The Contribution of Farm Animals to Human Health

Review TRENDS in Biotechnology Vol.22 No.6 June 2004

The contribution of farm animals to human health

Wilfried A. Kues and Heiner Niemann

Department of Biotechnology, Institut fu¨ r Tierzucht, Mariensee, D-31535 Neustadt, Germany

Farm animals and their products have a longstanding history. The pioneering work of Edward Jenner with and successful history of providing significant contri- cowpox in the 18th century paved the way for modern butions to human nutrition, clothing, facilitation of vaccination programs against smallpox, as well as other labour, research, development and medicine and have human and animal plagues. To date, more than 250 million thus been essential in improving life expectancy and people have benefited from drugs and vaccines produced human health. With the advent of transgenic technol- by recombinant technologies in bacteria and various types ogies the potential of farm animals for improving of mammalian cells and many more will benefit in the human health is growing and many areas remain to be future (New Medicines in Development for Biotechnology, explored. Recent breakthroughs in reproductive tech- 2002; www.phrma.org/newmedicines/biotech/). Further nologies, such as somatic cloning and in vitro embryo examples of the significant contribution of farm animals production, and their merger with molecular genetic to human health are the longstanding use of bovine and tools, will further advance progress in this field. Here, porcine insulin for treatment of diabetes as well as horse we have summarized the contribution of farm animals antisera against snake venoms and antimicrobial pep- to human health, covering the production of antimicro- tides. In addition, farm animals are models for novel bial peptides, dietary supplements or functional foods, surgical strategies, testing of biodegradable implants and animals used as disease models and the contribution of sources of tissue replacements, such as skin and heart animals to solving urgent environmental problems and valves. challenges in medicine such as the shortage of human Progress in transgenic technologies has allowed the cells, tissues and organs and therapeutic proteins. generation of genetically modified large animals for Some of these areas have already reached the level of applications in agriculture and biomedicine, such as the preclinical testing or commercial application, others production of recombinant proteins in the mammary gland will be further advanced only when the genomes of the and the generation of transgenic pigs with expression of animals concerned have been sequenced and anno- human complement regulators in xenotransplantation tated. Provided the necessary precautions are being research [1]. Further promising application perspectives taken, the transmission of pathogens from animals to will be developed when somatic cloning with genetically humans can be avoided to provide adequate security. modified donor cells is further improved and the genomes Overall, the promising perspectives of farm animals and of farm animals are sequenced and annotated. The first their products warrant further research and develop- transgenic livestock were born less than 20 years ago with ment in this field. the aid of microinjection technology [2]. Recently the first animals with knockout of one or even two alleles of a Farm animals have made significant contributions to targeted gene were reported (Table 1). Somatic nuclear human health and well-being throughout mankind’s transfer has been successful in 10 species, but the overall

Table 1. Milestones (live offspring) in transgenesis and reproductive technologies in farm animals

Year Milestone Strategy Refs 1985 First transgenic sheep and pigs Microinjection of DNA into one pronucleus of a zygote [2] 1986 Embryonic cloning of sheep Nuclear transfer using embryonic cells as donor cells [91] 1997 Cloning of sheep with somatic donor cells Nuclear transfer using adult somatic donor cells [92] 1997 Transgenic sheep produced by nuclear transfer Random integration of the construct [93] 1998 Transgenic cattle produced from fetal ﬁbroblasts and nuclear transfer Random integration of the construct [94] 1998 Generation of transgenic cattle by MMLV injection Injection of oocytes with helper viruses [95] 2000 Gene targeting in sheep Gene replacement and nuclear transfer [96] 2002 Trans-chromosomal cattle Additional artiﬁcial chromosome [15] 2002 Heterozygous knockout in pigs One allele of a-galactosyl-transferase knocked out [34,35] 2003 Homozygous gene knockout in pigs Both alleles of a-galactosyl-transferase knocked out [36] 2003 Transgenic pigs via lentiviral injection Gene transfer into zygotes via lentiviruses [97]

Corresponding author: Heiner Niemann ([email protected]). www.sciencedirect.com 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.04.003 Review TRENDS in Biotechnology Vol.22 No.6 June 2004 287

Table 2. Efﬁciency of somatic cloning of mammals

Species Number of animalsa % Viable offspring Comments Cattle ,3000 15–20 Up to 30% of the cloned calves showed abnormalities, such as increased birth weight Sheep ,400 5–8 Same problems as with cattle clones Goat ,400 3 Minor health problems reported Mouse ,300 ,2 Some adult mice clones showed obesity and a reduced life span Pig ,200 ,1 Some cloned piglets had reduced birth weights Cat 1 ,1 Rabbit 6 ,1 Mule 1 ,1 Horse 1 ,1 Rat 2 ,1 aEstimated total numbers of mammals derived from somatic cloning since 1997. efficiency is low and few cloned offspring have been born correct glycosylation patterns and post-translational worldwide (Table 2). Compared with microinjection of modifications, low running costs, rapid propagation of DNA constructs into pronuclei of zygotes, somatic nuclear the transgenic founders and high expression stability. transfer is superior for the generation of transgenic These attractive perspectives led to the development of the animals (Table 3). ‘gene pharming’ concept, which has been advanced to the Here, we have summarized the contribution of farm level of commercial application [5]. The most promising animals to human health covering (i) the production of site for production of recombinant proteins is the mam- pharmaceuticals; (ii) production of xenografts for over- mary gland, but other body fluids including blood, urine coming the severe shortage of human organs and tissues; and seminal fluid have also been explored [6]. The (iii) the use of farm animals as disease models; (iv) the mammary gland is the preferred production site mainly production of dietary supplements or functional foods; and because of the quantities of protein that can be produced (v) the contribution of farm animals to solving environ- and the ease of extraction or purification of the respective mental problems. protein. Based on the assumption of average expression levels, Farm animals for pharmaceutical production daily milk volumes and purification efficiency, 5400 cows Gene ‘pharming’: production of recombinant human would be needed to produce the 100 000 kg of human proteins in the mammary gland of transgenic animals serum albumin (HSA) that are required per year world- The conventional production of rare human therapeutic wide, 4500 sheep would be required for the production of proteins from blood or tissue extracts is an inefficient, 5000 kg a-antitrypsin (a-AT), 100 goats for 100 kg of expensive, labour and time consuming process, which in monoclonal antibodies, 75 goats for the 75 kg of antith- addition bears the risk of contamination with human rombin III (ATIII) and two pigs to produce 2 kg human pathogens. The production of human therapeutic proteins clotting factor IX. All these values are calculated on a by recombinant bacteria or cell cultures has alleviated yearly basis [3]. these problems and has made several therapeutic proteins Large amounts of numerous heterologous recombinant available for patients. However, these recombinant sys- proteins have been produced by targeting expression to the tems have several limitations. They are only suitable for mammary gland via mammary gland-specific promoter ‘simple’ proteins, the amount of protein produced is elements. Proteins were purified from the milk of limited, and post-translational modifications are often transgenic rabbits, pigs, sheep, goats and cattle. The incorrect leading to immune reactions against the protein. biological activity of the recombinant proteins was In addition, the technical prerequisites are challenging assessed and therapeutic effects have been characterized and production costs are high. [3,7]. Products such as ATIII, a-AT or tissue plasminogen Farm animals such as cattle, sheep, goats, pigs and activator (tPA) are advanced to clinical trials (Table 4) [5]. even rabbits [3,4] have several significant advantages for Phase III trials for ATIII have been completed and the the production of recombinant proteins over other sys- protein is expected to be on the market within the next 2–3 tems, including their potential for large-scale production, years. In February 2004 an application was submitted to

