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: