designed for use in humans. siderably higher, microarrays are still the most robust tools for In addition, the deciphering of the cynomolgus genome enabled routine tissue expression analysis, backed up by well-established us to establish a variety of chip-based applications for genotyping, bioinformatics tools for data processing and analysis. Since mRNA profiling, and copy-number variation analysis. pharmaceutical research mainly focuses on protein targets, micro- To decipher the genome of the Göttingen minipig, we based our arrays will remain valuable tools for studying gene expression at work on the efforts of a large pig genome consortium consisting of 38 institutions which deciphered the farm-pig genome “With the availability of the genomes for mouse, to advance research for animal production and breeding (http://piggenome.org/). rat, dog, minipig, and cynomolgus we can per- At the time of writing, an estimated form highly sophisticated tox-species selection 95–97% of the Göttingen minipig genome is known and assembled at Roche with which will not only reduce the number of animals about 18-fold sequence coverage. This genome version is already sufficient to required but also improve the prediction of drug deduce the minipig orthologues of virtu- safety in humans.” ally all human proteins, provided they are present in the minipig genome. When comparing conservation among protein-coding genes between different stages of preclinical drug development. It is likely that the human beings and the cynomolgus monkey, the overall sequence pig genome community will also appreciate the availability of these identity is about 93%. Based on available data, we estimate an arrays. Good compatibility for both organisms is more than likely average sequence similarity in the range of 85% for human and based on the genome project and the fact that pig and minipig minipigs. This highlights the need for careful validation of available belong to the same species. human assays and development of minipig-specific protein detec- In summary, minipig genomics support the decision-making tion kits and reagents. The availability of the genome is an impor- drug-safety process on rational grounds. Sequence and gene tant step towards this goal because the cross-reactivity of human expression information will largely inform the decision of whether assays becomes predictable. TNF-a is considered a biomarker the minipig is a responder species for the assessment of novel for systemic inflammation. If we wish to use a human ELISA kit drugs. Consequently, “trial-and-error” experiments are avoided to measure inflammatory responses in minipigs, for example, we which will ultimately lead to a significant reduction in animal usage can now align the TNF-a sequences of both species. In case the with a concomitant improvement of drug safety assessment. sequence identity is above 95%, the likelihood of cross-reactivity is Genomics in general represents a very useful tool for refining and high whilst identities below 85% are critical, especially when mono- reducing animal experiments concordant with the 3R principles. clonal antibodies are used for detection. Similarly, it is possible to With the availability of the genomes for mouse, rat, dog, minipig, predict whether a given therapeutic antibody cross-reacts with the and cynomolgus we can perform highly sophisticated tox-species minipig based on target identity. selection which will not only reduce the number of animals required Pharmacological activity as well as adverse events depend not but also improve the prediction of drug safety in humans. Finally, only on sequence similarity but also on gene expression in the novel minipig-specific tests will allow more reliable and specific target tissue. For this purpose we applied next generation RNA measurement of safety parameters. sequencing to profile mRNA expression in heart, liver, spleen, blood, kidney and lung. A highly sensitive analytical method of 1Non-clinical Safety and 2Translation Research Sciences, pRED this nature is required, especially for blood profiling. For example, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland

Newsletter 36 Autumn 2011 9 Stem Cell Research in Minipigs: a Potential Model for Mimicking Human Disease

Dr Vanessa Hall, University of Copenhagen, Faculty of Life Sciences, Department of Basic Animal and Veterinary Sciences

Stem cell research has developed into a huge research topic that humans 5 to 1. As Denmark has an enormous export production has grown and widened significantly in the past few years. This is of pork and pork products, Danes prize their pigs, which also play evident by the expansion of stem cell research conferences and an important economic role for the country’s GDP. The pig is also increasing numbers of delegates, as well as an increase in the considered a good, alternative model to the human. It is a large number of scientific publications. This expansion is largely due to mammal and more similar to human beings (in many organs, both promising developments in the transplantation of adult stem cells histologically and physiologically), than the well-characterised small for disease treatment, and a relatively new breakthrough: the pro- mammal, the mouse. duction of a new type of stem cell, the induced pluripotent stem The current focus of my research is to produce induced pluri- cell. These latter cells are stem cells that look and behave very potent stem cells from a transgenic minipig. This minipig was pro- similarly to embryonic stem cells. The advantage of induced pluri- duced by cloning (also known as somatic cell nuclear transfer), and potent stem cells is that they do not come from an early embryo. was designed to carry a human gene, which is inherited by a cohort In fact, they can be made from almost any type of adult cell in the of people who develop an early onset of Alzheimer’s disease. body. These cells are “reprogrammed” into embryonic stem cells by The hope is that this animal will develop Alzheimer’s in a manner introducing a number of key genes into the cells, which changes similar to humans. This transgenic animal model was first produced their cell identity and turns them into an embryonic stem cell. These in 2007 by a number of Danish researchers from the Foulum cells overcome the ethical barrier of embryonic stem cells whereby Research Centre (Faculty of Agricultural Sciences, University of an embryo must be sacrificed in order to produce them. This Aarhus), together with researchers from the Department of Human remains a controversial issue in many countries around the world Genetics (University of Aarhus) and the University of Copenhagen. and has now been apparently and potentially resolved. The main research principle is to study pathological features of the Why are embryonic and induced pluripotent stem cells so impor- disease in the stem cells and the cells derived from them (i.e. neu- tant for researchers? Because they have the ability to form any cell rons) in order to determine whether these cells carry clues about type of the body. This can be performed simply by adding a few the underlying mechanism of the disease. Can we see how these specific cues when culturing these cells. This means that these cells are unhealthy and die after we guide them from being stem cells are able to produce beating cardiomyocytes for treating car- cells into neurons? Therefore can we understand more about the diac disease, to produce insulin cells for treating diabetes, and to disease, which may lead to breakthroughs for better treatment in produce brain cells for potentially treating a number of brain-related the future? If these cells do display and recapitulate typical cha- diseases. Therefore, these cells have the capability to be used for racteristics of the disease, can these cells be used in screening for cell transplantation to replace diseased or lost cells in the body. new and better drug compounds and medicines? These questions In fact, there are phase 1 clinical trials currently in progress using remain the hallmark of the current research. Moreover, we are embryonic stem cells for treating spinal cord injury and for treating working with non-transgenic pigs and minipigs, to produce induced Stargardt’s macular dystrophy. These remarkable induced pluri- pluripotent stem cell lines from animals considered “healthy” to potent stem cells are the current object of focus of my research, determine whether these cells can form the specific neurons that which is primarily performed using the pig as a model. are lost in Alzheimer’s disease. Another key research question Many people often question why I use the pig as a research being investigated is whether these neurons display typical cha- model, since stem cell research is traditionally performed using racteristics of the neurons found in the brain. If we can produce either mice or human-derived cell lines. I think the best answer identical or similar neurons, these could be envisioned for future to this is that in Denmark we love pigs! There are considerably cell transplantation into the aging transgenic minipigs (or other more pigs in Denmark than human beings. In fact, pigs outnumber models of Alzheimer’s disease).

10 Newsletter 36 Autumn 2011 ➤ Stem Cell Research in Minipigs

Induced pluripotent stem cells (iPSC) have been produced successfully in the pig and minipig by a number of different research groups.

Cell line and cell background Pig breed Senior author and location iPSC from fetal fibroblasts Pig (breed unknown) Michael Roberts, University of Missouri (USA) iPSC from embryonic fibroblasts Tibetan minipig Duanquing Pei, Guangzhou Institutes of Biomedicine and Health (China) iPSC from adult ear fibroblasts Danish Landrace Lei Xiao, Shanghai Institutes for Biological Sciences (China) iPSC from adult ear fibroblasts White landrace x Large white Juan Carlos Izpisua Belmonte, Salk Institute for Biological Studies (USA) iPSC from mesenchymal stem cells Pig (breed unknown) Steven Stice, University of Georgia (USA)

One of the problems entailed in producing good animal models of the disease mechanisms, thereby limiting the extent of potential Alzheimer’s disease has been that, despite the ability to integrate breakthroughs for new and better treatments. the known human mutated genes which trigger the disease, these Alzheimer’s disease is the most common form of dementia and animals tend to lack the full pathological features of the disease. no cures are currently available. It is therefore important to look Traditionally, mouse models of Alzheimer’s disease have been to better, alternate disease models, which may mimic the disease produced. Zebra fish and rabbits have also been used, but less more closely to that observed in humans. The pig (being a large widely. Aging in mice is also significantly different from aging in mammal) is therefore a good alternative candidate to consider. humans. The lifespan of a mouse is roughly two years. Whether In so doing, we may just be able to gain a little more insight into these animals are a suitable organism for studying the process of Alzheimer’s disease, which could lead to the development of better aging is hotly debated by researchers. Given the fact that it has medicines and possibly breakthroughs, as well as pave the way for been difficult to produce an animal model displaying all the hall- developing a cure. mark features of the disease, it has been quite difficult to identify

Meeting calendar

Name Date Place

ACT 6-9 September Phoenix, AZ

Minipig Research Forum - Annual Meeting 28-29 November Frankfurt, Germany

5th Annual Preclinical Safety and Efficacy 30 November Testing for New Drugs – 1 December Zürich, Switzerland

BPS Winter Meeting 13-15 December London, UK

Newsletter 36 Autumn 2011 11 Use of pig, dog and NHP in biomedical research in Canada, the European Union (EU), Japan, and USA NC Ganderup Niels-Christian Ganderup [[email protected]], Chief Scientific Officer, Ellegaard Göttingen Minipigs A/S, Dalmose, Denmark.

ABSTRACT: Data for pigs, dogs, and non-human primates (NHP) is reported for the regions and, where possible, area of scientific use is provided. The total use (all regions combined) of pigs and NHPs has increased (15% and 33% respectively) while the use of dogs has decreased (2%) from 2002 to 2008. There are striking differences between the patterns of use of the three species among regions. Japan is unique in that dogs are used to a greater extent than pigs and NHPs. Dog use has decreased dramatically over two decades (38,915 in 1991 vs. 12,376 in 2007) and has not been replaced by pigs or NHPs. The USA has a constant use of the three species. Three observation can be made: (1) the use of dogs is fairly constant, (2) the use of pigs has gone down, (3) the use of NHP has increased. Canada and the EU have similar patterns of use of the three species; pigs being used most frequently, followed by the dog and NHP. In the EU the use of pigs has increased and the use of dogs has decreased, while the use of NHPs is constant. The ratio P = [pig/(dog+NHP)] of pig to dog and NHP varies with region and year: Canada [1.9-4.0], EU [1.4-3.0], Japan [0.01-0.16], USA [0.4-0.6]. E.g. for each one (1) combined dog and NHP 1.9 pig was used (Canada 2007). Thus, Canada and the EU has greater usage (and maybe acceptance) of the pig as a model in biomedical research.

DATA COLLECTION & PROVISOS: Data for the number of laboratory animals used in three regions (North America, European Union, Japan) of significant pharmaceutical industry activity is presented. Registration of this data is fragmented: type of information, level of detail as well as frequency of collection is inconsistent between regions. The latter is the reason why different years are reported for the different regions, and in the overview only selected years appear. Sources used are Canada: the Canadian Council on Animal Care’s website1. EU: Reports from the European Commission on the use of experimental animals in the European Union2. USA: Animal and Plant Health Inspectorate under the USDA3. Japan: Japanese Association for Laboratory Animal Science4. Provisos: the underlying reason why this is a valid approach is that a species is only used if it scientifically relevant and likely to generate the information sought. Implicitly this includes aspects such resemblance to human anatomy, physiology, biochemistry, as well as suitability and predictivity of the species selected. There are several limitations: these data do not specify purpose aim of the experimental (except EU); they do not distinguish between pigs and minipigs (except Canada); frequency of data collection varies greatly between the four regions, furthermore, the EU has expanded in size (from 12 to 27 countries) which in and of itself skews data, France reports data one year earlier than the rest of the EU, and finally not all countries have reported since Eurostat (EU’s Statistics Office) made it mandatory. Countries like China, India, Pakistan use a sizeable number of dogs, (mini)pigs and NHPs for biomedical purposes, but such data is, at best, difficult to access, and accuracy cannot be ascertained, hence they are not included. Despite of these shortcomings the comparison of usage of the three main non-rodent species still provides insights to the extent of their use in those regions.