Table 3. Advantages and disadvantages of two gene transfer methodologiesa

Microinjection Somatic nuclear transfer Integration efﬁciency þþþþ Integration site Random Random or targeted Gene deletion 2 þþþ Construction size .50 kb (Artiﬁcial chromosomes) , 30–50 kb Technical feasibility Technically demanding Technically demanding Mosaicism þþþ 2 Expression screen in vitro þþþþ Expression pattern Variable Controlled, consistent Multi-transgenics þþþþ aAbbreviations: 2, not possible; þ, weak advantage; þþ, moderate advantage; þþþ, strong advantage www.sciencedirect.com 288 Review TRENDS in Biotechnology Vol.22 No.6 June 2004

Table 4. Proteins produced in the mammary gland of transgenic farm animalsa

Protein Developmental phase Production species Therapeutic application Potential market introduction date AT III Phase III Goat Genetic heparin resistance 2005 TPA Phase II/III Goat Dissolving coronary clots . 2006 a-AT Phase II/III Goat and/or sheep Lung emphysema . 2007 Cystic fibrosis hFVIII Experimental Sheep Hemophilia A . 2008 HAS Phase I Cattle Blood substitute . 2008 Various antibodies Phase I/II Goat . 2007 a Abbreviations: a-AT, a-A1-antitrypsin; AT III, antithrombin III; hFVIII, human clotting factor VIII; HSA, human serum albumin; TPA, tissue plasminogen activator the European Market Authorization to allow Atrynw, the chromosome (HAC) containing the entire sequences of recombinant ATIII from the milk of transgenic dairy goats, the human immunoglobulin heavy and light chain loci to enter the market as a fully registered drug. The enzyme has been introduced into bovine fibroblasts, which a-glucosidase from the milk of transgenic rabbits has were then used in nuclear transfer. Transchromosomal Orphan drug registration and has been successfully used offspring were obtained that expressed human for the treatment of Pompe’s disease [8]. This is a rare immunoglobulin in their blood. This system could be glycogen storage disorder, which is fatal in children under a significant step forward in the production of human 2 years and currently application with recombinant therapeutic polyclonal antibodies [15]. Further studies a-glucosidase is the only way to treat this metabolic defect. will show whether the additional chromosome will be Biologically active human lactoferrin has been produced in maintained over future generations and how stable large amounts in the mammary glands of transgenic cows expression will be. and will probably be developed as a biopharmaceutical for prophylaxis and treatment of infectious diseases [9]. Production of a new class of antibiotics: cationic anti- Guidelines developed by the Food and Drug Adminis- microbial peptides tration (FDA) of the USA require monitoring of the With increasing antibiotic resistance in bacterial species, animals’ health, validation of the gene construct, charac- there is a growing need to develop new classes of anti- terization of the isolated recombinant protein, as well as microbial agents. Cationic anti-microbial peptides (AMP) performance of the transgenic animals over several have many of the desired features [16] because they generations. This has been taken into account when possess a broad spectrum of activity, kill gram-positive and developing ‘gene pharming’, for example by using only gram-negative bacteria rapidly, are unaffected by classical animals from prion disease-free countries (New Zealand) resistance genes and are active in animal models [17–19]. and keeping the animals in very hygienic conditions. AMPs belong to the innate immune defense, which acts as Successful drug registration of Atrynw will demonstrate a first barrier ahead of humoral and cellular immune the usefulness and solidity of this approach and will systems, and neutralizes bacteria by interacting specifi- accelerate registration of further products from this cally with their cell membranes. Their low transmem- process, as well as stimulate research and commercial brane potential of ,-100 mV, and the abundant anionic activity in this area. phospholipids are essential for this selective interaction. It When considering the ‘gene pharming’ concept, one has is proposed that AMPs physically disintegrate the cell to bear in mind that not every protein can be expressed at membrane, and in addition can interact with several the desired levels. Erythropoietin (EPO) could not be intracellular target molecules [16]. More than 500 such expressed in the mammary gland of transgenic cattle [10] peptides have been discovered in plants, insects, inverte- and was even detrimental to the health of rabbits brates, fish, amphibians, birds and mammals [16–20]. transgenic for EPO [11]. We have shown that human AMPs from livestock species would be superior anti- clotting factor VIII (hFVIII) cDNA constructs can be microbial drugs because they would lack cytotoxic effects expressed in the mammary gland of transgenic mice, that were found for insect peptides; the evolution of rabbits and sheep [1,12]. However, the yields of biologically resistance would not affect the human specific innate active recombinant hFVIII protein from ovine milk were immunity [20]. low because hFVIII was rapidly sequestered into ovine Prominent examples of cationic anti-microbial peptides milk. [13]. These results show that the technology needs from farm animals already in advanced clinical trials further improvements to achieve high-level expression (Phases II–III) are Iseganan (derived from protegrin-1 with large genes having complex regulation, such as that peptide of pig leucocytes; Intrabiotics; http://intrabiotics. coding for hFVIII, although higher levels of hFVIII have com/) and MBI-594 (similar to indolicidin peptide from been reported in transgenic swine [14]. With the advent of bovine neutrophils; Micrologix, http://mbiotech.com/). To transgenic crops that produce pharmacologically active date, there are no documented cases of antimicrobial proteins, there is an array of recombinant technologies peptide-resistance for AMPs and combinatorial available that will allow the most appropriate production approaches in peptides (20 possible amino acids in each system for a specific protein to be targeted. position) provide great potential for rational drug design An interesting new development is the generation of [21]. Recombinant production will keep the production transchromosomal animals (Table 1). A human artificial costs low. www.sciencedirect.com Review TRENDS in Biotechnology Vol.22 No.6 June 2004 289

