A transgenic animal is by definition an animal whose genetic composition has been altered to include selected genes from other animals or species by methods other than used in traditional breeding. In other words, an animal altered by the introduction of recombinant DNA through human intervention. The first transgenic animal was a mouse, created in 1981, carrying a gene which made the animal susceptible to cancer. The first transgenic farm-animal was a sheep created in 1985. Biotechnology firms and research institutes involved in pharmaceutical development and production use transgenic animals for three different ends.

• As a research model. An example is the modification of the genetic make-up of an animal in such a way that it develops a disease close to that of human beings. The animal is thus used as a model to see how a disease develops and reacts to drugs.
• As a test kit. An animal can be genetically modified to make a human protein. When this protein produced by an external source is applied to the transgenic animal, the immune response of the animal is an indication of the purity of the protein to be tested.
• As a pharmaceutical production unit. Via transgenic technology such as micro-injection, genes that code for the production of a human protein are inserted into the genome of an animal, which in turn produces the human protein.

Old and new transgenic techniques
The first successful pharmaceutical products employing genetically engineered organisms were protein drugs like human insulin (1982) and human growth hormone (1987). These drugs are manufactured in cell cultures employing genetically modified bacteria in bioreactors. However, bacteria and other micro-organisms cannot produce the more complex human proteins. For this, higher organisms like mammals are needed.
The use of transgenic livestock in protein production aims at overcoming several major barriers presented by cell-based systems. Potentially, this approach could provide large quantities of complex proteins in a cost-effective way. Compared to the facilities and chemicals required for cell culture production, capital investment required for animal production facilities is relatively low. The British company PPL Therapeutics (PPL) estimates that the basic costs for products developed by transgenic animals are four to five times lower than cell culture production. However, this does not take into account development costs. To date, the use of transgenic animals is developing fast for a range of pharmaceutical products. (see table)

R&D of medicine production by transgenic animals
Drug	Disease/Target	Animal	Company
alpha-lactalbumin	anti-infection	cow	PPL
alpha1 anti  trypsin (AAT)	deficiency leads to emphysema	sheep	PPL
CFTR	cystic fibrosis	sheep, mouse	PPL
human protein C	thrombosis	pig, sheep	PPL
tissue plasminogen activator (tPA)	thrombosis	mouse, goat	PPL
human calcitonin	osteoporosis	rabbit	PPL
factor VIII	hemophilia	pig  sheep	Pharming  PPL
factor IX	hemophilia	pig, cow  sheep	Pharming  PPL
fibrinogen	wound healing	cow  sheep	Pharming  PPL
alpha-glucosidase	Pompe disease	rabbit	Pharming
collagen I  collagen II	tissue repair  rheumatoid arthritis	cow  cow	Pharming
lactoferrin	GI tract infection,  infectious arthritis	cow	Pharming
antithrombin 3 (ATIII)	thrombosis	goat	GTC
glutamic acid decarboxylase	type 1 diabetes	mouse, goat	GTC
human serum albumin (HSA)	maintains blood volume	mouse, cow	GTC
msp-1	malaria	mouse	GTC
Pro542	HIV	mouse, goat	GTC

 
Actors and products
The main actors involved in pharming are three new biotechnology firms: Genzyme Transgenics Corporation (GTC) in the USA, PPL in the UK and Pharming Group in the Netherlands. GTC was the first company that successfully completed a second phase clinical trial for the pharming drug Antithrombin 3 (ATIII). The drug is used for treatment of thrombosis and is currently extracted from blood plasma. GTC will produce ATIII using transgenic goats and sees market opportunities for the protein in many healthcare areas. GTC expects to market the product by the year 2000.
The first pharming drug expected to be approved for sale, however, is human alpha glucosidase. This is a drug against Pompe disease, a rare neuro-muscular disease. It is produced by Pharming Group using transgenic rabbits. The fact that this drug is expected to be approved before ATIII, which was developed earlier, is due to the Orphan Drug Act. Orphan Drugs (OD) are drugs for rare diseases. OD status is granted by the Food and Drug Administration (FDA). By giving OD status, health authorities aim to stimulate pharmaceutical development for drugs with small markets. Although OD need to complete the clinical phase III trials successfully like any other medicine, these drugs do not have to be tested on thousands of people. Instead, OD need testing on only several tens of people to receive market-approval. For starting pharmaceutical companies OD can be a good opportunity to generate income quickly.
Another promising substance for commercial production by transgenic animals is Human Serum Albumine (HSA). To serve the world market of about 5,500 cows would be sufficient to replace the currently plasma-derived HSA, assuming one cow produces 80 kg of protein a year in its milk, as GTC claims.
However, transgenic cows producing HSA do not exist at the moment. GTC has developed only transgenic mice producing HSA in their milk. Nevertheless, GTC initiated a related collaboration with the UK-based company Advanced Cell Technology (ACT) for the development of cloned, transgenic cows. GTC has exclusive, worldwide rights to ACT`s cloning technology for the production of biopharmaceuticals in the milk of transgenic cows. In January 1998 ACT announced it had cloned three transgenic calves using the technique applied by the Roslin Institute for production of Dolly the sheep (see box).
Pharming Group was founded by Leiden University in the Netherlands, which still has shares in the company. Pharming Group is developing several pharmaceutical proteins. In agreement with the American Red Cross (ARC), Pharming Group will use ARC’s technology and patents to produce blood compounds such as factor VIII (hemophilia A), factor IX (hemophilia B) and fibrinogen (clotting protein) in the milk of cows and pigs. These plasma proteins are expected to be on the market within six to seven years.
Within 20 years the whole production of blood products, currently derived from pooled human blood, might be transferred to pharming production, according to Pharming Group’s vice president G. van Beynum. Health risks and accidents caused by the transmission of Human Immunodeficiency Virus (HIV) and hepatitis have stimulated the search for alternatives. At the development phase Pharming still requires screening for diseases transferable from animals to humans. This possibility has become obvious by the outbreak of diseases like Bovine Spongiform Encephalopathy (BSE).
PPL was established to commercialize the work of the Roslin Institute, a public pharmaceutical research institute in Scotland. PPL mainly uses sheep, but goats, cows and recently pigs and rabbits are also used for pharming. The company produces alpha 1 anti trypsin (AAT) in the milk of transgenic sheep. The first and best-known sheep to produce pharmaceuticals is called Tracey (see box), and was bought by the German pharmaceutical company Bayer for US$ 17 million. Currently PPL  owns a herd of 300 sheep, which are worth over US$ 100 million, and which produce AAT as a drug against cystic fibrosis. AAT has recently been granted OD status in the US.
Moreover, PPL holds a patent on the nuclear transfer cloning technique by which the clone Dolly was made. Although PPL vowed they will never clone human beings, the patent does not exclude humans. Dolly the sheep is the first mammal to be cloned from an adult cell. The ability of clones to produce healthy offspring is important for commercialization of the nuclear transfer technique which produced Dolly.
Not all new biotechnology firms have the same strategy for commercialization. PPL, for example, supplies technology to other companies. PPL can be considered as a technology platform for companies such as Bayer, Boehringer Ingelheim (Germany), Novo Nordisk (Denmark) and American Home Products (USA). Pharming Group, on the other hand, intends to become a pharmaceutical company itself. GTC takes a middle position because it markets some pharmaceuticals itself, but also contracts partners to market other products.
 