RESULTS & DISCUSSION: Data for pigs, dogs, and non-human primates (NHP) is reported by region (Canada, EU, Japan, USA) see bottom, and as a total for all regions with distribution among the three species (see right; numbers inside columns are per cent of total for a given year). The total use (all regions combined) of pigs and NHPs has increased by 15% and 33%, respectively, while the use of dogs has decreased negligibly (2%) from 2002 to 2008. No concrete reasons for those changes can be provided; one might speculate that the increase in use of NHP’s is driven by the increasing research and development efforts in biopharmaceuticals. Pigs and minipigs are used in greater numbers than both dogs and NHPs. There are striking differences between the patterns of use of the three species among regions. Canada and the EU have similar patterns of use of the three species, while Japan and the USA have altogether distinct patterns. In Canada the use of pigs fluctuates while a steady increase in dog use is observed. When looking at the second half of the 1990’ies and 2000’s respectively, it appears that more NHP’s are used in the second half the 2000’s. Again, biopharmaceuticals may be driving this increase. In the EU the use of pigs has increased and the use of dog and NHP is more or less constant. The EU has increased pressure to reduce the use of NHP’s which lead to a proposal for banning the use of Great Apes in 20075 and to the adoption of the new Directive 2010/63/EU6 published in 2010 whereby giving member states until 2013 to comply. The scientific and ethical principles motivating the directive has been commented by FELASA7 pointing out latent issues and shortcomings in the scientific basis of the directive. The directive prohibits the use of Great Apes for studies not pertinent to the continues existence of the species and thus does not directly impact the use of other NHPs in drug development, but the new directive increases requirements for scientific scrutiny before allowing the use of other NHPs, a trend which may well grow stronger making the use of NHPs in the EU less attractive to researchers and pharmaceutical industry alike. Pressure to move away from the dog, a companion animal, is rising in Europe which is evident through the increasing requirements for housing, welfare in relation to working with dogs. The minipig is not subject to such pressure as it is viewed as a food animal. This is not to say that it is of less ethical concern to use minipigs, it is not. Their 8 capacity to for pain and suffering is the same as e.g. the dog or the NHP as described in Webster et al . Japan is unique in REFERENCES that dogs are used to a much greater extent than pigs and NHPs. Dog use has decreased dramatically over two decades (1) CCAC. Canadian Council on Animal Care [www.ccac.ca/en_/publications/audf]. 15-1-2011. (38,915 in 1991 vs. 12,376 in 2007) and has not been replaced by pigs or NHPs. There is no clear trend in the use of NHPs (2) European Commission. Reports from the Commission to the Council and the European Parliament on the Statistics on the number of animals used for experimental and other scientific in Japan. From 1985 the use of NHPs fell to an all-time low in 1998, only to increase again after 1998 peaking at an all-time purposes in the member states of the European Union high in 2007. There is no obvious explanation for this pattern although biopharmaceuticals may, as mentioned before, play a [http://ec.europa.eu/environment/chemicals/lab_animals/reports_en.htm]. 2011. (3) United States Department of Agriculture (USDA) APHIA. Reports on Annual Use of Laboratory role. Pigs are only used in very small numbers in Japan, the reason for this is not known. Limited knowledge about the model Animals, Annual Reports of Enforcement [www.aphis.usda.gov/animal_welfare/pubs_reports.shtml]. and its merits combined with a traditional approach to species selection in experiments are possibilities. For the USA three 2011. (4) JLAS. Japanese Association for Laboratory Animal Science observations can be made: (1) with the exception of 2007 and 2008 the use of dogs is fairly constant, (2) the use of pigs has [http://www.jalas.jp/english/en_policy.html]. 2011. gone down 15% from 2002 to 2009, (3) the use of NHPs has increased by 35% over the same period; it is seen that an (5) Declaration of the European Parliament on primates in scientific experiments, EP reference number: DCL-0040/2007 / P6_TA-PROV(2007)00407, European Parliament, (2007). inversion of the relative proportions of pig and NHP use has taken happened from 2002 to 2009. No explanation for the (6) European Parliament. Directive 2010/63/EU of the European Parliament and of the council of 22 decreased pig use can be offered; the increase in NHP use likely driven by biopharmaceuticals. A more detailed analysis of September 2010 on the protection of animals used for scientific purposes. European Directive 2010. NHP use, similar to Carlsson et al.9, could address this question, but that is beyond the scope of this poster. (7) FELASA. FELASA statement on the European Parliament "Written declaration on primates in scientific experiments". FELASA official statement 2007. (8) Webster J, Bollen P, Grimm H, Jennings M. Ethical implications of using the minipig in regulatory toxicology studies. J Pharmacol Toxicol Methods 2010;62(3):160-166. CONCLUSION: pigs and minipigs are used in greater numbers than dogs and NHPs. In Canada and EU the patterns of (9) Carlsson HE, Schapiro SJ, Farah I, Hau J. Use of primates in research: a global overview. Am J relative use are comparable; furthermore the use of NHPs in the EU is constant, as opposed to Canada, where it has Primatol 2004;63(4):225-237. increased. In the USA pig use has decreased while the use of NHPs has increased; use of dogs is fairly constant. Japan has ACKNOWLEDGEMENTS seen a marked drop in the use of dogs, while the use of pigs and NHPs remains at low levels. The overall pattern suggests The author wishes to thank Dr Naoki Hayashi (OYC, Japan) for extracting data for experimental animal use in Japan. that the increase in use of NHPs could be explained by biopharmaceutical products becoming more numerous in discovery and development in pharmaceutical industry. Poster presented at The Swine in Biomedical Research conference July 2011, Chicago, IL, USA Use of pig, dog and NHP in biomedical research in Canada, the European Union (EU), Japan, and USA NC Ganderup Niels-Christian Ganderup [[email protected]], Chief Scientific Officer, Ellegaard Göttingen Minipigs A/S, Dalmose, Denmark.

ABSTRACT: Data for pigs, dogs, and non-human primates (NHP) is reported for the regions and, where possible, area of scientific use is provided. The total use (all regions combined) of pigs and NHPs has increased (15% and 33% respectively) while the use of dogs has decreased (2%) from 2002 to 2008. There are striking differences between the patterns of use of the three species among regions. Japan is unique in that dogs are used to a greater extent than pigs and NHPs. Dog use has decreased dramatically over two decades (38,915 in 1991 vs. 12,376 in 2007) and has not been replaced by pigs or NHPs. The USA has a constant use of the three species. Three observation can be made: (1) the use of dogs is fairly constant, (2) the use of pigs has gone down, (3) the use of NHP has increased. Canada and the EU have similar patterns of use of the three species; pigs being used most frequently, followed by the dog and NHP. In the EU the use of pigs has increased and the use of dogs has decreased, while the use of NHPs is constant. The ratio P = [pig/(dog+NHP)] of pig to dog and NHP varies with region and year: Canada [1.9-4.0], EU [1.4-3.0], Japan [0.01-0.16], USA [0.4-0.6]. E.g. for each one (1) combined dog and NHP 1.9 pig was used (Canada 2007). Thus, Canada and the EU has greater usage (and maybe acceptance) of the pig as a model in biomedical research.

DATA COLLECTION & PROVISOS: Data for the number of laboratory animals used in three regions (North America, European Union, Japan) of significant pharmaceutical industry activity is presented. Registration of this data is fragmented: type of information, level of detail as well as frequency of collection is inconsistent between regions. The latter is the reason why different years are reported for the different regions, and in the overview only selected years appear. Sources used are Canada: the Canadian Council on Animal Care’s website1. EU: Reports from the European Commission on the use of experimental animals in the European Union2. USA: Animal and Plant Health Inspectorate under the USDA3. Japan: Japanese Association for Laboratory Animal Science4. Provisos: the underlying reason why this is a valid approach is that a species is only used if it scientifically relevant and likely to generate the information sought. Implicitly this includes aspects such resemblance to human anatomy, physiology, biochemistry, as well as suitability and predictivity of the species selected. There are several limitations: these data do not specify purpose aim of the experimental (except EU); they do not distinguish between pigs and minipigs (except Canada); frequency of data collection varies greatly between the four regions, furthermore, the EU has expanded in size (from 12 to 27 countries) which in and of itself skews data, France reports data one year earlier than the rest of the EU, and finally not all countries have reported since Eurostat (EU’s Statistics Office) made it mandatory. Countries like China, India, Pakistan use a sizeable number of dogs, (mini)pigs and NHPs for biomedical purposes, but such data is, at best, difficult to access, and accuracy cannot be ascertained, hence they are not included. Despite of these shortcomings the comparison of usage of the three main non-rodent species still provides insights to the extent of their use in those regions.

RESULTS & DISCUSSION: Data for pigs, dogs, and non-human primates (NHP) is reported by region (Canada, EU, Japan, USA) see bottom, and as a total for all regions with distribution among the three species (see right; numbers inside columns are per cent of total for a given year). The total use (all regions combined) of pigs and NHPs has increased by 15% and 33%, respectively, while the use of dogs has decreased negligibly (2%) from 2002 to 2008. No concrete reasons for those changes can be provided; one might speculate that the increase in use of NHP’s is driven by the increasing research and development efforts in biopharmaceuticals. Pigs and minipigs are used in greater numbers than both dogs and NHPs. There are striking differences between the patterns of use of the three species among regions. Canada and the EU have similar patterns of use of the three species, while Japan and the USA have altogether distinct patterns. In Canada the use of pigs fluctuates while a steady increase in dog use is observed. When looking at the second half of the 1990’ies and 2000’s respectively, it appears that more NHP’s are used in the second half the 2000’s. Again, biopharmaceuticals may be driving this increase. In the EU the use of pigs has increased and the use of dog and NHP is more or less constant. The EU has increased pressure to reduce the use of NHP’s which lead to a proposal for banning the use of Great Apes in 20075 and to the adoption of the new Directive 2010/63/EU6 published in 2010 whereby giving member states until 2013 to comply. The scientific and ethical principles motivating the directive has been commented by FELASA7 pointing out latent issues and shortcomings in the scientific basis of the directive. The directive prohibits the use of Great Apes for studies not pertinent to the continues existence of the species and thus does not directly impact the use of other NHPs in drug development, but the new directive increases requirements for scientific scrutiny before allowing the use of other NHPs, a trend which may well grow stronger making the use of NHPs in the EU less attractive to researchers and pharmaceutical industry alike. Pressure to move away from the dog, a companion animal, is rising in Europe which is evident through the increasing requirements for housing, welfare in relation to working with dogs. The minipig is not subject to such pressure as it is viewed as a food animal. This is not to say that it is of less ethical concern to use minipigs, it is not. Their 8 capacity to for pain and suffering is the same as e.g. the dog or the NHP as described in Webster et al . Japan is unique in REFERENCES that dogs are used to a much greater extent than pigs and NHPs. Dog use has decreased dramatically over two decades (1) CCAC. Canadian Council on Animal Care [www.ccac.ca/en_/publications/audf]. 15-1-2011. (38,915 in 1991 vs. 12,376 in 2007) and has not been replaced by pigs or NHPs. There is no clear trend in the use of NHPs (2) European Commission. Reports from the Commission to the Council and the European Parliament on the Statistics on the number of animals used for experimental and other scientific in Japan. From 1985 the use of NHPs fell to an all-time low in 1998, only to increase again after 1998 peaking at an all-time purposes in the member states of the European Union high in 2007. There is no obvious explanation for this pattern although biopharmaceuticals may, as mentioned before, play a [http://ec.europa.eu/environment/chemicals/lab_animals/reports_en.htm]. 2011. (3) United States Department of Agriculture (USDA) APHIA. Reports on Annual Use of Laboratory role. Pigs are only used in very small numbers in Japan, the reason for this is not known. Limited knowledge about the model Animals, Annual Reports of Enforcement [www.aphis.usda.gov/animal_welfare/pubs_reports.shtml]. and its merits combined with a traditional approach to species selection in experiments are possibilities. For the USA three 2011. (4) JLAS. Japanese Association for Laboratory Animal Science observations can be made: (1) with the exception of 2007 and 2008 the use of dogs is fairly constant, (2) the use of pigs has [http://www.jalas.jp/english/en_policy.html]. 2011. gone down 15% from 2002 to 2009, (3) the use of NHPs has increased by 35% over the same period; it is seen that an (5) Declaration of the European Parliament on primates in scientific experiments, EP reference number: DCL-0040/2007 / P6_TA-PROV(2007)00407, European Parliament, (2007). inversion of the relative proportions of pig and NHP use has taken happened from 2002 to 2009. No explanation for the (6) European Parliament. Directive 2010/63/EU of the European Parliament and of the council of 22 decreased pig use can be offered; the increase in NHP use likely driven by biopharmaceuticals. A more detailed analysis of September 2010 on the protection of animals used for scientific purposes. European Directive 2010. NHP use, similar to Carlsson et al.9, could address this question, but that is beyond the scope of this poster. (7) FELASA. FELASA statement on the European Parliament "Written declaration on primates in scientific experiments". FELASA official statement 2007. (8) Webster J, Bollen P, Grimm H, Jennings M. Ethical implications of using the minipig in regulatory toxicology studies. J Pharmacol Toxicol Methods 2010;62(3):160-166. CONCLUSION: pigs and minipigs are used in greater numbers than dogs and NHPs. In Canada and EU the patterns of (9) Carlsson HE, Schapiro SJ, Farah I, Hau J. Use of primates in research: a global overview. Am J relative use are comparable; furthermore the use of NHPs in the EU is constant, as opposed to Canada, where it has Primatol 2004;63(4):225-237. increased. In the USA pig use has decreased while the use of NHPs has increased; use of dogs is fairly constant. Japan has ACKNOWLEDGEMENTS seen a marked drop in the use of dogs, while the use of pigs and NHPs remains at low levels. The overall pattern suggests The author wishes to thank Dr Naoki Hayashi (OYC, Japan) for extracting data for experimental animal use in Japan. that the increase in use of NHPs could be explained by biopharmaceutical products becoming more numerous in discovery and development in pharmaceutical industry. Poster presented at The Swine in Biomedical Research conference July 2011, Chicago, IL, USA Survey of minipig clinical biochemistry, coagulation, and urinalysis parameters in the European pharmaceutical industry

- current practice and challenges for the future development of biomarkers of toxicity in the minipig

N.C. Ganderup1, L. Earl2, C. Bouchez3, S. Mhedhbi4, A. Gibbs5, P. Glerup6, G. Itter7, P. Clausing8 1: Ellegaard Göttingen Minipigs, DK [[email protected]]. 2: Huntingdon Life Science, UK. 3: CiToxLAB CiT, France. 4: Galderma R&D, France. 5: Covance Laboratories, UK. 6: CitoxLAB Scantox, Denmark. 7: Sanofi-Aventis IPH, Germany. 8: Berlin, Germany

ABSTRACT: The use of minipigs in regulatory safety testing has increased significantly the past decade. With the increased use of minipigs establishing best practices for biomarkers (clinical-chemistry, haematology, coagulation parameters, and urinalysis) is essential to ensure predictive safety assessment studies, with patient safety and health as the ultimate goal. Much effort is invested in developing and validating biomarkers in minipigs and better understanding of current practices may prove more efficient and benefit both the researchers and research institutions. A survey of industry with the following objective was conducted: (1) collate information on current routine biomarkers and identify their value in the minipig; (2) identify biomarkers under development/consideration to address general and specific needs as markers of toxicity in the minipig, and (3) learn what the minipig user community thinks the prospects and challenges are for developing and using new biomarkers in the minipig. Many standard biomarkers used in toxicology are established in minipigs. Their development is primarily driven by the pharmaceutical industry’s needs in regulatory safety assessment studies and while there is published literature on biomarkers available they do not necessarily meet the needs of toxicologist. There appears to be an interest in creating a platform to share data and information about existing biomarkers as well as biomarkers under development. Such a database would benefit the use of minipigs in safety assessment as well as increase the accessibility and value of minipig biomarkers. This survey was conducted by Minipig Research Forum [MRF] Steering Group under the auspices of the MRF.