Xenotransplantation of porcine organs to human occurs within seconds or minutes. In the case of a patients discordant organ (e.g. in transplanting from pig to Solid organs human) naturally occurring antibodies react with anti- Today .250 000 people are alive only because of the genic structures on the surface of the porcine organ and successful transplantation of an appropriate human organ induce HAR by activating the complement cascade via the (allotransplantation). On average, 75–90% of patients antigen–antibody complex. Ultimately, this results in the survive the first year after transplantation and the formation of the membrane attack complex (MAC). average survival of a patient with a transplanted heart, However, the complement cascade can be shut down at liver or kidney is 10–15 years. This progress in organ various points by expression of regulatory genes that transplantation technology has led to an acute shortage of prevent the formation of the MAC. Regulators of the appropriate organs, and cadaveric or live organ donation complement cascade are CD55 (decay accelerating factor, cannot cover the demand in western societies. The 2001 DAF), CD46 (membrane cofactor protein, MCP) or CD59. figures from the United Network for Organ Sharing (www. MAC disrupts the endothelial cell layer of the blood unos.org) in the USA show that the ratio of patients with vessels, which leads to lysis, thrombosis, loss of vascular organ transplantation to those on the waiting list is ,1:4 integrity and ultimately to rejection of the transplanted (Table 5). A similar ratio is found in other countries such as organ [24]. the UK, France and Germany. A new person is added to the Induced xenoreactive antibodies are thought to be waiting list every 14 minutes. This has led to the sad and responsible for AVR, which occurs within days of a ethically challenging situation in which several thousand transplantation of a xenograft; disseminated intravascu- patients who could have survived if appropriate organs lar coagulation (DIC) is a predominant feature of AVR. had been available die every year. Despite severe immunosuppressive treatment, a disturbed To close the growing gap between demand and thrombocyte function and DIC were observed in a pig-to- availability of appropriate organs, porcine xenografts are primate xenotransplant model [25,26]. The endothelial cells of the graft’s microvasculature loose their anti- considered the solution of choice [22,23]. Today the thrombic properties, attract leucocytes, monocytes and domesticated pig is considered the optimal donor animal platelets leading to anemia and organ failure. The because (i) the organs are similar in size to human organs; underlying mechanism for DIC and thrombotic micro- (ii) porcine anatomy and physiology are not too different angiopathy is thought to be activation of the endothelial from that of humans; (iii) pigs have short reproduc- cells attributed to incompatibilities between human and tion cycles and large litters; (iv) pigs grow rapidly; porcine coagulation factors [25]. At least three incompat- (v) maintenance of high hygienic standards is possible at ibilities between human and porcine coagulation systems relatively low costs; and (vi) transgenic techniques for have been identified; the first is the failure of porcine modifying the immunogenicity of porcine cells and organs thrombomodulin (TM) to activate human anticoagulant are well established. protein C, the second is that the porcine tissue factor The process of generating and evaluating transgenic pathway inhibitor fails to inhibit human clotting factor Xa pigs as potential donors for xenotransplants involves a and the third is that porcine von Willebrand factor (vWF) variety of complex steps and is time-, labour- and resource- binds and activates human platelets [26]. Human throm- intensive. bomodulin (hTM) and heme-oxygenase 1 (hHO-1) are Essential prerequisites for successful xenotransplanta- crucially involved in the etiology of DIC and might be good tion are: targets for future transgenic studies to improve long-term (i) Overcoming the immunological hurdles. survival of porcine xenografts by creating multi-trans- (ii) Prevention of transmission of pathogens from the genic pigs. donor animal to the human recipient. The cellular rejection occurs within weeks after (iii) Compatibility of the donor organs with the human transplantation. In this process the blood vessels of the organ in terms of anatomy and physiology. transplanted organ are damaged by T-cells, which invade The immunological obstacles in a porcine-to-human the intercellular spaces and destroy the organ. This xenotransplantation are the hyperacute rejection rejection is observed after allotransplantation and is response (HAR), acute vascular rejection (AVR), cellular normally suppressed by life-long administration of immu- rejection and potentially chronic rejection [24]. The HAR nosuppressive drugs [24]. When using a discordant donor species such as the pig, Table 5. Overview of transplanted organs and demand for overcoming the HAR and AVR are the preeminent goals. a organ transplantation (USA) The most promising strategy for overcoming the HAR is Type of transplant Transplantations in 2001 Waiting patients the synthesis of human complement regulatory proteins Kidney 14 152 52 772 (RCAs) in transgenic pigs [22,23,27,28]. Following trans- Liver 5177 17 520 plantation, the porcine organ would produce the comp- Pancreas 468 1318 lement regulatory protein and can thus prevent the Kidney/Pancreas 884 2520 complement attack of the recipient. Pigs transgenic for Heart 2202 4163 Heart/Lung 27 209 DAF or MCP have been generated by microinjection of Lung 1054 3799 DNA constructs into pronuclei of zygotes. Hearts and Total 23 964 79 845 kidneys from these animals have been transplanted either aData from United Network for Organ Sharing, 2001 (www.unos.org). heterotopically, (in addition to the recipient’s own organ) or www.sciencedirect.com 290 Review TRENDS in Biotechnology Vol.22 No.6 June 2004

Table 6. Success rates of RCA-transgenic porcine organs after transplantation to primate recipientsa

RCA Organ/kind of transplant Recipient Immuno-suppression Survival (days) hDAF Heart/heterotropic Cynomologus þþþ , 135 hDAF Heterotopic Cynomologus þþ , 90 hDAF Orthopic Baboon þþþ , 28 hDAF Kidney/orthotopic Cynomologus þþ , 90 hCD59 Kidney/orthotopic, heterotopic Cynomologus þþþ , 20 hCD46 Heart, heterotopic Baboon þþ , 23 aAbbreviations: þ, weak immunosuppression; þþ, moderate immunosuppression; þþþ, heavy immunosuppression; RCA, regulator of complement activity orthotopically (life supportive) into non-human primates. that transgenic pigs will be available as organ donors Survival rates reached 23–135 days with porcine xeno- within the next five to ten years. Guidelines for the clinical grafts transgenic for one of the two complement regula- application of porcine xenotransplants are already avail- tors; survival rates were heavily affected by the strength of able in the USA and are currently being developed in the immune-suppressive protocol (Table 6) [29–31]. several other countries. Similarly, transgenic expression of hCD59 was compatible The ethical challenges of xenotransplantation have with an extended survival of porcine hearts in a perfusion been a matter of a worldwide intensive debate. A general model or following transfer into primates [32,33]. These consensus has been reached that the technology is data show that HAR can be overcome in a clinically ethically acceptable provided the individual well-being acceptable manner by expressing human complement does not compromise public health by producing and regulators in transgenic pigs [22]. transmitting new pathogens. Economically xenotrans- Another promising strategy towards successful xeno- plantation might be viable if the enormous costs caused transplantation is the knockout of the antigenic structures by patients suffering from severe kidney disease, needing on the surface of the porcine organ that cause HAR. These dialysis or those suffering from chronic heart diseases structures are known as 1,3-a-gal-epitopes and are could be avoided by a functional kidney or heart xenograft. primarily produced by activity of 1,3-a-galactosyltransferase (a-gal). Piglets in which one allele of a-gal locus had Xenogenic cells and tissue been knocked out by homologous recombination in Another promising area of application for transgenic primary donor cells that were employed in nuclear animals will be the supply of xenogenic cells and tissue. transfer were recently generated [34,35]. The birth of Several intractable diseases, disorders and injuries are four healthy piglets with disruption of both allelic loci for associated with irreversible cell death and/or aberrant a-gal has meanwhile also been published. Applying toxin A cellular function. Despite numerous attempts, primary from Clostridium difficile to cells that already carried one human cells cannot yet be expanded well enough in deleted a-gal allele selected a cell clone, which carried an culture. In the future, human embryonic stem cells could inactivating point mutation on the second allele. This cell be a source for specific differentiated cell types that can be clone was then used in nuclear transfer [36]. The used in cell therapy [48,49]. Xenogenic cells, in particular usefulness of these animals for xenotransplantation has from the pig, hold great promise for successful cell recently been reported [37]. therapies for human patients because cells can be Further improvements in the success of xenotransplan- implanted at the optimal therapeutic location (i.e. immu- tation will arise from the possibility of inducing a noprivileged sites, such as the brain), genetically or permanent tolerance across xenogenic barriers [38,39].A otherwise modified before transplantation to enhance particularly promising strategy for long-term graft accep- cell function, banked and cryopreserved, or combined tance is the induction of a permanent chimerism via with different cell types in the same graft [50]. intraportal injection of embryonic stem cells [40]. Xenogenic cell therapy has been advanced to preclinical Prevention of transmission of zoonoses from the donor studies. Porcine islet cells have been transplanted to animal to the human recipient is crucial for clinical diabetic patients and were shown to be at least partially application of porcine xenografts. This aspect gained functional over a limited period of time [51]. Porcine fetal particular significance when a few years ago it was neural cells were transplanted into the brain of shown that porcine endogenous retroviruses (PERV) can patients suffering from Parkinson’s and Huntington’s be produced by porcine cell lines and can even infect disease [52,53]. In a single autopsied patient the graft human cell lines in vitro [41]. However, until today no survived for .7 months and the transplanted cells formed infection has been found in patients that had received dopaminergic neurons and glial cells. Pig neurons various forms of living porcine tissues (e.g. islet cells, extended axons from the graft site into the host brain insulin, skin, extracorporal liver) for up to 12 years [42]. [52]. Further examples for the potential use of porcine Recent intensive research has shown that porcine neural cells are in cases of stroke and focal epilepsy [54]. endogenous retroviruses probably do not present a risk Olfactory ensheathing cells (OECs) or Schwann cells for recipients of xenotransplants provided all necessary derived from hCD59 transgenic pigs promoted axonal precautions are taken [43–46]. In addition, a strain of regeneration in rat spinal cord lesion [55]. Thus, cells from miniature pigs has been identified that does not produce genetically modified pigs could restore electrophysiologi- infective PERV [47]. Although xenotransplantation poses cally functional axons across the site of a spinal cord numerous further challenges to research, it is expected transsection. Xenogenic porcine cells could also be useful www.sciencedirect.com Review TRENDS in Biotechnology Vol.22 No.6 June 2004 291 as a novel therapy for liver diseases. On transplantation of prion locus has been reported; however the cloned lambs porcine hepatocytes to Watanabe heritable hyperlipidemic carrying the kockout locus died several days after birth (WHHL) rabbits (a model for familial hypercholesterole- [65]. Prion knockout animals could be an appropriate mia) the xenogenic cells migrated out of the vessels and model for studying the epidemiology of spongiform integrated into the hepatic parenchyma. The integrated encephalopathies in humans and are crucial for porcine hepatocytes provided functional low density developing strategies to eliminate prion carriers from a lipoprotein (LDL) receptors and thus reduced cholesterol farm animal population. levels by 30–60% for at least 100 days [56]. The pig could be a useful model to study defects of A clone of bovine adrenocortical cells restored adrenal growth hormone releasing hormone (GHRH), which is function upon transplantation to adrenalectomized severe characterized by a variety of conditions such as Turner combined immunodeficient (SCID) mice indicating that syndrome, hypochrondroplasia, Crohn’s disease, intra- functional endocrine tissue can be derived from a single uterine growth retardation or renal insufficiency. Appli- somatic cell [57]. Bovine neuronal cells were collected from cation of recombinant GHRH in an injectable form and its transgenic fetuses, and when transplanted into the brain myogenic expression has been shown to alleviate these of rats resulted in significant improvements in symptoms problems in a porcine model [66]. of Parkinson’s disease [58]. Furthermore, xenotransplan- An important aspect of large animal models for human tation of retinal pigment epithelial cells holds promise for diseases is the recent finding that somatic cloning per se treating retinal diseases such as macular degeneration, does not result in shortening of the telomeres. Telomeres which is associated with photoreceptor losses. Porcine or are highly repetitive DNA sequences at the end of the bovine fetal cardiomyocytes or myoblasts might provide a chromosomes that are crucial for their structural integrity therapeutic approach for the treatment of ischemic heart and function and are thought to be related to lifespan. disease. Similarly, xenogenic porcine cells might be Telomere shortening is usually correlated with severe valuable for the repair of skin or cartilage damage [50]. limitations of the regenerative capacity of cells, the onset In light of the emergence of significantly improved of cancer, ageing and chronic disease with significant protocols for genetic modification of donor animals and impact on human lifespan [67–70]. Expression of the new powerful immunosuppressive drugs xenogenic cell enzyme telomerase, which is primarily responsible for the therapy will evolve as an important therapeutic option for formation and rebuilding of telomeres, is suppressed in the treatment of human diseases. most somatic tissues postnatally. Although telomeres in cloned sheep (Dolly) derived from epithelial cells were Farm animals as models for human diseases shorter than those of naturally bred age matched control In mouse genetics the generation of