Transgenic sheep   •  Tracey was the first transgenic sheep to produce human protein in its milk (AAT), PPL produced Tracy in 1991 using  micro-injection for the transfer of human genes.  •  Dolly was the first mammal ever to be produced using a cell of an adult sheep, a technique not considered possible before. At the Roslin Institute in Scotland, the cell material was extracted from the udder of a 6-year old sheep and transplanted into an emptied egg cell. Dolly was born in 1996. In 1998, Dolly gave birth to a lamb called Bonny, proving that clones are able to produce healthy offspring.  •  In 1997, PPL announced that Polly, a genetically engineered lamb, had been produced by the same method of nuclear transfer that had produced Dolly. In addition to her usual complement of sheep genes, she also contained a human gene which had been added to the cells while they were still a cell culture. Polly produces a pharmaceutical protein in her milk.

 
Animal husbandry and welfare
The production of pharmaceuticals by transgenic animals is not likely to have a big impact on animal husbandry in general. Since the number of transgenic animals which will provide pharmaceutical proteins for the world market is limited to several thousands, pharming is expected to become a separate area of animal production. According to P. Brascamp of Wageningen Agricultural University in the Netherlands, pharming will be part of the pharmaceutical production chain.
Animal welfare groups, and those concerned about ethical implications of biotechnology, question the development and use of transgenic animals. Cloning can involve invasive procedures to harvest eggs. A number of cloned animals have developed malformed internal organs. Animal welfare groups fear that any herd of cloned animals used in agriculture would be vulnerable to diseases because of the reduced gene pool from which they were drawn. Animal welfare groups like Compassion in World Farming along with the British Union for the Abolition of Vivisection therefore have lobbied the British government to ban animal cloning.

Pharming for developing countries
Most of the pharmaceutical industry’s revenues are derived from sales of drugs in the industrialized countries. By far the majority of these drugs are targeted towards "diseases of affluence" such as heart diseases, ulcers and depression. Could pharming provide treatments for diseases such as malaria, tuberculosis and cholera? According to the Netherlands’ biotechnology industry association Niaba, the return of investment for the drug development of such diseases would be too low, given the low-income markets for such drugs. On the other hand, if deals could be made between United Nations (UN) organizations such as the World Health Organization (WHO) or the United Nations Industrial Development Organization (UNIDO) and pharmaceutical or pharming companies, drugs could be provided at lower prices under certain conditions. Such a deal was made between Glaxo Wellcome (UK) and the Joint United Nations Program on HIV/AIDS (UNAIDS) to provide the Anti AIDS drug AZT for 30 per cent of the market price (see Monitor No. 34).
Perhaps the most promising for developing countries is GTC`s research on malaria. 300 to 500 million people are infected with malaria and 2 million people die of this disease annually. GTC developed a malaria antigen, MSP-1, in the milk of transgenic mice. This was part of GTC’s collaboration with the American National Institute of Allergy and Infectious Disease (NIAID) for the production of transgenic recombinant malarial proteins for use in malaria vaccines.
Jeroen Breekveldt/ Joost Jongerden

Working Group Technology and Agrarian Development, Wageningen Agricultural University, Nieuwe Kanaal 11, 6709 PA Wageningen, the Netherlands.
E-mail jeroen.breekveldt@tao.tct.wau.nl joost.jongerden@tao.tct.wau.nl

Sources
http://www.prnewswire.com

http://www.genzyme.com

press releases Pharming Group

Nature Biotechnology, October 1997

Personal communications with G.M.A. van Beynum (Pharming Group) and P. Brascamp (Wageningen Agricultural University).