INTRODUCTION: The role of biomarkers in pharmaceutical development is of importance to regulators and industry alike. This is MATERIALS AND METHODS: A series of questions were formulated and condensed into survey. The survey evidenced by the attention they receive from both “sides of the table”. Applications and challenges in biomarker qualification is discussed by the consisted of ten questions with reply options and/or blank fields. For coagulation, biochemistry, and urinalysis (Q1-3) respondents pharmaceutical industry (Guerreio et al. 2003; ; Lee et al. 2005; Lühe et al. 2005; Gaughan 2006; Marrer and Dieterle 2007). Regulatory agencies could reply whether they were performing the analysis under Non-GLP conditions (Non-GLP), under GLP conditions (GLP), or not (mainly the US FDA) offer their perspective on how to qualify genomic biomarkers in an industry guidance document (US FDA) and in scientific performed at all (No) for well-known parameters in toxicity studies. For the remaining questions (4-10) blank fields were provided literature (Goodsaid and Frueh 2007). Furthermore, US FDA also provides a list of valid genomic biomarkers (US FDA 2010). The take home for free from replies. Survey Monkey ® was used as survey engine to send out, collect and manage the results of the survey. message is that qualified biomarkers are of value in the development of new medicines and concerted efforts will lead to faster qualification. Several areas were investigated with the specific objectives to identify: The use of minipigs in regulatory safety testing has increased significantly the past decade. With the increased use of minipigs establishing • Which routine and new clinical biochemistry, coagulation and urinalysis parameters used within the European pharmaceutical best practices for biomarkers such as clinical biochemistry, coagulation parameters, and urinalysis is essential to ensure predictive safety industry in drug development? assessment studies, with patient safety and health as the definitive objectives. Much effort is invested in developing and validating biomarkers, a • Which analytes/parameters are being/have been validated as biomarkers in toxicity studies in the minipig? better understanding of current practices may prove more efficient and benefit both the researchers and research institutions. • Which analytes/parameters have caused difficulties in assay development and what has been the outcome? More specifically, the purpose of this survey can be summarized as follows: (1) collate information on the current routine clinical • Commercially available assay kits which have been internally validated for use in toxicity studies? biochemistry, coagulation and urinalysis analytes/parameters and identify the value of said in the minipig. (2) Identify new biomarkers about to be • What challenges and prospects direct the development of biomarkers of toxicity in the minipig? developed to address general and specific needs as markers of toxicity in the minipig. (3) Learn what the minipig user community thinks the Experienced minipigs users, academic researchers and specialists in the field were asked to complete the questionnaire. 335 prospects and challenges are for developing and using new biomarkers in the minipig. This survey was conducted by the European Minipig emails were sent out and 18 responded giving a response rate of 5 %. All responses are presented anonymous. Research Forum Steering Group. Respondents are thanked for your valuable input. The Minipig Research Forum in Europe is the user group which seeks to provide information to minipig users to enable them to use other RESULTS (BELOW LEFT): Responses to each of the ten questions (1-10) posed in the survey are provided below. people’s experience to refine and progress their research in an effective and ethical way and provides a forum to enable experienced users to give For relevant parameters the number of respondents (Respondents) for each individual parameter is provided. For Q1-3 the per the rest of us the benefit of their experience. Learn more at www.minipigresearchforum.org. cent distribution of parameters conducted under “Non-GLP”, “GLP”, or not at all (No) conditions is presented. Free form responses (Q4-10) are collated and have undergone only minor editing and removal of duplicate responses.

4: WHICH PARAMETERS (OTHER THAN THE ROUTINE IDENTIFIED ABOVE) HAVE BEEN INVESTIGATED AS Table 1: Published biomarkers employed in minipigs ESULTS POTENTIAL BIOMARKERS OF TOXICITY AND WHY WAS IT FELT NECESSARY TO DEVELOP THE ASSAY ISCUSSION R ? D Biomarkers in (mini)pigs are described in the published literature, Respondents gave the following examples. Replies are in no particular order; any repetitions Q1: WHICH COAGULATION PARAMETERS DO YOU Questions 1-3 are covered first, followed by key include biomarkers of disease states and toxicity; but biomarkers have been removed. CURRENTLY MEASURE AS ROUTINE HAEMATOLOGY points identified in the qualitative questions (Q4-10). used in food safety and nutrition are known. ANALYTES/PARAMETERS ON TOXICITY STUDIES IN • Serum amyloid A, Troponin I, faecal occult blood (to check blood loss in the gastrointestinal Examples of biomarkers from the published literature Biomarker Reference MINIPIGS? tract), insulin, S-100 and NSE (indicators of brain damage), urinary electrolytes, osteocalcin, Gerhardy et al. Non- Respon Serum biomarkers of cartilage and bone Parameter GLP No are presented (Table 1) pointing the reader towards 2009 GLP dents thyroid hormones (T3, T4, TSH; to check thyroid toxicity), methaemoglobin, bone-specific metabolism to predict osteochondrosis lesions Fibrinogen 1 6 8 15 alkaline phosphatase, testosterone, histamine, haptoglobin, thrombin-antithrombin complexes pertinent publications discussing biomarker validation Cardiac troponin I (cTnI) in normal pigs. Frantz et al. 2010 Activated partial Xia et al. 2009 thromboplastin time 1 8 6 15 (TAT), C-reactive protein (due to compound specific toxicities), Factor Xa Parameter 7 C3. from an industry and regulatory perspective. Angiotensin II subtype 1 receptor (AT(1)R) is (APTT) 5: WHAT CHALLENGES DID YOU FACE IN DEVELOPING THE ASSAY FOR THE PARAMETERS IN THE Among coagulation, biochemistry and urinalysis introduced as a diagnostic biomarker of Prothrombin time 2 7 6 15 QUESTION ABOVE renovascular disease. Tissue plasminogen ? there is a clear trend (Table 2) that these investigation 1 0 11 12 8-hydroxydeoxyguanosine as a non-invasive Loft et al. 1993 activator (tPA) Respondents replied with a number of challenges. They are listed in no particular order; are not conducted under Non-GLP conditions, that is D-Dimer 0 0 11 11 repetitions have been removed. urinary biomarker of oxidative DNA damage in Thrombin-antithrombin 0 0 11 11 to say, they are either performed under GLP or not at humans, pigs, and other laboratory animal • Lack of species specific kits. complexes (TAT) all. This correlated with the demographics of survey species. • Differences in plasma/serum from different minipig sources. % of total parameters 6% 27% 67% respondents being related predominantly to Lactate dehydrogenase as a relevant biomarker Bacqueville et al. • Main challenge (assuming adequate sample collection & appropriate time points) is the 2007 pharmaceutical development where GLP is the norm. to assess skin injury in ex vivo porcine skin organ adoption of appropriate reference materials for pig (i.e. to prove we are measuring what we culture. Q2: WHICH BLOOD BIOCHEMISTRY PARAMETERS DO think we are measuring). The approach is to investigate pig matrix interference and work with Urinalysis of deposits after centrifugation has the Formation of malondaldehyde as a potential in Ichinose et al. YOU CURRENTLY MEASURE AS ROUTINE CLINICAL the given quality controls. highest proportion of GLP operational standards, 1994 BIOCHEMISTRY ANALYTES/PARAMETERS ON TOXICITY vitro biomarker of carbon tetrachloride induced STUDIES IN MINIPIGS? • Rare use of immunoluminometric assay followed by urinalysis. If this difference is because the hepatotoxicity Non- Respo Wolf et al. 2005 Parameter GLP No • Finding a suitable thyroid-stimulating hormone ELISA kit and performing complete validation. analytical methods applied are very standardized and In vitro exposure of benzo(a)pyrene on porcine GLP ndents Producing internal quality control. urinary bladder epithelial cells indicated CYP1A1 Alkaline phosphatase 2 7 2 11 easy to use; whether this is indicative of a tick-box- Glucose 2 7 2 11 • Finding suitable Troponin I ELISA kit and performing complete validation. Producing internal mRNA expression as a potential biomarker approach, or, if there is an underlying scientific Urea 2 7 2 11 quality control. Polychlorinated biphenyls mediated induction of Van der Burght et Sodium 2 7 2 11 rationale to explain this pattern is unclear. CYP1A activity as biomarker for Ah-receptor al. 2000 Potassium 2 7 2 11 6: WHAT WAS THE OUTCOME OF THE ASSAY DEVELOPMENT AND/OR PERCEIVED VALUE OF THE mediated responses. Alanine aminotransferase 1 7 2 10 BIOMARKER? Given the ease with which blood can be obtained Aspartate aminotransferase 1 7 2 10 Alkylresorcinols may be a useful biomarker of Linko et al. 2006 Respondents gave some examples of outcome and predictive value. They are listed in no (compared to e.g. urine) and the number of Gamma glutamyltransferase 2 5 3 10 wholegrain wheat and rye intake Creatinine 2 6 2 10 particular order; any repetitions have been removed. parameters which can be monitored in this matrix it is Total protein 1 7 2 10 Biomarkers for various parameters relevant for Stahl et al. 2007; Albumin 1 7 2 10 • Most assays have been validated and have performed well as biomarkers. Some assays noteworthy, that less than half (48%) of all blood commercial pig (meat) production. Te Pas et al. 2009 Chloride 1 7 2 10 haven't worked so well e.g. parathyroid hormone. biochemistry parameters are performed under GLP. Haptoglobin as a biomarker to monitor various Saco et al. 2010 Calcium 1 7 2 10 • For thrombin-antithrombin complexes, successful validation of this assay and use of this Total bilirubin 1 6 2 9 Matrix effects by serum/plasma/whole blood can make production parameters including use of Aujeszky Direct bilirubin – Total biomarker serves as a measure of in vivo coagulation activation and reflects thrombin modified live vaccine in production pigs. 1 6 2 9 assay development more challenging may partly be to cholesterol formation. Triglycerides 2 5 2 9 reason for this low number as establishing and Albumin/globulin ratio 0 7 2 9 • Generally good, but large standard deviation of measurements. Creatine 1 4 3 8 • Thyroid hormone biomarkers: challenging to set-up and validate. Good indicator of thyroid validating new methods is costly it is done only when Table 2: Summary of operational standards for coagulation, Gamma globulins 0 4 4 8 absolutely necessary; developing a new biomarker is biochemistry and urinalysis. Inorganic phosphorus 0 6 2 8 toxicity. The details of the qualitative questions are reviewed. Key points Lactate dehydrogenase 2 2 3 7 • Troponin I: challenging to set-up and validate. Good indicator of cardiac toxicity. driven by need-to-do, rather than nice to know. HDL-cholesterol 2 1 4 7 are the lack of species specific assays/kits, new cardiac and LDL-cholesterol 2 1 4 7 7: WHICH COMMERCIALLY AVAILABLE ASSAY KITS DO YOU USE FOR MEASURING THE PARAMETERS? Another explanation may also be that the kidney biomarkers of toxicity are sought after, as are methods to Lactate 2 1 4 7 (OTHER THAN ROUTINE CLINICAL BIOCHEMISTRY ANALYTES) PLEASE DESCRIBE ANY PROBLEMS WITH development was abandoned because of hurdles, e.g. help ensure quality control including positive controls. Sharing of Alpha-1 globulins 0 3 4 7 DEVELOPING VALIDATING A MINIPIG SPECIFIC ASSAY Alpha-2 globulins 0 3 4 7 / . technical, which could not be overcome. information (including data) on new biomarkers, as well as Magnesium 0 4 3 7 Respondents replied with a number of commercial kits which have been used. They are listed in advertising their existence is pointed out as pivotal to move With regards to coagulation parameters there Creatine kinase 1 3 2 6 no particular order; any repetitions have been removed. forward this field in an efficient manner. This path may well also Glutamic dehydrogenase 1 2 3 6 appears to be a standard set used in minipig studies, Amylase 1 1 4 6 • SERUM AMYLOID ASSAY kit (AbCys SA). be the best way to persuade kit manufacturers to develop minipig Phospholipids 1 1 4 6 • Siemens Insulin TKIN1. as all respondents analysed fibrinogen, activated specific kits so the technologies become available in a convenient Uric acid 1 1 4 6 partial thromboplastin time, and prothrombin time and cost effective way Bile acids 1 1 4 6 • LIAISON Sangtec. Lipase 0 1 4 5 • ELISA kits: the problem is the lack of commercial Quality Control, the lack of positive control, under GLP. Parameter Non-GLP GLP No Leucine aminopeptidase 0 0 5 5 Urinalysis, deposits after the low sensitivity, the absence of shared historical data. 2% 80% 18% 5’Nucleotidase 0 0 5 5 For the groups above a detailed follow-up with centrifugation Insulin 1 0 4 5 • BTI mid-tact human osteocalcin BT-480. individual respondents may shed light on the reasons Urinalysis 11% 63% 26% Free fatty acids 0 1 4 5 • LIAISON Sangtec. Iron 0 1 4 5 for the observed distributions and apparent Blood Biochemistry 12% 48% 39% Total iron binding capacity 0 1 4 5 • Quidel Corp BAP 8012. differences. Coagulation 6% 27% 67% Transferrin 0 0 5 5 • Neogen Histamine 409010. Beta globulins 0 3 1 4 % of total parameters 12% 48% 39% • Where possible species-specific commercially available kits have been used; if not possible, human or multi-species kits are often used, and validations of said would be described as fit ONCLUSIONS AND ECOMMENDATIONS Many standard biomarkers used in toxicology are Q3: WHICH URINALYSIS PARAMETERS DO YOU CURREN- for purpose. C R : TLY MEASURE AS ROUTINE CLINICAL BIOCHEMISTRY ANA- established in minipigs. Biomarkers in use in the minipig user community is primarily driven by the pharmaceutical 8: PLEASE COMMENT ON THE PROSPECTS FOR THE DEVELOPMENT OF BIOMARKERS OF TOXICITY IN THE LYTES/PARAMETERS ON TOXICITY STUDIES IN MINIPIGS? industry’s needs in regulatory safety assessment studies and while there is published literature on biomarkers Non- Respon MINIPIG. Parameter GLP No GLP dents Responses are listed in no particular order; any repetitions have been removed. available they do not necessarily meet the needs of toxicologist. There seems to be an interest in creating a platform pH 2 7 1 10 for minipigs users to share data and information about existing biomarkers as well as biomarkers under development. Specific gravity 2 7 1 10 • Hopefully prospects are good as more and more companies seem to be interested in using Protein 2 7 1 10 the minipig as a non-rodent used in safety assessment. Respondent has seen only one assay Identifying and overcoming existing or potential hurdles to minipig users can be done by initiating a survey with the Glucose 2 7 1 10 Ketone 2 6 2 10 fail. same design targeting dogs and non-human primates (NHP). If the overall picture observed is similar for dogs and Bilirubin (bile pigments) 2 6 2 10 • The development of new cardiac and kidney biomarkers could be of great help in the non- NHP’s, as it is for minipigs, this would indicate that hurdles faced by the minipig is the same as dogs and NHPs, or Appearance 1 7 1 9 clinical toxicological study. Volume 1 7 1 9 vice versa, i.e. it would help pinpoint specific focus areas were work must be done to bring the tool box on par with Blood pigments 2 5 2 9 • For the immediate future we will follow the known mainstream toxicities in developing what is available for use in dogs and NHPs. Urobilinogen 0 6 2 8 predictive biomarkers. What would be ideal is a cheap tool, such as clinical chemistry Urea 1 3 2 6 Sodium 0 4 1 5 metabolomics, where key simple changes are flagged to guide development of further REFERENCES (Bacqueville, D., Boisson, M., Mavon, A., 2007. Lactate Dehydrogenase is a Relevant Endpoint Biomarker to Study Irritation and Solar Radiation- Induced Cellular Damages in Pig Skin Organ Culture. Meeting Poster Abstract: Potassium 0 4 1 5 biomarker analysis. 68th Annual Meeting of the Society for Investigative Dermatology (SID 2007), Hyatt Regency Century Plaza Hotel, Los Angeles, California (USA), 9-12 May 2007. Calcium 0 3 2 5 Frantz, N.Z., Friesen, K.G., Andrews, G.A., et al., 2010. Use of serum biomarkers to predict the development and severity of osteochondrosis lesions in the distal portion of the femur in pigs. American journal of veterinary Magnesium 0 3 2 5 • Once available they will provide a convenient solution, saving time and effort. research, August 01, 71(8):946-952. Chloride 0 4 1 5 Gerhardy, C., Reiniche, A., Boucheix, O., et al.,2009. Cardiac biomarker evaluation following Isoprenaline administration in the minipig. Toxicology Letters, Vol. 189, S157. 9: WHAT DO YOU FEEL ARE THE CHALLENGES WE FACE IN DEVELOPING PREDICTIVE BIOMARKERS OF Ichinose, T., Miller, M.G., Shibamoto, T., 1994. Determination of Free Malonaldehyde Formed in Liver Microsomes upon CCl sub(4) Oxidation. Journal of Applied Toxicology [J. Appl. Toxicol.]. Vol. 14, no. 6, 453 p. Nov 1994. Inorganic phosphorus 0 3 2 5 TOXICITY IN THE MINIPIG? Linko, A.M., Alastair B., Kamal-Eldin, A., et al., 2006. Kinetics of the appearance of cereal alkylresorcinols in pig plasma. The British journal of nutrition, 95(2):282-287 N-acetyl D-glucosaminidase 0 3 2 5 Loft, S., Fischer-Nielsen, A., Jeding, I.B., et al., 1993. 8-Hydroxydeoxyguanosine as a urinary biomarker of oxidative DNA damage. Journal of Toxicology and Environmental Health, vol. 40, no. 2-3. 1993. Gamma glutamyl transferase 0 2 3 5 Responses are listed in no particular order; repetitions have been removed. Saco Y, Fraile L, Gimenez M, et al., 2010. Haptoglobin serum concentration is a suitable biomarker to assess the efficacy of a feed additive in pigs. Animal vol. 4 issue: 9 pages: 1561-1567. Lactate dehydrogenase 0 2 3 5 Stahl, T.S., Zamzow, J.B., Wang, D., et al. 2007. Relationship of Isoprostanes, Biomarker of Oxidative Stress, and Pig Productivity. 2007 Joint Annual Meeting of the American Dairy Science Association, Poultry Science • Species specificity. Association, Asociacion Mexicana de Produccion Animal and the American Society of Animal Science, San Antonio, Texas (USA), 8-12 Jul 2007 Creatinine 0 3 2 5 Te Pas, M., Keuning, E., Kruijt, L., et al., 2009. Biomarker development for recovery from stress in pig muscles. 60th Annual Meeting of the European Association for Animal Production, Fira de Barcelona, Barcelona, 24-27 Aug Uric acid 1 1 3 5 • To develop biomarker with good predictivity and suitable for translational toxicology. 2009. Amylase 0 1 3 4 Van der Burght, A.S.A.M., Tysklind, M., Andersson, P.L., et al., 2000. Structure dependent induction of CYP1A by polychlorinated biphenyls in hepatocytes of male castrated pigs. Chemosphere, vol. 41, no. 10, pp. 1697-1708. • Still an evolving science - the more we approach investigation of biomarkers in minipigs the Wolf, A., Kutz, A., Plottner, S., et al., 2005. The Effect of Benzo(a)pyrene on Porcine Urinary Bladder Epithelial Cells analyzed for the Expression of Selected Genes and Cellular Toxicological Endpoints. Toxicology, vol. 207, no. Per cent of total parameters 11% 63% 26% more we will have to offer and add to the wider scientific and contract research community. 2, 255 p. 14 Feb 2005. DEPOSIT AFTER CENTRIFUGATION EXAMINED FOR: Xia, J., Seckin, E., Xiang, Y., et al., 2008. Positron-emission tomography imaging of the angiotensin II subtype 1 receptor in swine renal artery stenosis. Hypertension, 51(2):466-473. Leucocytes 0 6 2 8 • If the future use diminishes that will reduce incentive to develop new biomarkers. US FDA 2010. Valid Genomic Biomarkers in the Context of Approved Drug Labels. http://www.fda.gov/Drugs/ScienceResearch/ResearchAreas/Pharmacogenetics/ucm083378.htmt accessed November 26th 2010. Erythrocytes 0 6 2 8 US FDA. Guidance for industry. E16 Genomic Biomarkers Related to Drug Response: Context, Structure, and Format of Qualification Submissions. 10: DO YOU HAVE ANY SUGGESTIONS FOR OVERCOMING THE CHALLENGES? Guerreio, N., Staedtler, F., Grenet, O., 2003. Toxicogenomics in Drug Development. Toxicologic Pathology, 31:471–479. Crystals 1 6 1 8 Lühe, A., Suter, L., Ruepp, S., et al., 2005. Toxicogenomics in the pharmaceutical industry: Hollow promises or real benefit? Mutation Research 575 (2005) 102–115. Epithelial cells 0 6 1 7 Suggestions are listed in no particular order; repetitions have been removed. Marrer, E., and Dieterle, F., 2007. Promises of Biomarkers in Drug Development – A Reality Check. Chemical Biology & Drug Design, 69: 381–394. Spermatozoa and precursors 0 5 2 7 • Persuade kit manufacturers to provide species specific kits. Lee, J.W., Weiner, R.S., Sailstad, J.M., et al. 2005. Method Validation and Measurement of Biomarkers in Nonclinical and Clinical Samples in Drug Development: A Conference Report. Pharmaceutical Research, Vol. 22, No. 4, Casts 0 6 1 7 pp. 499-511. • Share information and experience in order to identify and cross-qualify new safety biomarkers. Goodsaid, F., and Frueh, F., 2007. Biomarker Qualification Pilot Process at the US Food and Drug Administration. The AAPS Journal 2007; 9 (1) Article 10 (http://www.aapsj.org). Other abnormal components 0 6 1 7 Gaughan, A., 2006. Bridging the divide: the need for translational informatics. Pharmacogenomics, 7(1), pp.117-22. % of total parameters 2% 80% 18% • Advertising as much as possible when the development of a new biomarker is achieved. Poster presented at The Swine in Biomedical Research conference July 2011, Chicago, IL, USA Survey of minipig clinical biochemistry, coagulation, and urinalysis parameters in the European pharmaceutical industry