knockout animals is a animals, telomere lengths in cloned mice and cattle were standard procedure and several thousand strains carrying not different from those determined in age matched gene knockouts or transgenes have been developed [Mouse controls even when senescent donor cells had been Knockout and Mutation Database (MKMD); http:// employed [71–75]. Recent studies in our laboratory have research.bmn.com/mkmd]. Models have been developed revealed that telomere length is established already early for several human diseases. However, mouse physiology, in preimplantation development by a specific genetic anatomy and life span differ significantly from those in programme and is dependent on telomerase activity [76]. humans, making the rodent model inappropriate for several human diseases. Farm animals, such as pigs, Dietary modifications of animal products sheep or even cattle could be more appropriate models to Application of gene and biotechnology for nutrition and study human diseases in particular non-insulin-depen- biomedicine is more developed for plants than farm dent diabetes, cancer and neurodegenerative disorders, animals [77]. The term nutriceuticals in the farm animal which require longer observation periods than those context means that gene and biotechnology are used to possible in mice [59–61]. With the aid of the microinjection enhance farm animal products to improve diet and have technology an important pig model for the rare human eye concomitant medical applications. Because these products disease Retinitis pigmentosa (PR) has been developed [62]. have a proven pharmacological effect on the body, they are Patients with PR develop night blindness early in life regarded as ‘drugs’ and must be tested for efficacy and attributed to a loss of photoreceptors. The transgenic pigs safety. Functional foods are those designed to provide express a mutated rhodopsin gene and show a great specific and beneficial physiological effects on human similarity with the human phenotype. Treatment models health and welfare and should prevent diet-related with value for human patients are being developed [63]. diseases. Functional foods from animals could be used to The development of the somatic cloning technology and lower cholesterol levels in an effort to battle cardiovascular the merger with targeted genetic modifications and diseases, to reduce high blood pressure by adding conditional gene expression will enhance the possibilities angiotensin converting enzyme (ACE) inhibitors or to for creating useful models for human diseases in large increase immunity by adding specific immunostimulatory animals. A good example is the knockout of the prion gene peptides [78]. However, data showing that nutriceuticals that would make sheep and cattle non-susceptible to are really beneficial for human health are rare. spongiform encephalopathies (scrapie and BSE). Mice Regarding the production of improved quality animal models showed that the knockout of the prion protein is the products, interesting observations have been made in only secure way to prevent infection and transmission of several beef cattle breeds, such as Belgian Blue and the disease [64]. The first successful targeting of the ovine Piedmontese. These breeds were accidentally bred for www.sciencedirect.com 292 Review TRENDS in Biotechnology Vol.22 No.6 June 2004 mutations of the myostatin gene, which renders it non- environmental pollution. These pigs carry a bacterial functional or less functional than the wild-type gene [79]. phytase gene under the transcriptional control of a The mutated genes cause muscle hypertrophy and led to salivary gland specific promoter, which allows the pigs to improved meat quality. This observation makes targeted digest plant phytate. Without the bacterial enzyme, the modifications of the myostatin gene an interesting option phytate phosphorus passes undigested into manure and for the meat industry. A diet rich in non-saturated fatty pollutes the environment. With the bacterial enzyme, the acids is correlated with a reduced risk of stroke and fecal phosphorus output was reduced up to 75% [86]. These coronary diseases. One transgenic approach to this is the environmentally friendly pigs are expected to enter the generation of pigs with increased amounts of non- commercial production chains within the next few years. saturated fatty acids. Pigs producing a higher ratio of unsaturated versus saturated fatty acids in their muscles Precautions and perspectives are currently under development in Japan. Throughout mankind’s history farm animals have made An attractive example for targeted genetic modification significant contributions to human health and well-being. could be dairy production [80,81]. Apart from conventional The convergence of the recent advances in reproductive dairy products, it could be possible to produce fat-reduced technologies with the tools of molecular biology opens a or even fat-free milk or milk with a modified lipid new dimension for this area [1]. Major prerequisites will be composition via modulation of enzymes involved in lipid the continuous refinement of reproductive biotechnologies metabolism; to increase curd and cheese production by and a rapid completion of livestock genome sequencing enhancing expression of the casein gene family in the and annotation. The technology developed in the decipher- mammary gland; to create ‘hypoallergenic’ milk by knock- ing of the human genome will improve and accelerate out of the b-lactoglobulin gene; to generate lactose-free sequencing of genomes from livestock [87]. We anticipate milk via knockout of the a-lactalbumin locus that is the genetically modified animals will play a significant role in key molecule in milk sugar synthesis; to produce ‘infant the biomedical arena, in particular via the production of milk’ in which human lactoferrin is abundantly available valuable pharmaceutical proteins and the derivation of or to produce milk with a highly improved hygienic xenografts, within the next 5–7 years. Agricultural standard via an increased level of lysozyme or other application might be further away (.10 years) given the anti-microbiological substances in the udder. Lactose- complexity of some of the economically important traits reduced or lactose-free milk could make dairy products and the public skepticism of genetic modification related to suitable for consumption by a large proportion of the food production [1]. world’s population who do not possess an active lactase A crucial aspect of animal-derived products is the enzyme in their gut system. However, one has to bear in prevention of transmission of pathogens from animals to mind that lactose is the main osmotically active substance humans. This requires sensitive and reliable diagnostic in milk and a lack thereof could interfere with milk and screening methods for the various types of pathogenic secretion. A lactase construct has been expressed in the organisms. The recent findings (see above) that the risk of mammary gland of transgenic mice and reduced lactose PERV transmission is negligible are promising and show contents by 50–85% without altering milk secretion [82]. that with targeted and intensive research such important However, mice with a homozygous knockout for a- questions can be answered within a limited period of time, lactalbumin could not feed their offspring because of the paving the way for preclinical testing of xenografts. high viscosity of the milk [83]. These diverging findings Furthermore, it should be kept in mind that the biomedical demonstrate the feasibility of obtaining significant altera- applications of farm animals will require strict standards tions of milk composition by applying the appropriate of ‘genetic security’ and reliable and sensitive methods for strategy. the molecular characterization of the products. A major The physicochemical properties of milk are mainly affected by the ratio of casein variants. Therefore, casein is contribution towards the goal of well-defined products will a prime target for the improvement of milk composition. come from array technology (cDNA, peptide or protein Mouse models have been developed for most of the above arrays), which establishes ‘fingerprint’ profiles at the modifications indicating the feasibility of obtaining sig- transcriptional and/or protein level [88,89]. Meanwhile nificant alterations in milk composition but at the same improvements of RNA isolation and unbiased amplifica- time showing that unwanted side effects cannot be ruled tion of tiny amounts of mRNA (picogram) enable research- out [83,84]. Only one full-scale study in livestock has been ers to analyse RNA from single embryos [90]. With the aid reported as yet [85]. The recent report showed that the of this technology one can gain in-depth insight into the casein ratio can be altered by overexpression of b-and proper functioning of a transgenic organism and thereby k-casein in cattle clearly underpinning the potential for ensure the absence of unwanted side effects [88,89]. This improvements in the functional properties of bovine would also be required to maintain the highest possible milk [85]. levels of animal welfare in cases of genetic modification.

