In June 2006, an announcement was made that the world's first medicine derived from a genetically engineered goat had been recommended for approval in Europe.¹ The product, ATryn, is an anticlotting agent used for patients with a rare congenital antithrombin deficiency. Antithrombin is a protein in human plasma that has anticoagulant and anti-inflammatory properties. The active substance in Atryn, antithrombin alfa, is a copy of the natural blood protein that is produced by recombinant DNA technology.

Drug development using transgenic animals is a new form of farming. Thus the term pharming has been adopted to describe the process. The term transgenic, however, was first introduced in 1981.² Pharming has been gaining application among biotechnology firms since the development of transgenic mice in 1982. The techniques developed in the mouse have since been applied to a number of agricultural species.³

Many companies and universities in the United States are involved in research and development, and clinical trials are ongoing for several new drugs. The production of recombinant protein in the milk of transgenic goats is currently being tested for the production of a number of therapeutic antibodies and for a human serum albumin.⁴

Goats are not the only animals used. A biotechnology firm in this country just described a new technique to create genetically modified chickens that can be used to produce treatments for human diseases.⁵ Mice, rats, rabbits, pigs, and sheep also can be used.

Transgenic animals are engineered to carry genes from other species. Trans is a Latin preposition meaning "across, over, or beyond." The Federation of European Laboratory Animal Associations defines the term transgenic animal as an animal in which there has been a deliberate modification of its genome, the genetic makeup of an organism responsible for inherited characteristics. For those with little or no background in genetics, the federation includes the following explanation:

The nucleus of all cells in every living organism contains genes made up of deoxyribonucleic acid (DNA). These genes store information that regulates how our bodies form and function. Genes can be altered artificially, so that some characteristics of an animal are changed. For example, an embryo can have an extra, functioning gene from another source artificially introduced into it, or a gene introduced which can knock out the functioning of another particular gene in the embryo. Inserted genes are called transgenes. Animals that have their DNA manipulated in this way are known as transgenic animals.⁶

The FDA defines a transgenic animal as an animal that is altered by the introduction of recombinant DNA through human intervention.⁷ There are 2 classes: those with heritable germ-line DNA alterations and those with somatic (nonreproductive) nonheritable alterations. A germ-line cell divides to produce gametes, which are male or female cells whose union is necessary in sexual reproduction. Human germ-line manipulations are those made to the genes of germinal or reproductive cells (the egg and the sperm). These manipulations mean altering the fertilized egg, the first cell in the embryo-to-be, so that the genetic changes will be copied into every cell of the future adult, including his or her reproductive cells.

Examples of heritable alterations include animals with germ-line DNA altered through methods requiring ex vivo manipulation of gametes, early embryonic stages, or embryonic stem cell lines. Examples of nonheritable alterations include animals with somatic-cell DNA alterations achieved through gene therapy approaches such as direct plasmid DNA injection or virally mediated gene transfer. In essence, new genetic material may be introduced either into the germ line or into all or some somatic cells.

Transgene, mentioned above, refers to a segment of recombinant DNA that is either introduced into somatic cells or integrated stably into the germ line of its animal host strain and is transmissible to subsequent generations. Recombinant DNA is any DNA molecule formed by joining DNA segments from different sources. Plasmids are small, circular extrachromosomal DNA molecules that can replicate independently of the genome.⁸

The Transgenic Technique

The underlying principle in the production of transgenic animals is the introduction of a foreign gene or genes into an animal. The foreign genes must be transmitted through the germ line, so that all cells—including germ cells—of the animal contain the same modified germ material. Germ cells are cells whose function is to transmit genes to an organism's offspring.

There are 3 basic methods of producing transgenic animals: (1) DNA microinjection, (2) retrovirus-mediated gene transfer, and (3) embryonic stem cell-mediated gene transfer. Gene transfer by microinjection is the predominant method. Because the insertion of DNA results in a random process, transgenic animals are mated to ensure that their offspring acquire the desired transgene. The mouse was the first animal to undergo successful gene transfer using the DNA microinjection method.

Pharming

A number of medical applications are derived from engineering transgenic animals. They include the following:

•Xenotransplantation—Pigs can be used to provide transplant organs. This application is currently hampered by a pig protein that can cause donor rejection. Research is underway, however, to remove the pig protein and substitute one from humans.

•Nutritional supplements and pharmaceuticals— Products such as insulin, growth hormone, and blood anticlotting factors can be obtained from the milk of transgenic cows, goats, or sheep. Goats are especially useful, because they have a relatively short generational interval (145 days, as opposed to 279 days for cows). Goats also have a large average milk output.

•Human gene therapy—A normal copy of a gene is added to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5000 named genetic diseases is large.⁹

To produce the first medicine just introduced in Europe, the gene encoding the protein responsible for anticlotting factors was fused to milk-specific elements to generate a transgene. The transgene was then introduced in the germ line of goats by pronuclear microinjection of one-cell embryos. Microinjected embryos were then transferred to the surrogate mother. Often up to 5% to 10% of the offspring resulting from these microinjections carry the transgene. After integration into the germ line, the mammary gland-specific transgene became a dominant genetic characteristic predictably inherited by offspring of the founder animal.¹⁰

The animal becomes a production facility with many advantages. It is reproducible, has a flexible production capacity through the number of animals bred, and maintains its own fuel supply. Perhaps best of all, the drug is delivered from the animal in a very convenient form—in the milk. The milk must be collected and the desired proteins recovered. The proteins are further processed to produce the therapeutic drug.

Final Thoughts

The introduction of ATryn into the European market demonstrates the capability of transgenic recombinant drug production. Other products are sure to follow. Companies that are successful in the United States must address acceptable animal health and testing programs and acceptable animal husbandry practices. They must provide purity, potency, and efficacy through validated purification processes in preclinical and clinical studies.¹¹

This is no easy task, as evidenced by a lack of uniformity of standards and little in the way of recent regulatory guidance from the FDA.

Mr. Sherman is president of Sherman Consulting Services Inc.

For a list of references, send a stamped, self-addressed envelope to: References Department, Attn. A. Rybovic, Pharmacy Times, Ascend Media Healthcare, 103 College Road East, Princeton, NJ 08540; or send an email request to: arybovic@ascendmedia.com.