- current practice and challenges for the future development of biomarkers of toxicity in the minipig

N.C. Ganderup1, L. Earl2, C. Bouchez3, S. Mhedhbi4, A. Gibbs5, P. Glerup6, G. Itter7, P. Clausing8 1: Ellegaard Göttingen Minipigs, DK [[email protected]]. 2: Huntingdon Life Science, UK. 3: CiToxLAB CiT, France. 4: Galderma R&D, France. 5: Covance Laboratories, UK. 6: CitoxLAB Scantox, Denmark. 7: Sanofi-Aventis IPH, Germany. 8: Berlin, Germany

ABSTRACT: The use of minipigs in regulatory safety testing has increased significantly the past decade. With the increased use of minipigs establishing best practices for biomarkers (clinical-chemistry, haematology, coagulation parameters, and urinalysis) is essential to ensure predictive safety assessment studies, with patient safety and health as the ultimate goal. Much effort is invested in developing and validating biomarkers in minipigs and better understanding of current practices may prove more efficient and benefit both the researchers and research institutions. A survey of industry with the following objective was conducted: (1) collate information on current routine biomarkers and identify their value in the minipig; (2) identify biomarkers under development/consideration to address general and specific needs as markers of toxicity in the minipig, and (3) learn what the minipig user community thinks the prospects and challenges are for developing and using new biomarkers in the minipig. Many standard biomarkers used in toxicology are established in minipigs. Their development is primarily driven by the pharmaceutical industry’s needs in regulatory safety assessment studies and while there is published literature on biomarkers available they do not necessarily meet the needs of toxicologist. There appears to be an interest in creating a platform to share data and information about existing biomarkers as well as biomarkers under development. Such a database would benefit the use of minipigs in safety assessment as well as increase the accessibility and value of minipig biomarkers. This survey was conducted by Minipig Research Forum [MRF] Steering Group under the auspices of the MRF.

INTRODUCTION: The role of biomarkers in pharmaceutical development is of importance to regulators and industry alike. This is MATERIALS AND METHODS: A series of questions were formulated and condensed into survey. The survey evidenced by the attention they receive from both “sides of the table”. Applications and challenges in biomarker qualification is discussed by the consisted of ten questions with reply options and/or blank fields. For coagulation, biochemistry, and urinalysis (Q1-3) respondents pharmaceutical industry (Guerreio et al. 2003; ; Lee et al. 2005; Lühe et al. 2005; Gaughan 2006; Marrer and Dieterle 2007). Regulatory agencies could reply whether they were performing the analysis under Non-GLP conditions (Non-GLP), under GLP conditions (GLP), or not (mainly the US FDA) offer their perspective on how to qualify genomic biomarkers in an industry guidance document (US FDA) and in scientific performed at all (No) for well-known parameters in toxicity studies. For the remaining questions (4-10) blank fields were provided literature (Goodsaid and Frueh 2007). Furthermore, US FDA also provides a list of valid genomic biomarkers (US FDA 2010). The take home for free from replies. Survey Monkey ® was used as survey engine to send out, collect and manage the results of the survey. message is that qualified biomarkers are of value in the development of new medicines and concerted efforts will lead to faster qualification. Several areas were investigated with the specific objectives to identify: The use of minipigs in regulatory safety testing has increased significantly the past decade. With the increased use of minipigs establishing • Which routine and new clinical biochemistry, coagulation and urinalysis parameters used within the European pharmaceutical best practices for biomarkers such as clinical biochemistry, coagulation parameters, and urinalysis is essential to ensure predictive safety industry in drug development? assessment studies, with patient safety and health as the definitive objectives. Much effort is invested in developing and validating biomarkers, a • Which analytes/parameters are being/have been validated as biomarkers in toxicity studies in the minipig? better understanding of current practices may prove more efficient and benefit both the researchers and research institutions. • Which analytes/parameters have caused difficulties in assay development and what has been the outcome? More specifically, the purpose of this survey can be summarized as follows: (1) collate information on the current routine clinical • Commercially available assay kits which have been internally validated for use in toxicity studies? biochemistry, coagulation and urinalysis analytes/parameters and identify the value of said in the minipig. (2) Identify new biomarkers about to be • What challenges and prospects direct the development of biomarkers of toxicity in the minipig? developed to address general and specific needs as markers of toxicity in the minipig. (3) Learn what the minipig user community thinks the Experienced minipigs users, academic researchers and specialists in the field were asked to complete the questionnaire. 335 prospects and challenges are for developing and using new biomarkers in the minipig. This survey was conducted by the European Minipig emails were sent out and 18 responded giving a response rate of 5 %. All responses are presented anonymous. Research Forum Steering Group. Respondents are thanked for your valuable input. The Minipig Research Forum in Europe is the user group which seeks to provide information to minipig users to enable them to use other RESULTS (BELOW LEFT): Responses to each of the ten questions (1-10) posed in the survey are provided below. people’s experience to refine and progress their research in an effective and ethical way and provides a forum to enable experienced users to give For relevant parameters the number of respondents (Respondents) for each individual parameter is provided. For Q1-3 the per the rest of us the benefit of their experience. Learn more at www.minipigresearchforum.org. cent distribution of parameters conducted under “Non-GLP”, “GLP”, or not at all (No) conditions is presented. Free form responses (Q4-10) are collated and have undergone only minor editing and removal of duplicate responses.