Towards environmentally friendly farm animals References Phosphorus pollution by animal production is a serious 1 Niemann, H. and Kues, W.A. (2003) Application of transgenesis on livestock for agriculture and biomedicine. Anim. Reprod. Sci. 79, problem in agriculture and excess phosphate from manure 291–317 promotes eutrophication. Phytase transgenic pigs have 2 Hammer, R.E. et al. (1985) Production of transgenic rabbits, sheep and been developed to address the problem of manure-based pigs by microinjection. Nature 315, 680–683 www.sciencedirect.com Review TRENDS in Biotechnology Vol.22 No.6 June 2004 293

3 Rudolph, N.S. (1999) Biopharmaceutical production in transgenic system for the study of discordant xenotransplantation. Transplan- livestock. Trends Biotechnol. 17, 367–374 tation 71, 132–142 4Bo¨sze, Zs. et al. (2003) The transgenic rabbit as model for human 31 Zaidi, A. et al. (1998) Life-supporting pig to primate renal xeno- diseases and as a source of biologically active recombinant proteins. transplantation using genetically modified donors. Transplantation Transgenic Res. 12, 541–553 65, 1584–1590 5 Ziomek, C.A. (1998) Commercialization of proteins produced in the 32 Fodor, W.L. et al. (1994) Expression of a functional human complement mammary gland. Theriogenology 49, 139–144 inhibitor in a transgenic pig as a model for the prevention of 6 Dyck, M.K. et al. (2003) Making recombinant proteins in animals – xenogeneic hyperacute organ rejection. Proc. Natl. Acad. Sci. U. S. A. different systems, different applications. Trends Biotechnol. 21, 91, 11153 – 11157 394–399 33 Niemann, H. et al. (2001) CMV early promoter induced expression of 7 Meade, H.M. et al. (1999) Expression of recombinant proteins in the hCD59 in porcine organs provides protection against hyperacute milk of transgenic animals. In: Gene Expression Systems. Fernandez, rejection. Transplantation 72, 1898–1906 J.M., Hoeffler, J.P., (eds.) Academic Press, San Diego, U. S. A., pp. 399– 34 Dai, Y. et al. (2002) Targeted disruption of the alpha1,3-galactosyl- 427 transferase gene in cloned pigs. Nat. Biotechnol. 20, 251–255 8 Van der Hout, J.M.P. et al. (2001) Enzyme therapy for Pompe disease 35 Lai, L. et al. (2002) Production of alpha-1,3-galactosyltransferase with recombinant human a-glucosidase from rabbit milk. J. Inherit. knockout pigs by nuclear transfer cloning. Science 295, 1089–1092 Metab. Dis. 24, 266–274 36 Phelps, C.J. et al. (2003) Production of alpha 1,3-galactosyltransferase- 9 van Berkel, P.H. et al. (2002) Large scale production of recombinant deficient pigs. Science 299, 411–414 human lactoferrin in the mik of trangenic cows. Nat. Biotechnol. 20, 37 Yamada, K. et al. (2003) An initial report of alpha-gal deficient pig-to- 484–487 baboon renal xenotransplantation: evidence for the benefit of co- 10 Hyttinen, J.M. et al. (1994) Generation of transgenic dairy cattle from transplanting vascularized donor thymic tissue. Xenotransplantation transgene-analyzed and sexed embryos produced in vitro. Biotechnol- 10, 480 ogy (N. Y.) 12, 606–608 38 Greenstein, J. and Sachs, D.H. (1997) The use of tolerance for 11 Massoud, M. et al. (1996) The deleterious effects of human erythro- transplantation across xenogeneic barriers. Nat. Biotechnol. 15, poietin gene driven by the rabbit whey acidic protein gene promoter in 235–238 transgenic rabbits. Reprod. Nutr. Dev. 36, 555–563 39 Auchincloss, H. Jr and Sachs, D.H. (1998)) Xenogenic transplantation. 12 Hiripi, L. et al. (2003) Expression of active human blood clotting factor Annu. Rev. Immunol. 16, 433–470 ¨ VIII in the mammary gland of transgenic rabbits. DNA Cell Biol. 22, 40 Fandrich, F. et al. (2002) Preimplantation-stage stem cells induce long- term allogeneic graft acceptance without supplementary host con- 21–25 ditioning. Nat. Med. 8, 171–177 13 Niemann, H. et al. (1999) Expression of human blood clotting factor 41 Patience, C. et al. (1997) Infection of human cells by an endogenous VIII in the mammary gland of transgenic sheep. Transgenic Res. 8, retrovirus of pigs. Nat. Med. 3, 282–286 237–247 42 Paradis, K. et al. (1999) Search for cross-species transmission of 14 Paleyanda, R.K. et al. (1997) Transgenic pigs produce functional porcine endogenous retrovirus in patients treated with living pig human factor VIII in milk. Nat. Biotechnol. 