4: WHICH PARAMETERS (OTHER THAN THE ROUTINE IDENTIFIED ABOVE) HAVE BEEN INVESTIGATED AS Table 1: Published biomarkers employed in minipigs ESULTS POTENTIAL BIOMARKERS OF TOXICITY AND WHY WAS IT FELT NECESSARY TO DEVELOP THE ASSAY ISCUSSION R ? D Biomarkers in (mini)pigs are described in the published literature, Respondents gave the following examples. Replies are in no particular order; any repetitions Q1: WHICH COAGULATION PARAMETERS DO YOU Questions 1-3 are covered first, followed by key include biomarkers of disease states and toxicity; but biomarkers have been removed. CURRENTLY MEASURE AS ROUTINE HAEMATOLOGY points identified in the qualitative questions (Q4-10). used in food safety and nutrition are known. ANALYTES/PARAMETERS ON TOXICITY STUDIES IN • Serum amyloid A, Troponin I, faecal occult blood (to check blood loss in the gastrointestinal Examples of biomarkers from the published literature Biomarker Reference MINIPIGS? tract), insulin, S-100 and NSE (indicators of brain damage), urinary electrolytes, osteocalcin, Gerhardy et al. Non- Respon Serum biomarkers of cartilage and bone Parameter GLP No are presented (Table 1) pointing the reader towards 2009 GLP dents thyroid hormones (T3, T4, TSH; to check thyroid toxicity), methaemoglobin, bone-specific metabolism to predict osteochondrosis lesions Fibrinogen 1 6 8 15 alkaline phosphatase, testosterone, histamine, haptoglobin, thrombin-antithrombin complexes pertinent publications discussing biomarker validation Cardiac troponin I (cTnI) in normal pigs. Frantz et al. 2010 Activated partial Xia et al. 2009 thromboplastin time 1 8 6 15 (TAT), C-reactive protein (due to compound specific toxicities), Factor Xa Parameter 7 C3. from an industry and regulatory perspective. Angiotensin II subtype 1 receptor (AT(1)R) is (APTT) 5: WHAT CHALLENGES DID YOU FACE IN DEVELOPING THE ASSAY FOR THE PARAMETERS IN THE Among coagulation, biochemistry and urinalysis introduced as a diagnostic biomarker of Prothrombin time 2 7 6 15 QUESTION ABOVE renovascular disease. Tissue plasminogen ? there is a clear trend (Table 2) that these investigation 1 0 11 12 8-hydroxydeoxyguanosine as a non-invasive Loft et al. 1993 activator (tPA) Respondents replied with a number of challenges. They are listed in no particular order; are not conducted under Non-GLP conditions, that is D-Dimer 0 0 11 11 repetitions have been removed. urinary biomarker of oxidative DNA damage in Thrombin-antithrombin 0 0 11 11 to say, they are either performed under GLP or not at humans, pigs, and other laboratory animal • Lack of species specific kits. complexes (TAT) all. This correlated with the demographics of survey species. • Differences in plasma/serum from different minipig sources. % of total parameters 6% 27% 67% respondents being related predominantly to Lactate dehydrogenase as a relevant biomarker Bacqueville et al. • Main challenge (assuming adequate sample collection & appropriate time points) is the 2007 pharmaceutical development where GLP is the norm. to assess skin injury in ex vivo porcine skin organ adoption of appropriate reference materials for pig (i.e. to prove we are measuring what we culture. Q2: WHICH BLOOD BIOCHEMISTRY PARAMETERS DO think we are measuring). The approach is to investigate pig matrix interference and work with Urinalysis of deposits after centrifugation has the Formation of malondaldehyde as a potential in Ichinose et al. YOU CURRENTLY MEASURE AS ROUTINE CLINICAL the given quality controls. highest proportion of GLP operational standards, 1994 BIOCHEMISTRY ANALYTES/PARAMETERS ON TOXICITY vitro biomarker of carbon tetrachloride induced STUDIES IN MINIPIGS? • Rare use of immunoluminometric assay followed by urinalysis. If this difference is because the hepatotoxicity Non- Respo Wolf et al. 2005 Parameter GLP No • Finding a suitable thyroid-stimulating hormone ELISA kit and performing complete validation. analytical methods applied are very standardized and In vitro exposure of benzo(a)pyrene on porcine GLP ndents Producing internal quality control. urinary bladder epithelial cells indicated CYP1A1 Alkaline phosphatase 2 7 2 11 easy to use; whether this is indicative of a tick-box- Glucose 2 7 2 11 • Finding suitable Troponin I ELISA kit and performing complete validation. Producing internal mRNA expression as a potential biomarker approach, or, if there is an underlying scientific Urea 2 7 2 11 quality control. Polychlorinated biphenyls mediated induction of Van der Burght et Sodium 2 7 2 11 rationale to explain this pattern is unclear. CYP1A activity as biomarker for Ah-receptor al. 2000 Potassium 2 7 2 11 6: WHAT WAS THE OUTCOME OF THE ASSAY DEVELOPMENT AND/OR PERCEIVED VALUE OF THE mediated responses. Alanine aminotransferase 1 7 2 10 BIOMARKER? Given the ease with which blood can be obtained Aspartate aminotransferase 1 7 2 10 Alkylresorcinols may be a useful biomarker of Linko et al. 2006 Respondents gave some examples of outcome and predictive value. They are listed in no (compared to e.g. urine) and the number of Gamma glutamyltransferase 2 5 3 10 wholegrain wheat and rye intake Creatinine 2 6 2 10 particular order; any repetitions have been removed. parameters which can be monitored in this matrix it is Total protein 1 7 2 10 Biomarkers for various parameters relevant for Stahl et al. 2007; Albumin 1 7 2 10 • Most assays have been validated and have performed well as biomarkers. Some assays noteworthy, that less than half (48%) of all blood commercial pig (meat) production. Te Pas et al. 2009 Chloride 1 7 2 10 haven't worked so well e.g. parathyroid hormone. biochemistry parameters are performed under GLP. Haptoglobin as a biomarker to monitor various Saco et al. 2010 Calcium 1 7 2 10 • For thrombin-antithrombin complexes, successful validation of this assay and use of this Total bilirubin 1 6 2 9 Matrix effects by serum/plasma/whole blood can make production parameters including use of Aujeszky Direct bilirubin – Total biomarker serves as a measure of in vivo coagulation activation and reflects thrombin modified live vaccine in production pigs. 1 6 2 9 assay development more challenging may partly be to cholesterol formation. Triglycerides 2 5 2 9 reason for this low number as establishing and Albumin/globulin ratio 0 7 2 9 • Generally good, but large standard deviation of measurements. Creatine 1 4 3 8 • Thyroid hormone biomarkers: challenging to set-up and validate. Good indicator of thyroid validating new methods is costly it is done only when Table 2: Summary of operational standards for coagulation, Gamma globulins 0 4 4 8 absolutely necessary; developing a new biomarker is biochemistry and urinalysis. Inorganic phosphorus 0 6 2 8 toxicity. The details of the qualitative questions are reviewed. Key points Lactate dehydrogenase 2 2 3 7 • Troponin I: challenging to set-up and validate. Good indicator of cardiac toxicity. driven by need-to-do, rather than nice to know. HDL-cholesterol 2 1 4 7 are the lack of species specific assays/kits, new cardiac and LDL-cholesterol 2 1 4 7 7: WHICH COMMERCIALLY AVAILABLE ASSAY KITS DO YOU USE FOR MEASURING THE PARAMETERS? Another explanation may also be that the kidney biomarkers of toxicity are sought after, as are methods to Lactate 2 1 4 7 (OTHER THAN ROUTINE CLINICAL BIOCHEMISTRY ANALYTES) PLEASE DESCRIBE ANY PROBLEMS WITH development was abandoned because of hurdles, e.g. help ensure quality control including positive controls. Sharing of Alpha-1 globulins 0 3 4 7 DEVELOPING VALIDATING A MINIPIG SPECIFIC ASSAY Alpha-2 globulins 0 3 4 7 / . technical, which could not be overcome. information (including data) on new biomarkers, as well as Magnesium 0 4 3 7 Respondents replied with a number of commercial kits which have been used. They are listed in advertising their existence is pointed out as pivotal to move With regards to coagulation parameters there Creatine kinase 1 3 2 6 no particular order; any repetitions have been removed. forward this field in an efficient manner. This path may well also Glutamic dehydrogenase 1 2 3 6 appears to be a standard set used in minipig studies, Amylase 1 1 4 6 • SERUM AMYLOID ASSAY kit (AbCys SA). be the best way to persuade kit manufacturers to develop minipig Phospholipids 1 1 4 6 • Siemens Insulin TKIN1. as all respondents analysed fibrinogen, activated specific kits so the technologies become available in a convenient Uric acid 1 1 4 6 partial thromboplastin time, and prothrombin time and cost effective way Bile acids 1 1 4 6 • LIAISON Sangtec. Lipase 0 1 4 5 • ELISA kits: the problem is the lack of commercial Quality Control, the lack of positive control, under GLP. Parameter Non-GLP GLP No Leucine aminopeptidase 0 0 5 5 Urinalysis, deposits after the low sensitivity, the absence of shared historical data. 2% 80% 18% 5’Nucleotidase 0 0 5 5 For the groups above a detailed follow-up with centrifugation Insulin 1 0 4 5 • BTI mid-tact human osteocalcin BT-480. individual respondents may shed light on the reasons Urinalysis 11% 63% 26% Free fatty acids 0 1 4 5 • LIAISON Sangtec. Iron 0 1 4 5 for the observed distributions and apparent Blood Biochemistry 12% 48% 39% Total iron binding capacity 0 1 4 5 • Quidel Corp BAP 8012. differences. Coagulation 6% 27% 67% Transferrin 0 0 5 5 • Neogen Histamine 409010. Beta globulins 0 3 1 4 % of total parameters 12% 48% 39% • Where possible species-specific commercially available kits have been used; if not possible, human or multi-species kits are often used, and validations of said would be described as fit ONCLUSIONS AND ECOMMENDATIONS Many standard biomarkers used in toxicology are Q3: WHICH URINALYSIS PARAMETERS DO YOU CURREN- for purpose. C R : TLY MEASURE AS ROUTINE CLINICAL BIOCHEMISTRY ANA- established in minipigs. Biomarkers in use in the minipig user community is primarily driven by the pharmaceutical 8: PLEASE COMMENT ON THE PROSPECTS FOR THE DEVELOPMENT OF BIOMARKERS OF TOXICITY IN THE LYTES/PARAMETERS ON TOXICITY STUDIES IN MINIPIGS? industry’s needs in regulatory safety assessment studies and while there is published literature on biomarkers Non- Respon MINIPIG. Parameter GLP No GLP dents Responses are listed in no particular order; any repetitions have been removed. available they do not necessarily meet the needs of toxicologist. There seems to be an interest in creating a platform pH 2 7 1 10 for minipigs users to share data and information about existing biomarkers as well as biomarkers under development. Specific gravity 2 7 1 10 • Hopefully prospects are good as more and more companies seem to be interested in using Protein 2 7 1 10 the minipig as a non-rodent used in safety assessment. Respondent has seen only one assay Identifying and overcoming existing or potential hurdles to minipig users can be done by initiating a survey with the Glucose 2 7 1 10 Ketone 2 6 2 10 fail. same design targeting dogs and non-human primates (NHP). If the overall picture observed is similar for dogs and Bilirubin (bile pigments) 2 6 2 10 • The development of new cardiac and kidney biomarkers could be of great help in the non- NHP’s, as it is for minipigs, this would indicate that hurdles faced by the minipig is the same as dogs and NHPs, or Appearance 1 7 1 9 clinical toxicological study. Volume 1 7 1 9 vice versa, i.e. it would help pinpoint specific focus areas were work must be done to bring the tool box on par with Blood pigments 2 5 2 9 • For the immediate future we will follow the known mainstream toxicities in developing what is available for use in dogs and NHPs. Urobilinogen 0 6 2 8 predictive biomarkers. What would be ideal is a cheap tool, such as clinical chemistry Urea 1 3 2 6 Sodium 0 4 1 5 metabolomics, where key simple changes are flagged to guide development of further REFERENCES (Bacqueville, D., Boisson, M., Mavon, A., 2007. Lactate Dehydrogenase is a Relevant Endpoint Biomarker to Study Irritation and Solar Radiation- Induced Cellular Damages in Pig Skin Organ Culture. Meeting Poster Abstract: Potassium 0 4 1 5 biomarker analysis. 68th Annual Meeting of the Society for Investigative Dermatology (SID 2007), Hyatt Regency Century Plaza Hotel, Los Angeles, California (USA), 9-12 May 2007. Calcium 0 3 2 5 Frantz, N.Z., Friesen, K.G., Andrews, G.A., et al., 2010. Use of serum biomarkers to predict the development and severity of osteochondrosis lesions in the distal portion of the femur in pigs. American journal of veterinary Magnesium 0 3 2 5 • Once available they will provide a convenient solution, saving time and effort. research, August 01, 71(8):946-952. Chloride 0 4 1 5 Gerhardy, C., Reiniche, A., Boucheix, O., et al.,2009. Cardiac biomarker evaluation following Isoprenaline administration in the minipig. Toxicology Letters, Vol. 189, S157. 9: WHAT DO YOU FEEL ARE THE CHALLENGES WE FACE IN DEVELOPING PREDICTIVE BIOMARKERS OF Ichinose, T., Miller, M.G., Shibamoto, T., 1994. Determination of Free Malonaldehyde Formed in Liver Microsomes upon CCl sub(4) Oxidation. Journal of Applied Toxicology [J. Appl. Toxicol.]. Vol. 14, no. 6, 453 p. Nov 1994. Inorganic phosphorus 0 3 2 5 TOXICITY IN THE MINIPIG? Linko, A.M., Alastair B., Kamal-Eldin, A., et al., 2006. Kinetics of the appearance of cereal alkylresorcinols in pig plasma. The British journal of nutrition, 95(2):282-287 N-acetyl D-glucosaminidase 0 3 2 5 Loft, S., Fischer-Nielsen, A., Jeding, I.B., et al., 1993. 8-Hydroxydeoxyguanosine as a urinary biomarker of oxidative DNA damage. Journal of Toxicology and Environmental Health, vol. 40, no. 2-3. 1993. Gamma glutamyl transferase 0 2 3 5 Responses are listed in no particular order; repetitions have been removed. Saco Y, Fraile L, Gimenez M, et al., 2010. Haptoglobin serum concentration is a suitable biomarker to assess the efficacy of a feed additive in pigs. Animal vol. 4 issue: 9 pages: 1561-1567. Lactate dehydrogenase 0 2 3 5 Stahl, T.S., Zamzow, J.B., Wang, D., et al. 2007. Relationship of Isoprostanes, Biomarker of Oxidative Stress, and Pig Productivity. 2007 Joint Annual Meeting of the American Dairy Science Association, Poultry Science • Species specificity. Association, Asociacion Mexicana de Produccion Animal and the American Society of Animal Science, San Antonio, Texas (USA), 8-12 Jul 2007 Creatinine 0 3 2 5 Te Pas, M., Keuning, E., Kruijt, L., et al., 2009. Biomarker development for recovery from stress in pig muscles. 60th Annual Meeting of the European Association for Animal Production, Fira de Barcelona, Barcelona, 24-27 Aug Uric acid 1 1 3 5 • To develop biomarker with good predictivity and suitable for translational toxicology. 2009. Amylase 0 1 3 4 Van der Burght, A.S.A.M., Tysklind, M., Andersson, P.L., et al., 2000. Structure dependent induction of CYP1A by polychlorinated biphenyls in hepatocytes of male castrated pigs. Chemosphere, vol. 41, no. 10, pp. 1697-1708. • Still an evolving science - the more we approach investigation of biomarkers in minipigs the Wolf, A., Kutz, A., Plottner, S., et al., 2005. The Effect of Benzo(a)pyrene on Porcine Urinary Bladder Epithelial Cells analyzed for the Expression of Selected Genes and Cellular Toxicological Endpoints. Toxicology, vol. 207, no. Per cent of total parameters 11% 63% 26% more we will have to offer and add to the wider scientific and contract research community. 2, 255 p. 14 Feb 2005. DEPOSIT AFTER CENTRIFUGATION EXAMINED FOR: Xia, J., Seckin, E., Xiang, Y., et al., 2008. Positron-emission tomography imaging of the angiotensin II subtype 1 receptor in swine renal artery stenosis. Hypertension, 51(2):466-473. Leucocytes 0 6 2 8 • If the future use diminishes that will reduce incentive to develop new biomarkers. US FDA 2010. Valid Genomic Biomarkers in the Context of Approved Drug Labels. http://www.fda.gov/Drugs/ScienceResearch/ResearchAreas/Pharmacogenetics/ucm083378.htmt accessed November 26th 2010. Erythrocytes 0 6 2 8 US FDA. Guidance for industry. E16 Genomic Biomarkers Related to Drug Response: Context, Structure, and Format of Qualification Submissions. 10: DO YOU HAVE ANY SUGGESTIONS FOR OVERCOMING THE CHALLENGES? Guerreio, N., Staedtler, F., Grenet, O., 2003. Toxicogenomics in Drug Development. Toxicologic Pathology, 31:471–479. Crystals 1 6 1 8 Lühe, A., Suter, L., Ruepp, S., et al., 2005. Toxicogenomics in the pharmaceutical industry: Hollow promises or real benefit? Mutation Research 575 (2005) 102–115. Epithelial cells 0 6 1 7 Suggestions are listed in no particular order; repetitions have been removed. Marrer, E., and Dieterle, F., 2007. Promises of Biomarkers in Drug Development – A Reality Check. Chemical Biology & Drug Design, 69: 381–394. Spermatozoa and precursors 0 5 2 7 • Persuade kit manufacturers to provide species specific kits. Lee, J.W., Weiner, R.S., Sailstad, J.M., et al. 2005. Method Validation and Measurement of Biomarkers in Nonclinical and Clinical Samples in Drug Development: A Conference Report. Pharmaceutical Research, Vol. 22, No. 4, Casts 0 6 1 7 pp. 499-511. • Share information and experience in order to identify and cross-qualify new safety biomarkers. Goodsaid, F., and Frueh, F., 2007. Biomarker Qualification Pilot Process at the US Food and Drug Administration. The AAPS Journal 2007; 9 (1) Article 10 (http://www.aapsj.org). Other abnormal components 0 6 1 7 Gaughan, A., 2006. Bridging the divide: the need for translational informatics. Pharmacogenomics, 7(1), pp.117-22. % of total parameters 2% 80% 18% • Advertising as much as possible when the development of a new biomarker is achieved. Poster presented at The Swine in Biomedical Research conference July 2011, Chicago, IL, USA Reformulating Agricultural Organophosphorus Pesticides to Reduce Global Suicide Rates: a Göttingen Minipig Model

Michael Eddleston,1,2 Jonathan M Street,1 Ian Self,3 Adrian Thompson,1 Tim King,5 Nicola Williams,6 Gregorio Naredo,7 Kosala Dissanayake,1 Franz Worek,8 Harald John,8 Sionagh Smith,3,4 Horst Thiermann,8 John B Harris,9 and R Eddie Clutton.3

The article is still under review

Organophosphorus (OP) insecticide self-poisoning is responsible severe poisoning. These results indicated that the solvent co- for about one-quarter of global suicides. Treatment classically formulant cyclohexanone was essential for full toxicity. This has not focuses on the fact that OP compounds inhibit the enzyme acetyl- previously been considered. cholinesterase, causing overstimulation of cholinergic receptors in The main manufacturer of dimethoate, Cheminova, then pro- central and autonomic nervous systems and in the neuromuscular vided an experimental formulation of dimethoate EC that did not junction. Poisoned patients die from respiratory failure. However, contain cyclohexanone. This showed markedly less mammalian drugs that reactivate the acetyl-cholinesterase enzyme have been toxicity in this model than the usual agricultural formulation. found to provide little benefit to OP-poisoned humans. These results indicate that solvents play a crucial role in Part of the reason may be that humans ingest formulated dimethoate toxicity. If companies were to reformulate agricultural ‘emulsifiable concentrate’ (EC) pesticides, containing solvents and dimethoate and other toxic OP insecticides, they would probably surfactants as well as the OP active ingredient (AI), rather than be much less toxic to humans. Safer formulations of dimethoate pure OP AI. The authors of this paper therefore studied the role of and other OP insecticides should rapidly reduce global suicide solvent co-formulants in OP toxicity, developing a novel Göttingen numbers. Further studies are required to determine how to change minipig model of agricultural OP poisoning with the widely used the formulations to increase human safety while maintaining agri- insecticide dimethoate. This species was selected based on the cultural efficacy. similarity of its cardiorespiratory physiology and drug metabolism with humans and on its size, which allows collection of multiple 1 Clinical Pharmacology Unit and 7 Endocrinology Unit, University/ blood samples and the use of human monitors. BHF Centre for Cardiovascular Science, 3 Department of Gottingen minipigs under terminal anaesthesia were orally poi- Anaesthesia, and 4 Veterinary Pathology Unit, Royal (Dick) soned with a clinically relevant dose of the agricultural EC formula- School of Veterinary Sciences, and 5 Roslin Institute, University tion of dimethoate, the dimethoate active ingredient (AI) alone, the of Edinburgh, UK 2 National Poisons Information Service solvents cyclohexanone and xylene, or a control. The severity of - Edinburgh, Royal Infirmary, Edinburgh, UK 6 Centre for poisoning was recorded by monitoring the heart, lung and nerve Statistics in Medicine, Wolfson College, University of Oxford, function and by measuring the poisons’ effects in the blood. UK 8 Bundeswehr Institute of Pharmacology and Toxicology, Poisoning with agricultural dimethoate EC40, but not saline Munich, Germany 9 Medical Toxicology Centre and Institute of control, caused respiratory arrest within 30 minutes, very low blood Neuroscience, Newcastle University, Newcastle upon Tyne, UK pressure, and nerve dysfunction, that was highly similar to human poisoning. Mean arterial lactate concentration rose to 15.6 [1.1] in poisoned pigs compared to 1.4 [0.4] in control pigs. By contrast, only moderate toxicity resulted from poisoning with dimethoate AI alone or the major solvent cyclohexanone, compared to dimethoate EC40. Combining dimethoate AI with cyclohexanone reproduced

16 Newsletter 36 Autumn 2011 Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research

Authors: Wiebke Garrels, Heiner Niemann. Corresponding author: Wilfried A. Kues

Abstract Whitelaw, 2003; Niemann and Kues, 2007; Robl et al., 2007). A The first transgenic pigs were produced by the microinjection of bottleneck for porcine transgenesis is the lack of authentic pluri- foreign DNA into zygotic pronuclei in 1985. Since then, the me- potent stem cells that are suitable for blastocyst complementation thodological repertoire for porcine transgenesis was expanded to experiments (Brevini et al., 2008; Kues et al., 2010a). The semi- somatic cell nuclear transfer, lentiviral transgenesis and, recently, nal development of induced pluripotent stem cells (iPS) in mice cytoplasmic plasmid injection. The major impact of transgenic pigs and humans (Takahashi and Yamanaka, 2006) provides a new and minipigs took place in the fields of humanised pig models approach to this end. The results of the first attempts to generate and biomedical disease models, whereas agricultural applications porcine iPS cells were published recently (Esteban et al., 2009; did not find broad acceptance. The recent release of the porcine Wu et al., 2009; Ezashi et al., 2009), yet the potential of current whole genome sequence and parallel developments of highly spe- porcine iPS cells to contribute to chimera formation seems to be cific enzymes and RNAs now make it possible to perform precise limited (West et al., 2010). genetic modifications and fully exploit the advantages of this large This paper briefly discusses the current progress of transgenic animal model. We anticipate that genetically modified pigs and pig models for biomedical research. Comprehensive overviews minipigs will increasingly complement the commonly used small- about transgenic pigs and livestock are available elsewhere (Clark animal models in biomedical research, since several aspects of and Whitelaw, 2003; Robl et al., 2007; Kues and Niemann, 2011; disease progression, physiology, metabolism and aging cannot Whyte and Prather, 2011). properly be mirrored in small-animal models. Basic and biomedical applications Introduction of transgenic pigs The production of transgenic pigs is labour-intensive and cost- In the last few years, an expanded methodological repertoire for intensive and depends on advanced techniques in molecular biolo- porcine gene transfer has been developed (Table 1), resulting in an gy and the micromanipulation of gametes and zygotes. At present, increasing number of transgenic approaches (Whyte and Prather, progress in reproductive techniques and gene-transfer methods 2011). At least 90% of genetically modified pigs are generated has allowed targeted modifications of the porcine genome (glos- for biomedical studies (Fig. 1A). Sequencing and annotation of sary box), albeit the overall success rates are still low (Clark and the porcine genome are important milestones for accelerating the

Fig.1. Increasing scientific interest in transgenic pig models A) Scientific interest in porcine transgenesis. Depicted are the numbers of total citations per year, as extracted from Thomson Reuters ISI Web of Knowledge for topic search terms “transgenic” and “pig model”. B) Transgenic boar exhibiting ubiquitous expression of the Venus fluorophor gene (Garrels et al., 2011). The boar is shown under specific excitation condi- tions of Venus, in front of the boar an autofluorescent toy is visible. Almost all somatic and germ cells are fluorescent.

Key words: Domestic animals, disease model, humanised, genome, large animal model Newsletter 36 Autumn 2011 17 ➤ Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research

Table 1. Progress of technologies for transgenesis in pigs and minipigs

Development Strategy Reference

First transgenic pigs PNI Hammer et al., 1985

Somatic cloning of transgenic pigs SCNT using transgenic donor cells Park et al., 2001

Sperm-mediated gene transfer SMGT Lavitrano et al., 2002; Chang et al., 2002

Knock-out in pigs Homologous recombination in somatic Dai et al., 2002; Lai et al., 2002 cells and SCNT Homozygous gene knockout Homozygous knockout Phelps et al., 2003

Lentiviral transgenesis Perivitelline injection of lentiviruses Hofmann et al., 2003; Whitelaw et al., 2004

SMGT / ICSI combination SMGT and ICSI Kurome et al, 2006

Conditional transgenesis PNI Kues et al., 2006

Episomal transgenesis SMGT and episomal plasmid Manzini et al., 2006; Giovannoni et al., 2010

Gene knock-down Knock-down of PERV genes with siRNA and Dieckhoff et al., 2008; Ramsoondar et al., SCNT 2009 Transposon transgenesis Sleeping Beauty transposition in zygotic genome Garrels et al., 2010; Kues et al., 2010b by CPI Transposon transgenesis Sleeping Beauty transposition in somatic cells Jacobsen et al., 2011; Carlson et al. 2011 and SCNT Targeted gene knockout Zinc finger nuclease-catalysed gene deletion in Whyte et al., 2011; Yang et al., 2011; primary cells and SCNT Hauschild et al., 2011 Targeted integration Recombination-mediated cassette exchange in Garrels et al., 2011 primary cells and SCNT

generation of transgenic models, even if the porcine genome with porcine alpha-galactosyltransferase knockout organs (kidney assembly still has gaps (annotated porcine genome data can be or heart) transplanted to baboons (Kuwaki et al., 2005; Yamada found at: www.ensembl.org and www.pubmed.org). Since pig et al., 2005). and minipig physiology, anatomy, pathology, genome organisa- Extensive research has been conducted to reduce the risk of tion, body weight and life span are more similar to humans than porcine endogenous retrovirus (PERV) transmission to human are rodents, the domesticated pig represents a more appropriate patients (Switzer et al., 2001; Irgang et al., 2003). RNA interfe- biomedical model (Table 2). rence (RNAi) is a promising method for knocking down the PERV For certain biomedical therapies, such as xenotransplanta- expression. RNAi is based on small RNAs, either small interfering tion (transplantation of organs from one species to another (e.g. RNA (siRNA) or short hairpin RNAs (shRNA). In the cytoplasm, porcine-to-human)), transgenic pigs are the only reasonable spe- small RNA molecules are incorporated into an RNA-induced cies (Niemann and Kues, 2003). Xenotransplantation seems to silencing complex (RISC) and targets binding to a complementary be one option for closing the widening gap between demand and transcript sequence, resulting in mRNA degradation (Plasterk, availability of appropriate human organs (Yang and Sykes, 2007). 2002; Dallas and Vlassow, 2006). The efficacy of RNAi for redu- The prerequisites for potential porcine–human xenotransplanta- cing PERV expression has been demonstrated in cloned piglets tion are: (i) overcoming immunological hurdles; (ii) preventing the (Dieckhoff et al., 2008; Ramsoondar et al., 2009). transmission of porcine pathogens to human recipients; and (iii) the For several approaches, a conditional gene expression is desi- compatibility of porcine organs with human physiology. rable over a constitutive transgenic expression. Initial animal mod- The suppression of hyperacute rejection of porcine xenografts els carrying the first generation of conditional promoter elements has been achieved by transgenic expression of human regula- suffered from high basal-expression levels and pleiotropic effects tors of complement activity (RCA) (Tucker et al., 2002) and a (Miller et al., 1989). Recent expression systems responsive to gene knockout of the porcine alpha, 1,3-galactosyltransferase exogenous tetracycline resulted in more tightly controlled expres- gene (Dai et al., 2002; Lai et al., 2002; Phelps et al., 2003). sion. In pigs, a tetracycline-controlled transgenic expression was Maximal survival rates of up to 3–6 months have been achieved achieved with a bicistronic expression cassette (Kues et al., 2006)

18 Newsletter 36 Autumn 2011 ➤

that was designed to give ubiquitous expression of human RCAs. tion. Recently, the first immunodeficient pigs were cloned by SCNT Crossbreeding of lines with two cassettes was necessary to over- (Mendicino et al., 2010; Ramsoondar et al., 2011), promising to come epigenetic silencing and to achieve tetracycline-sensitive serve as large-animal models for cell transplantation experiments. RCA expression. Conventional gain-of-function transgenesis is based on random Transgenic pigs have been shown to mimic human diseases integration of the transgene at sites of spontaneous double-strand such as atherosclerosis, non-insulin-dependent diabetes, cystic breaks of chromosomal DNA. The frequency of DNA double- fibrosis, cancer, ophthalmological and neurodegenerative disorders strand breaks at a defined locus can be considerably increased by (Kues and Niemann, 2004; Kragh et al., 2010; Rogers et al., introducing specifically designed endonuclease enzymes (Urnov 2008; Yang et al., 2010; Luo et al., 2011). An important exam- et al., 2005; Arnould et al., 2007). The artificial endonucleases ple is the minipig cystic fibrosis model, which develops disease are based on the DNA recognition sites of zinc finger transcription phenotypes that are highly similar to human patients (Rogers et factors, meganuclei or transcription factor like elements (TALE), al., 2008), whereas transgenic mouse models failed to exhibit and they can be designed to bind highly specifically to a single, lung, pancreatic and intestinal obstructions. Huntington’s disease predetermined sequence in the genome. Double-strand break- is a neurodegenerative disorder characterised by the expression repair pathways often create small deletions and, thus, designed of mutated huntingtin with expanded polyglutamine tracts. The endonucleases allow efficient gene knockouts. The proof-of-prin- misfolded protein accumulates in neurons and is suspected of trig- ciple to generate knockout pigs by synthetic zinc finger nucleases gering apoptosis. Whereas genetic mouse models often failed to has been demonstrated by the inactivation of enhanced green replicate overt neurodegeneration and apoptosis, a minipig model fluorescent protein (EGFP), peroxisome proliferator-activated expressing the N-terminal huntingtin with a polyglutamine tract receptor (PPAR gamma) and alpha-galactosyltransferase (Whyte seems to do so (Yang et al., 2010). et al., 2011; Yang et al., 2011; Hauschild et al., 2011) in primary Truncation mutations in the elongation of a very long-chain fatty- somatic cells and the subsequent use of knockout cells for SCNT, acids-4 (ELOVL4) gene cause macular dystrophy. Photoreceptor respectively. Thus current lack of authentic porcine ES cells can be topography in the pig retina is more similar to that in humans as it circumvented for the purpose of generating knockout pigs. includes cone-rich, macula-like area centralis, whereas mice lack DNA-based transposons are mobile genetic elements that a macular. Transgenic pigs expressing disease-causing ELOVL4 move in the genome via a “cut-and-paste” mechanism. Most DNA mutations were generated by PNI and SCNT (Sommer et al., transposons are simply organised: they encode a transposase 2011). A detailed analysis showed photoreceptor loss, disorga- protein flanked by inverted terminal repeats (ITRs), which carry nised inner and outer segments, and diminished electroretinogra- transposase binding sites, and it has been possible to separate phy responses, suggesting that the transgenic pigs mirror macular the transposase coding sequence from ITR sequences. Any DNA degeneration and provide a unique model for therapeutic interven- flanked by ITRs will be recognised by the transposase and will