15, 971–975 tissue. Science 285, 1236–1241 15 Kuroiwa, Y. et al. (2002) Cloned transchromosomic calves producing 43 Patience, C. et al. (1998) No evidence of pig DNA of retroviral infection human immunoglobulin. Nat. Biotechnol. 20, 889–894 in patients with short-term extracorporal connection of pig kidneys. 16 Hancock, R.E.E. and Scott, M.G. (2000) The role of antimicrobial Lancet 352, 699–701 peptides in animal defense. Proc. Natl. Acad. Sci. U. S. A. 97, 44 Dinsmore, L.E. et al. (2000) No evidence for infection of human cells 8856–8861 with porcine endogenous retrovirus (PERV) after exposure to porcine 17 Goldstein, B.P. (1998) Activity of nisin against Streptococcus pneuno- fetal neuronal cells. Transplantation 70, 1382–1389 niae, in vitro, and in a mouse infection model. J. Antimicrob. 45 Switzer, W.M. et al. (2001) Lack of cross-species transmission of Chemother. 42, 277–278 porcine endogenous retrovirus infection to nonhuman primate 18 Gamelli, R. et al. (1998) Improvement in survival with peptidyl recipients of porcine cells, tissues and organs. Transplantation 71, membraned interactive molecule D4B treatment after burn wound 959–965 infection. Arch. Surg. 133, 715–720 46 Martin, U. et al. (2002) Absence of PERV specific humoral immune 19 Kirikae, T. et al. (1998) Protective effects of a human 18-kilodalton response in baboons after transplantation of porcine cells of organs. cationic antimicrobial protein (CAP18)-derived peptide against mur- Transplant. Int. 15, 361–368 ine endotoxemia. Infect. Immun. 66, 1861–1868 47 Oldmixon, B.A. et al. (2002) Porcine endogenous retrovirus trans- 20 Zhang, G. et al. (2001) Porcine antimicrobial peptided: new prospects mission characteristics of an inbred herd of miniature swine. J. Virol. for ancient molecules of host defense. Vet. Res. 31, 277–296 76, 3045–3048 21 Lee, K.H. (2002) Development of short antimicrobial peptides derived 48 Rossant, J. (2001) Stem cells from the mammalian blastocyst. Stem from host defense peptides or by combinatorial libraries. Curr. Pharm. Cells 19, 477–482 Des. 8, 795–813 49 Reubinoff, B.E. et al. (2000) Embryonic stem cell lines from human 22 Bach, F.H. (1998) Xenotransplantation: Problems and prospects. blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, Annu. Rev. Med. 49, 301–310 399–404 23 Platt, J.L. and Lin, S.S. (1998) The future promises of xenotransplan- 50 Edge, A.S.B. et al. (1998) Xenogeneic cell therapy: Current progress tation. In: Xenotransplantation. Ann. NY Acad. Sci. 862, 5–18. and future developments in porcine cell transplantation. Cell 24 White, D. (1996) Alteration of complement activity: a strategy for Transplant. 7, 525–539 xenotransplantation. Trends Biotechnol. 14, 3–5 51 Groth, C.G. et al. (1994) Transplantation of porcine fetal pancreas to 25 Cowan, P.J. et al. (2000) Renal xenografts from triple-transgenic pigs diabetic patients. Lancet 344, 1402–1404 are not hyperacutely rejected but cause coagulopathy in non- 52 Deacon, T. et al. (1997) Histological evidence of fetal pig neutral cell immunosuppressed baboons. Transplantation 69, 2504–2515 survival after transplantation into a patient with Parkinson’s disease. 26 Gaca, J.G. et al. (2002) The role of the porcine von Willebrand factor: Nat. Med. 3, 350–353 Baboon platelet interactions in pulmonary xenotransplantation. 53 Fink, J.S. et al. (2000) Porcine xenografts in Parkinson’s disease and Transplantation 74, 1596–1603 Huntington’s disease patients: preliminary results. Cell Transplant. 9, 27 Cozzi, E. and White, D.J.G. (1995) The generation of transgenic pigs as 273–278 potential organ donors for humans. Nat. Med. 1, 964–966 54 Bjo¨rklund, A. (1991) Neural transplantation – An experimental tool 28 White, D. (1996) Alteration of complement activity: a strategy for with clinical possibilities. Trends Neurosci. 14, 319–322 xenotransplantation Trends. Biotechnol. 14, 3–5 55 Imaizumi, T. et al. (2000) Xenotransplantation of transgenic pig 29 Cozzi, E. et al. (2000) Progress in xenotransplantation. Clin. Nephrol. olfactory ensheathing cells promotes axonal regeneration in rat spinal 53, 13–18 cord. Nat. Biotechnol. 18, 949–953 30 Diamond, L.E. et al. (2001) A human CD46 transgenic pig model 56 Gunsalus, J.R. et al. (1997) Reduction of serum cholesterol in www.sciencedirect.com 294 Review TRENDS in Biotechnology Vol.22 No.6 June 2004

Watanabe rabbits by xenogeneic hepatocellular transplantation. Nat. 77 Manzur, B.J. (2001) Developing transgenic grains with improved oils, Med. 3, 48–53 proteins and carbohydrates. Novartis Found. Symp. 236, 233–239 57 Thomas, M. et al. (1997) Adrenocortical tissue formed by transplan- 78 German, B. et al. (1999) The development of functional foods: lessions tation of normal clones of bovine adrenocortical cells in scid mice from the gut. Trends Biotechnol. 17, 492–499 replaces the essential functions of the animals’ adrenal glands. Nat. 79 Grobet, L. et al. (1997) A deletion in the bovine myostatin gene causes Med. 3, 978–983 the double-muscled phenotype in cattle. Nat. Genet. 17, 71–74 58 Zawada, W.M. et al. (1998) Somatic cell cloned transgenic bovine 80 Yom, H.C. and Bremel, R.D. (1993) Genetic engineering of milk neurons for transplantation in parkinson rats. Nat. Med. 4, 569–574 composition: modification of milk components in lactating transgenic 59 Milan, D. et al. (2000) A mutation in PRKAG3 associated with excess animals. Am. J. Clin. Nutr. 58 (Suppl.), 299S–306S glycogen content in pig skeletal muscle. Science 288, 1248–1251 81 Karatzas, C.N. and Turner, J.D. (1997) Toward altering milk 60 Palmarini, M. and Fan, H. (2001) Retrovirus-induced ovine pulmonary composition by genetic manipulation: current status and challenges. adenocarcinoma, an animal model for lung cancer. J. Natl. Cancer Inst. J. Dairy Sci. 80, 2225–2232 93, 1603–1614 82 Jost, B. et al. (1999) Production of low-lactose milk by extopic 61 Theuring, F. et al. (1997) Transgenic animals as models of neurode- expression of intestinal lactase in the mouse mammary gland. Nat. generative disease in humans. Trends Biotechnol. 15, 320–325 Biotechnol. 17, 160–164 62 Petters, R.M. et al. (1997) Genetically engineered large animal model 83 Stinnakre, M.G. et al. (1994) Creation and phenotypic analysis of for studying cone photoreceptor survival and degeneration in retinitis a-lactalbumin-deficient mice. Proc. Natl. Acad. Sci. U. S. A. 91, pigmentosa. Nat. Biotechnol. 15, 965–970 6544–6548 63 Mahmoud, T.H. (2003) Lensectomy and vitrectomy decrease the rate of 84 Kumar, S. et al. (1994) Milk composition and lactation of b-casein- photoreceptor loss in rhodopsin P347L transgenic pigs. Graefes Arch. deficient mice. Proc. Natl. Acad. Sci. U. S. A. 91, 6138–6142 Clin. Exp. Ophtalmol. 241, 298–308 85 Brophy, B. et al. (2003) Cloned transgenic cattle produce milk with 64 Weissmann, C. et al. (2002) Transmission of prions. Proc. Natl. Acad. higher levels of b-casein and k-casein. Nat. Biotechnol. 21, 157–162 Sci. U. S. A. 99 (Suppl. 4), 16378–16383 86 Golovan, S.P. et al. (2001) Pigs expressing salivary phytase produce 65 Denning, C. et al. (2001) Deletion of the alpha (1,3)galactosyl low-phosphorus manure. Nat. Biotechnol. 19, 741–745 transferase (GGTA1) and the prion protein (PrP) gene in sheep. Nat. 87 O’Brien, S.J. et al. (1999) The promise of comparative genomics in Biotechnol. 19, 559–562 mammals. Science 286, 458–481 66 Draghia-Akli, R. et al. (1999) Myogenic expression of an injectable 88 Hughes, T.R. et al. (2000) Widespread aneuploidy revealed by DNA protease-resistant growth hormone-releasing hormone augments microarray expression profiling. Nat. Genet. 25, 333–337 long-term growth in pigs. Nat. Biotechnol. 17, 1179–1183 89 Templin, M.F. et al. (2002) Protein microarray technology. Trends 67 Djojosubroto, M.W. et al. (2003) Telomeres and telomerase in aging, Biotechnol. 20, 160–166 regeneration and cancer. Mol. Cells 15, 164–175 90 Brambrink, T. et al. (2002) Application of cDNA arrays to monitor 68 Rudolph, K.L. et al. (1999) Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell 96, 701–712 mRNA profiles in single preimplantation mouse embryos. Biotechni- 69 Rudolph, K.L. et al. (2001) Telomere dysfunction and evolution of ques 33, 376–378 intestinal carcinoma in mice and humans. Nat. Genet. 28, 155–159 91 Willadsen, S.M. (1986) Nuclear transplantation in sheep embryos. 70 Cawthon, R.M. et al. (2003) Association between telomere length in Nature 320, 63–65 blood and mortality in people aged 60 years or older. Lancet 361, 92 Wilmut, I. et al. (1997) Viable offspring derived from fetal and adult 393–395 mammalian cells. Nature 385, 810–813 71 Shiels, P.G. et al. (1999) Analysis of telomere lengths in cloned sheep. 93 Schnieke, A.E. et al. (1997) Human factor IX transgenic sheep Nature 399, 316–317 produced by transfer of nuclei from transfected fetal fibroblasts. 72 Wakayama, T. et al. (2000) Cloning of mice to six generations. Nature Science 278, 2130–2133 407, 318–319 94 Cibelli, J.B. et al. (1998) Cloned transgenic calves produced from 73 Tian, X.C. et al. (2000) Normal telomere lengths found in cloned cattle. nonquiescent fetal fibroblasts. Science 280, 1256–1258 Nat. Genet. 26, 272–273 95 Chan, A.W.S. et al. (1998) Transgenic cattle produced by reverse- 74 Lanza, R.P. et al. (2000) Extension of cell life-span and telomere length transcribed gene transfer in oocytes. Proc. Natl. Acad. Sci. U. S. A. 95, in animals cloned from senescent somatic cells. Science 288, 665–669 14028–14033 75 Betts, D. et al. (2001) Reprogramming of telomerase activity and 96 McCreath, K.J. et al. (2000) Production of gene-targeted sheep by rebuilding of telomere length in cloned cattle. Proc. Natl. Acad. Sci. nuclear transfer from cultured somatic cells. Nature 405, U. S. A. 98, 1077–1082 1066–1069 76 Scha¨tzlein, S. et al. (2004) Telomere length is reset during early 97 Hofmann, A. et al. (2003) Efficient transgenesis in farm animals by mammalian embryogenesis. Proc. Natl. Acad. Sci. U. S. A. (accepted) lentiviral vectors. EMBO Rep. 4, 1054–1060