Table 2. Selected pig and minipig models for biomedicine and pharmaceutical research

Model Comment Reference Xenotransplantation knockout of alpha-galactosyltransferase Lai et al., 2002; Dai et al., 2002 Xenotransplantation expression of tumour necrosis factor ligand Klose et al., 2005 Xenotransplantation expression of human leukocyte antigen Weiss et al., 2009 Xenotransplantation PERV-knock down Dieckhoff et al., 2008 Xenotransplantation expression of human thrombomodulin Petersen et al., 2009 Xenotransplantation expression of human A20 (anti-apoptotic gene) Oropeza et al., 2009 Cystic fibrosis pig knockout of cystic fibrosis transmembrane conductance receptor Rogers et al., 2008 Diabetes model expression of mutated hepatocyte nuclear factor-1 Umeyama et al., 2009 Diabetes model expression of mutated insulin 2 Renner et al., 2010 Immunodeficient pig knockout of light chain Ramsoondar et al., 2010 Immunodeficient pig knockout of joining gene cluster Mendicino et al., 2010 Huntington model expression of mutated huntingtin with polyglutamine tract Yang et al., 2010 Alzheimer model expression of mutated human amyloid precursor protein Kragh et al., 2010 Breast cancer knockout of BRCA1 gene Luo et al., 2011 Macular degeneration introduced deletion in ELOVL4 gene Sommer et al., 2011

Newsletter 36 Autumn 2011 19 ➤ Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research

Fig.2. Applications of transposon transgenesis Depicted is one integration site of a Venus transposon on chromosome X (red arrow). By means of targeted cassette exchange (via the Cre/loxP system), the Venus reporter gene can be replaced by a gene of choice (I), thus introducing a transgene in a pretested locus (Garrels et al., 2011) suitable for expression, and avoiding integration into heterochromatic regions or inser- tional mutagenesis. Alternatively, by supplying the SB transposase in trans, a remobilisation (II) of the transposon can be induced. The annotated pig genome sequence was extracted from www.ensembl.org.

become enzymatically integrated into nuclear DNA. In a two- expression. Importantly, transposon-tagged loci can be read- component system, the transposon is integrated solely by the dressed by recombination-mediated cassette exchange (RMCE) in trans-supplementation activity of transposase. The first transposon cell culture. Via SCNT, the RMCE cells can be used to generate sufficiently active for use in vertebrates was the Sleeping Beauty vital piglets carrying a targeted integration into a “safe harbour” (SB) transposon (Ivics et al., 1997; Clark et al., 2007). Many locus (Garrels et al., 2011). drawbacks of classical transgenic methods can be overcome Since integrated transposons can be remobilised in the pre- by transposition-catalysed gene delivery, which increases the sence of a transposase enzyme, these animals can provide the efficiency of chromosomal integration and facilitates single-copy basis for performing whole genome mutagenesis screens in the (monomeric) insertion events. An additional advantage of trans- pig. For the SB transposon, the phenomenon of local hopping poson-catalysed transgenesis is that the integration of monomeric after mobilisation has been described. The majority of secondary transgene units is directed to accessible euchromatic regions. integrations take place at a distance of up to 5 megabases from Transposon transgenic pigs have been generated (Kues et al., the original integration. Figure 2 depicts one integration site on 2010b; Garrels et al., 2011) by CPI (Iqbal et al., 2009), as well as the gene-rich X chromosome. The neighbouring porcine genes by SCNT (Jakobsen et al., 2010; Carlson, 2011; Garrels, 2011). are the von Hippel-Lindau binding gene (VBP1) and a novel gene, Ubiquitous expression of a fluorescent Venus protein, a derivative both about 10,000 base pairs away from the integration site. After of the commonly used EGFP, was found in somatic and germ mobilisation, the integration site can be screened for integration cells (differentiated spermatozoa) in own experiments (Fig. 1B, events in neighbouring genes, such as the VBP1. The VBP1 gene Garrels et al., 2011) for all integrations sites, strongly supporting is of potential interest as an animal model, and the gene product is the hypothesis that transposase preferentially integrates DNA into assumed to form a complex with the von Hippel-Lindau tumor sup- euchromatic regions. The robust transgenic expression of Venus is pressor (VHL). The von Hippel-Lindau syndrome is a dominantly strictly copy-number dependent and facilitates cell-tracking experi- inherited cancer syndrome predisposing carriers to several malig- ments in cell-therapy approaches. The identification of integrations nant and benign tumours. Thus, transposon transgenic pigs can be sites revealed that most transposon integration sites were found in employed for performing unbiased and biased mutagenic events. intergenic regions of the porcine genome (Fig. 2). This approach It is anticipated that mutagenic screens with more advanced con- made it possible to identify loci, which are suitable for transgenic structs will be applied in the near future.

20 Newsletter 36 Autumn 2011 ➤

Conclusions transposon system. Transgenic Res. 20, Jan. 9 (epub ahead of Methodological improvements for gene transfer into the pig print). genome and a rapidly increasing list of biomedical pig models have Clark J, Whitelaw B (2003) A future for transgenic livestock. Nat been developed in recent years. Together with more accurate Rev Genet 4:825-833 genome data and highly specific designed enzymes and RNAs, Clark, K.J:, Carlson, D.F., Fahrenkrug, S.C. 2007. Pigs taking precise genetic modifications have become feasible. It is anticipa- wings with transposons and recombinases. Genome Biol. 8, Suppl ted that authentic pluripotent cells of the pig will be generated in 1: S13. the near future. Thus, porcine transgenesis will become a routine Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, tool for generating relevant humanised porcine models. The most Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell- obvious application of transgenic pigs will be as disease models Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL (2002) and biomedical therapies, which are not well-reflected in small Targeted disruption of the a1,3-galactosyltransferase gene in rodent models. The progress expected in porcine transgenesis cloned pigs. Nat Biotechnol 20: 251-255 (increased success rates and decreasing costs), however, will Dallas A, Vlassow A (2006) RNAi: A novel antisense technology make the pig an attractive complementary model for advanced and its therapeutic potential. Med Sci Monit 12 RA67-74 approaches in biomedical research. Dieckhoff B, Petersen B, Kues WA, Kurth R, Niemann H, Denner J (2008) Knockdown of porcine endogenous retrovirus Acknowledgments (PERV) expression by PERV-specific shRNA in transgenic pigs. The expert technical support of Ms S. Holler, Ms Barg-Kues, Xenotransplantation 15: 36-45 Ms Herrmann and Ms Ziegler, and the financial support of the Esteban, M.A., J. Xu, J. Yang, M. Peng, D. Qin, W. Li, Z. Jiang, Deutsche Forschungsgemeinschaft (DFG) are gratefully acknow- J. Chen, K. Deng, M. Zhong, J. Cai, L. Lai, and D. Pei. 2009. ledged. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J Biol Chem. 284:17634-17640. Conflicts of interest Ezashi, T., B.P.V.L. Telugu, A.P. Alexenko, S. Sachdev, S. The authors declare no conflicts of interest. Sinha, and R.M. Roberts. 2009. Derivation of induced pluri- potent stem cells from pig somatic cells. Proceedings of the Wiebke Garrels, Heiner Niemann National Academy of Sciences of the United States of America. Friedrich-Loeffler-Institute 106:10993-10998. Mariensee, DE-31535 Neustadt, Germany Garrels, W., Mates, L., Holler,S., Niemann, H., Izsvak, Z., Ivics, Z., Kues, W.A: 2010. Generation of transgenic pigs by the Wilfried A. Kues Sleeping Beauty transposition in zygotes. Reprod. Dom. Anim. 45, Friedrich-Loeffler-Institute 65 (abstract). Institute of Farm Animal Genetics Garrels, W., Mates, L., Holler, S., Dalda, A., Taylor, U., Mariensee, DE-31535 Neustadt, Germany Petersen B, Niemann H, Izsvak Z, Ivics Z, Kues WA (2011). 0049 – (0)5034 871 120 Germline transgenic pigs by Sleeping Beauty transposition in por- 0049 – (0)5034 871 101 cine zygotes and targeted integration in the pig genome. PloS One 6, e23573, doi:10.1371/journal.pone.0023573. Giovannoni, R., Vargiolu, A., Manzini, S., De Cecco, M., Seruggia, D., Busnelli, M., Cerrito, M.G., Smolenski, R., Forni, M.., Bacci, M.L., Lavitrano, M. 2010. CMV-based episomal vector References is progressively lost in vivo in multi-transgenic pigs produced by SMGT. Transgenic Research 19, 330 (abstract) Arnould S, Perez C, Cabaniols JP, Smith J, Gouble A, Grizot S, Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert Epinat JC, Duclert A, Duchateau P, Pâques F (2007) Engineered KM, Palmiter RD, Brinster RL (1985) Production of transgenic I-CreI derivatives cleaving sequences from the human XPC gene rabbits, sheep and pigs by microinjection. Nature 315: 680-683 can induce highly efficient gene correction in mammalian cells. J Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath Mol Biol 371:49-65 JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer Brevini, T.A., S. Antonini, G. Pennarossa, and F. Gandolfi. R, Cost GJ, Niemann H. 2011. Efficient generation of a biallelic 2008. Recent progress in embryonic stem cell research and its knockout in pigs using zinc-finger-nucleases. Proc Natl Acad Sci application in domestic species. Reprod Domest Anim. 43 Suppl U S A 108, 12013-7. Epub 2011 Jul 5 2:193-199. Hofmann A, Kessler B, Ewerling S, Weppert M, Vogg B, Ludwig Chang K, Qian J, Jiang M, Liu YH, Wu MC, Chen CD, Lai CK, H, Stojkovic M, Boelhauve M, Brem G, Wolf E, Pfeifer A (2003) Lo HL, Hsiao CT, Brown L, Bolen J Jr, Huang HI, Ho PY, Shih Efficient transgenesis in farm animals by lentiviral vectors. EMBO PY, Yao CW, Lin WJ, Chen CH, Wu FY, Lin YJ, Xu J, Wang K Rep 4:1054-1060 (2002) Effective generation of transgenic pigs and mice by linker Iqbal, K., Barg-Kues, B., Broll, S., Bode, J., Niemann, H., based sperm-mediated gene transfer. BMC Biotechnol 2:5 Kues, W.A. 2009. Cytoplasmic injection of circular plasmids allows Carlson, DF, Garbe JR, Tan W, Martin MJ, Dobrinsky JR, targeted expression in mammalian embryos. BioTechniques 47, Hackett PB, Clark KR, Fahrenkrug SC. 2011 Strategies for selec- 959-968. tion marker-free swine transgenesis using the Sleeping Beauty Irgang M, Sauer IM, Karlas A, Zeilinger K, Gerlach J, Kurth R,

Newsletter 36 Autumn 2011 21 ➤ Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research

Neuhaus P, Denner J (2003) Porcine endogenous retroviruses: no Giovannoni R, Della Casa G, Seren E, Rossi G. 2002. Efficient infection in patients treated with a bioreactor based on porcine liver production by sperm-mediated gene transfer of human decay cells. J Clin Virology 28:141-154 accelerating factor (hDAF) transgenic pigs for xenotransplantation. Ivics Z, Hackett PB, Plasterk RH, Izsvák Z (1997) Molecular Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14230-5 reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, Luo Y, Li J, Liu Y, Lin L, Du Y, Li S., Yang H, Vaitja G, Callesen and its transposition in human cells. Cell 91: 501-510 H, Bolund L, Sorensen CB 2011. High efficiency of BRCA knock- Jakobsen, J., Li, J., Kragh, P.M., Moldt, B., Lin, L., Liu, Y., out using rAAV-mediated gene targeting: developing a pig model Schmidt, M., Winther, K.D., Schyth, B.D., Holm, I.E., Vajta, for breast cancer. Transgenic Res. G., Bolund, L., Callesen, H., Jorgensen, A.L., Nielsen, A.L., Manzini, S, Vargiolu A, Stehle IM, Bacci ML, Cerrito MG, Mikkelsen, J.G., 2011. Pig transgenesis by Sleeping Beauty DNA Giovannoni R, Zannoni A, Bianco MR, Forni M, Donini P, Papa M, transposition. Transgenic Research 20, 533-545. Lipps HJ, Lavitrano M, 2006. Genetically modified pigs produced Klose R, Kemter E, Bedke T, Bittmann I, Kelsser B, Endres R, with a nonviral episomal vector. Proc Natl Acad Sci USA 103, Pfeffer K, Schwinzer R, Wolf E. 2005. Expression of biologically 17672-17677. active human TRAIL in transgenic pigs. Transplantation. 2005 Jul Mendicino, M., Ramsoondar, J., Phelps, C.,Vaught, T., Ball, S. 27;80(2):222-30 LeLoith, T, Monahan, J., Chen, S., Dandro, A., Boone, J., Jobst, Kragh PM, Nielsen AL, Li J, Du Y, Lin L, Schmidt M, Bøgh IB, P., Vance, A., Wertz, N.,. Bergman, Z., Sun, X.-Z., Polejaeva, Holm IE, Jakobsen JE, Johansen MG, Purup S, Bolund L, Vajta G, I., Butler, J., Dai, Y., Ayares, D., Wells, K. 2010. Generation of Jørgensen AL. 2009. Hemizygous minipigs produced by random antibody- and B cell-deficient pigs by targeted disruption of the gene insertion and handmade cloning express the Alzheimer’s J-region gene segment of the heavy chain locus. Trans. Research. disease-causing dominant mutation APPsw. Transgenic Res. (epub ahead of print) DOI 10.1007/s11248-010-9444-z 2009 Aug;18(4):545-58. Miller KF, Bolt DJ, Pursel VG, Hammer RE, Pinkert CA, Kues WA, Niemann H (2004) The contribution of farm animals Palmiter RD, Brinster RL (1989) Expression of human or bovine to human health. Trends Biotechnol 22: 286-294 growth hormone gene with a mouse metallothionein-1 promoter in Kues WA, Schwinzer R, Wirth D, Verhoeyen E, Lemme E, transgenic swine alters the secretion of porcine growth hormone Herrmann D, Barg-Kues B, Hauser H, Wonigeit H, Niemann H and insulin-like growth factor-I. J Endocrinol 120: 481-488 (2006) Epigenetic silencing and tissue independent expression Niemann H, Kues WA (2003) Application of transgenesis in of a novel tetracycline inducible system in double-transgenic pigs. livestock for agriculture and biomedicine. Anim Reprod Sci 79: FASEB Journal Express doi: 10.1096/fj.05-5415fje; printed short 291-317 version: FASEB J 20: 1200-1202 Niemann H, Kues, WA (2007) Transgenic farm animals: an Kues, W.A., Nowak-Imialek M, Haridoss S., Niemann H. update. Reprod Fertil Develop 19: 762-770 2010a. Strategies for the derivation of pluripotent cells from farm .Oropeza M, Petersen B, Carnwath JW, Lucas-Hahn A, Lemme animals. Reprod. Dom Anim 45, Suppl 3, 25-31. E, Hassel P, Herrmann D, Barg-Kues B, Holler S, Queisser Kues, W.A., Garrels, W., Mates, L., Holler,S., Niemann, H., AL, Schwinzer R, Hinkel R, Kupatt C, Niemann H. 2009. Izsvak, Z., Ivics, Z. 2010b. Production of transgenic pigs by the Transgenic expression of the human A20 gene in cloned pigs Sleeping Beauty transposon system. Transgenic Research 19, provides protection against apoptotic and inflammatory stimuli. 336 (abstract). Xenotransplantation. 2009 Kues, W.A., Niemann H. 2011. Advances in transgenic farm Park KW, Cheong HT, Lai L, Im GS, Kühholzer B, Bonk A, animals. Prev. Vet. Med. (epub ahead of print), doi:10.1016/j. Samuel M, Rieke A, Day BN, Murphy CN, Carter DB, Prather RS. prevetmed.2011.04.009 2001. Production of nuclear transfer-derived swine that express Kurome M, Ueda H, Tomii R, Naruse K, Nagashima H. the enhanced green fluorescent protein. Anim Biotechnol 12, 2006. Production of transgenic-clone pigs by the combination of 173-181. ICSI-mediated gene transfer with somatic cell nuclear transfer. Petersen B, Ramackers W, Tiede A, Lucas-Hahn A, Herrmann Transgenic Res. 15, 229-40. D, Barg-Kues B, Schuettler W, Friedrich L, Schwinzer R, Kuwaki K, Tseng YL, Dor FJ, Shimizu A, House SL, Sanderson Winkler M, Niemann H. 2009. Pigs transgenic for human TM, Lanceros CJ, Rabharasuth DD, Cheng J, Moran K, Hisashi thrombomodulin have elevated production of activated protein C. Y, Mueller N, Yamadoa K, Greenstein JL, Hawley RJ, Patience Xenotransplantation. 2009 Nov-Dec;16(6):486-95. C, Awwad M, Fishman JA, Robson SC, Schuurman HJ, Sachs Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, DH, Cooper DK (2005) Heart transplantation in baboons using Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma 1, 3-glactosyltransferase knockout pigs as donors: initial experi- SB, Lamborn AE, Garst AS, Moore M, Demetris AJ, Rudert WA, ments. Nat Med 11: 29-31 Bottino R, Bertera S, Trucco M, Starzl TE, Dai Y, Ayares DL Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, (2003) Production of alpha 1,3-galactosyltransferasedeficient Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter pigs. Science 299: 411–414 DB, Hawley RJ, Prather RS (2002) Production of a1, 3-galacto- Plasterk RH (2002) RNA silencing: the genomes immune sys- syltransferase knockout pigs by nuclear transfer cloning. Science tem. Science 296:1263-1265 295:1089-1092 Ramsoondar J, Vaught T, Ball S, Mendicino M, Monahan J, Jobst Lavitrano M, Bacci ML, Forni M, Lazzereschi D, Di Stefano P, Vance A, Duncan J, Wells K, Ayares D. Production of transgenic C, Fioretti D, Giancotti P, Marfé G, Pucci L, Renzi L, Wang H, pigs that express porcine endogenous retrovirus small interfering Stoppacciaro A, Stassi G, Sargiacomo M, Sinibaldi P, Turchi V, RNAs. Xenotransplantation. 2009 May-Jun;16(3):164-80.

22 Newsletter 36 Autumn 2011 ➤

Ramsoondar J, Mendicino M, Phelps C, Vaught T, Ball S, induced pluripotent stem cells produce chimeric offspring. Stem Monahan J, Chen S, Dandro A, Boone J, Jobst P, Vance A, Wertz Cells Dev. 19:1211-1220. N, Polejaeva I, Butler J, Dai Y, Ayares D, Wells K. 2011. Targeted Whitelaw CB, Radcliffe PA, Ritchie WA, Carlisle A, Ellard FM, disruption of the porcine immunoglobulin kappa light chain locus. Pena RN, Rowe J, Clark AJ, King TJ, Mitrophanous KA (2004) Transgenic Res. 2011 Jun;20(3):643-53. Efficient generation of transgenic pigs using equine infectious Renner S, Fehlings C, Herbach N, Hofmann A, von Waldthausen anaemia virus (EIAV) derived vector. FEBS Lett 571: 233-236 DC, Kessler B, Ulrichs K, Chodnevskaja I, Moskalenko V, Wu, Z., J. Chen, J. Ren, L. Bao, J. Liao, C. Chun, L. Rao, Amselgruber W, Göke B, Pfeifer A, Wanke R, Wolf E. 2010. H. Li, Y. Gu, H. Dai, H. Zhu, X. Teng, L. Cheng, and L. Xiao. Glucose intolerance and reduced proliferation of pancreatic beta- 2009. Generation of pig-induced pluripotent stem cells with a cells in transgenic pigs with impaired glucose-dependent insulino- drug-inducible system. Journal of Molecular Cell Biology. 1:46-54 tropic polypeptide function. Diabetes. 2010 May;59(5):1228-38. Whyte JJ, Prather RS. 2011. Genetic modifications of pigs for Robl JM, Wang Z, Kasinathan P, Kuroiwa Y (2007) Transgenic medicine and agriculture. Mol Reprod Dev. 2011 Jun 10. doi: animal production and animal biotechnology. Theriogenology 10.1002/mrd.21333. [Epub ahead of print] 67:127-133 (epub ahead of print, doi: 10.1016/j.theriogenol- Whyte JJ, Zhao J, Wells KD, Samuel MS, Whitworth KM, ogy.2006.09.034) Walters EM, Laughlin MH, Prather RS. 2011. Gene targeting with Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina zinc finger nucleases to produce cloned eGFP knockout pigs. Mol T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Reprod Dev. 78, 2. Wohlford-Lenane CL, Davis GJ, Hanfland RA, Smith TL, Samuel Yamada K, Yazawa K, Shimizu A, Iwanaga T, Hisashi Y, Nuhn M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, M, O’Malley P, Nobori S, Vagefi PA, Patience C, Fishman J, Brogden KA, Shilyansky J, McCray PB Jr, Zabner J, Prather RS, Cooper DK, Hawley RJ, Greenstein J, Schuurman HJ, Awwad M, Welsh MJ. (2008). Disruption of the CFTR gene produces a model Sykes M, Sachs DH (2005) Marked prolongation of porcine renal of cystic fibrosis in newborn pigs. Science. xenograft survival in baboons through the use of a1, 3-galacto- Sommer JR, Estrada JL, Collins EB, Bedell M, Alexander CA, syltransferase gene-knockout donors and the cotransplantation of Yang Z, Hughes G, Mir B, Gilger BC, Grob S, Wei X, Piedrahita vascularized thymic tissue. Nat Med 11: 32-34 JA, Shaw PX, Petters RM, Zhang K 2011 Production of ELOVL4 Yang YG, Sykes M (2007) Xenotransplantation: current status transgenic pigs: a large animal model for Stargardt-like macular and a perspective on the future. Nat Rev Immunol 7: 519-531 degeneration. Yang D, Wang CE, Zhao B, Li W, Ouyang Z, Liu Z, Yang H, Br J Ophthalmol. 2011 Aug 26. [Epub ahead of print] Fan P, O’Neill A, Gu W, Yi H, Li S, Lai L, Li XJ. 2010. Expression Switzer WM, Michler RE, Shangmugam V, Matthews A, of Huntington’s disease protein results in apoptotic neurons in Hussain AI, Wright A, Sandstrom P, Chapman L, Weber C, the brains of cloned transgenic pigs. Hum Mol Genet. 2010 Oct Safley S, Denny RD, Navarro A, Evans V, Norin AJ, Kwiatkowski 15;19(20):3983-94 P, Heneine W (2001) Lack of cross-species transmission of Yang D, Yang H, Li W, Zhao B, Ouyang Z, Liu Z, Zhao Y, porcine endogenous retrovirus infection to nonhuman primate Fan N, Song J, Tian J, Li F, Zhang J, Chang L, Pei D, Chen YE, recipients of porcine cells, tissues and organs. Transplantation Lai L. 2011. Generation of PPARγ mono-allelic knockout pigs via 71: 959-965 zinc-finger nucleases and nuclear transfer cloning. Cell Res. 2011 Takahashi K, Yamanaka S (2006) Induction of pluripotent stem Jun;21(6):979-82. Epub 2011 Apr 19 cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-676 Tucker A, Belcher C, Moloo B, Bell J, Mazzulli T, Humar Y, Ellegaard Göttingen Minipigs Hughes A, McArdle P, Talbot A (2002) The production of trans- genic pigs for potential use in clinical xenotransplantation: baseline DVDs available: clinical pathology and organ size studies. Xenotransplantation 9: 203-208 • The Göttingen Minipig – Handling and dosing Umeyama K, Watanabe M, Saito H, Kurome M, Tohi S, This DVD contains an English, German and Matsunari H, Miki K, Nagashima H. 2009. Dominant-negative French version. A unique tool for those who mutant hepatocyte nuclear factor 1alpha induces diabetes in work with the Göttingen Minipig. transgenic-cloned pigs. Transgenic Res. 2009 Oct;18(5):697-706 Price each ¤ 90 excl. shipping and handling. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC • The Göttingen Minipig – Histology (2005) Highly efficient endogenous human gene correction using This DVD allows users to familiarize designed zinc-finger nucleases. Nature 435: 646–651 themselves with the normal histology of Weiss EH, Lilienfeld BG, Müller S, Müller E, Herbach N, the Göttingen Minipig. Kessler B, Wanke R, Schwinzer R, Seebach JD, Wolf E, Brem Price each ¤ 65 excl. shipping G. HLA-E/human beta2-microglobulin transgenic pigs: protection and handling. against xenogenic human anti-pig natural killer cell cytotoxicity. Transplantation. 2009 Jan 15;87(1):35-43. Please contact us for further information at West, F.D., S.L. Terlouw, D.J. Kwon, J.L. Mumaw, S.K. Dhara, [email protected] or phone +45 5818 5818. K. Hasneen, J.R. Dobrinsky, and S.L. Stice. 2010. Porcine

Newsletter 36 Autumn 2011 23 Register now for the annual meeting of the Minipig Research Forum

The annual meeting of the Minipig Research learning from the many presenters, the par- Forum will take place on 28-29 November ticipants exploit this opportunity to exchange 2011. This year’s venue is Lindner Hotel & minipig knowledge, ideas and experiences. Residence Main Plaza in Frankfurt (Germany), situated just 6 km from Frankfurt Airport. You can now register for the meeting at the website www.minipigresearchforum.org The main topics for the meeting will be Juvenile and Embryofoetal Development Besides access to an interesting two-day pro- Studies and neurobehavioural assessment in gramme, the ¤300 registration fee includes minipigs. lunch and meeting dinner on the first day and take-away lunch on day 2. By joining the meeting you will have a unique opportunity to meet minipig users from all For further information please contact the over Europe. At the previous meetings we have Minipig Research Forum: experienced that besides listening to and [email protected]

The Minipig in Biomedical Research

The Minipig in Biomedical Research is a comprehensive resource book discusses the regulatory uses of minipigs in the evalua- for research scientists concerning the potential and use of the tion of human and veterinary pharmaceuticals, medical devices, minipig in basic and applied biomedical research and in the devel- and other classes of xenobiotics. It describes features of normal opment of drugs and chemicals. Written by acknowledged experts minipig health, normal laboratory values, and common diseases. in the field and drawing on the authors’ global contacts and experi- It also carefully elucidates ethical and legal considerations in ence with regulatory authorities and the pharmaceutical industry the breeding, housing, and trans- and other industries, this accessible manual covers a wide range port of minipigs. The result is an of topics including the biological, scientific, and practical uses of all-inclusive and up-to-date manual the minipig in the laboratory. It also covers the minipig’s origins, about the experimental uses of the anatomy, genetics, immunology, and physiology, as well as minipig minipig that describes ‘How to’ and welfare, health, and husbandry; practical dosing and examination ‘Why’ and ’What to expect under procedures; surgical techniques; and all areas of toxicity testing and normal circumstances’, combining the uses of the minipig as a disease model. Regulatory aspects of enthusiasm and experience with a minipig use are also considered. critical assessment of the minipig’s values and potential problems. The reader will find extensive theoretical and practical information in the pharmacology, ADME and toxicology chapters, which will Further details about the book and help scientists and managers in deciding which species to use in how to order it are available at the basic research; drug discovery and pharmacology; and toxicology CRC Press website: www.crcpress. studies of chemicals, biotechnology products and devices. The com.

24 Newsletter 36 Autumn 2011