"Milking" Transgenic Animal-Derived Drugs
In June 2006, an announcement wasmade that the world's first medicinederived from a genetically engineeredgoat had been recommended forapproval in Europe.1 The product, ATryn,is an anticlotting agent used for patientswith a rare congenital antithrombin deficiency.Antithrombin is a protein inhuman plasma that has anticoagulantand anti-inflammatory properties. Theactive substance in Atryn, antithrombinalfa, is a copy of the natural blood proteinthat is produced by recombinant DNAtechnology.
Drug development using transgenicanimals is a new form of farming. Thusthe term pharming has been adopted todescribe the process. The term transgenic,however, was first introduced in1981.2 Pharming has been gaining applicationamong biotechnology firms sincethe development of transgenic mice in1982. The techniques developed in themouse have since been applied to anumber of agricultural species.3
Many companies and universities inthe United States are involved in researchand development, and clinical trialsare ongoing for several new drugs.The production of recombinant protein inthe milk of transgenic goats is currentlybeing tested for the production of a numberof therapeutic antibodies and for ahuman serum albumin.4
Goats are not the only animals used. Abiotechnology firm in this country justdescribed a new technique to creategenetically modified chickens that canbe used to produce treatments forhuman diseases.5 Mice, rats, rabbits, pigs,and sheep also can be used.
Transgenic animals are engineered tocarry genes from other species. Trans isa Latin preposition meaning "across,over, or beyond." The Federation ofEuropean Laboratory Animal Associationsdefines the term transgenic animalas an animal in which there has been adeliberate modification of its genome,the genetic makeup of an organismresponsible for inherited characteristics.For those with little or no background ingenetics, the federation includes the followingexplanation:
The nucleus of all cells in every livingorganism contains genes made up ofdeoxyribonucleic acid (DNA). Thesegenes store information that regulateshow our bodies form and function.Genes can be altered artificially, so thatsome characteristics of an animal arechanged. For example, an embryo canhave an extra, functioning gene fromanother source artificially introduced intoit, or a gene introduced which can knockout the functioning of another particulargene in the embryo. Inserted genes arecalled transgenes. Animals that havetheir DNA manipulated in this way areknown as transgenic animals.6
The FDA defines a transgenic animalas an animal that is altered by the introductionof recombinant DNA throughhuman intervention.7 There are 2 classes:those with heritable germ-line DNA alterationsand those with somatic (nonreproductive)nonheritable alterations. Agerm-line cell divides to producegametes, which are male or female cellswhose union is necessary in sexualreproduction. Human germ-line manipulationsare those made to the genes ofgerminal or reproductive cells (the eggand the sperm). These manipulationsmean altering the fertilized egg, the firstcell in the embryo-to-be, so that thegenetic changes will be copied into everycell of the future adult, including his orher reproductive cells.
Examples of heritable alterationsinclude animals with germ-line DNAaltered through methods requiring exvivo manipulation of gametes, earlyembryonic stages, or embryonic stemcell lines. Examples of nonheritablealterations include animals with somatic-cell DNA alterations achieved throughgene therapy approaches such as directplasmid DNA injection or virally mediatedgene transfer. In essence, new geneticmaterial may be introduced either intothe germ line or into all or some somaticcells.
Transgene, mentioned above, refers toa segment of recombinant DNA that iseither introduced into somatic cells orintegrated stably into the germ line of itsanimal host strain and is transmissible tosubsequent generations. RecombinantDNA is any DNA molecule formed byjoining DNA segments from differentsources. Plasmids are small, circularextrachromosomal DNA molecules thatcan replicate independently of thegenome.8
The Transgenic Technique
The underlying principle in the productionof transgenic animals is the introductionof a foreign gene or genes into ananimal. The foreign genes must be transmittedthrough the germ line, so that allcells—including germ cells—of the animalcontain the same modified germmaterial. Germ cells are cells whosefunction is to transmit genes to an organism'soffspring.
There are 3 basic methods of producingtransgenic animals: (1) DNA microinjection,(2) retrovirus-mediated genetransfer, and (3) embryonic stemcell-mediated gene transfer. Gene transferby microinjection is the predominantmethod. Because the insertion of DNAresults in a random process, transgenicanimals are mated to ensure that theiroffspring acquire the desired transgene.The mouse was the first animal to undergosuccessful gene transfer using theDNA microinjection method.
Pharming
A number of medical applicationsare derived from engineeringtransgenic animals.They include the following:
•Xenotransplantation—Pigs can be used to providetransplant organs.This application is currentlyhampered by a pigprotein that can causedonor rejection. Researchis underway, however, toremove the pig proteinand substitute one fromhumans.
•Nutritional supplementsand pharmaceuticals—Products such as insulin, growth hormone,and blood anticlotting factorscan be obtained from the milk oftransgenic cows, goats, or sheep.Goats are especially useful, becausethey have a relatively short generationalinterval (145 days, as opposedto 279 days for cows). Goats alsohave a large average milk output.
•Human gene therapy—A normalcopy of a gene is added to thegenome of a person carrying defectivecopies of the gene. The potentialfor treatments for the 5000 namedgenetic diseases is large.9
To produce the first medicine justintroduced in Europe, the gene encodingthe protein responsible for anticlottingfactors was fused to milk-specific elementsto generate a transgene. Thetransgene was then introduced in thegerm line of goats by pronuclear microinjectionof one-cell embryos. Microinjectedembryos were then transferredto the surrogate mother. Often up to 5%to 10% of the offspring resulting fromthese microinjections carry the transgene.After integration into the germ line,the mammary gland-specific transgenebecame a dominant genetic characteristicpredictably inherited by offspring ofthe founder animal.10
The animal becomes a production facilitywith many advantages. It is reproducible,has a flexible production capacitythrough the number of animals bred, andmaintains its own fuel supply. Perhapsbest of all, the drug is delivered from theanimal in a very convenient form—in themilk. The milk must be collected and thedesired proteins recovered. The proteinsare further processed to produce the therapeuticdrug.
Final Thoughts
The introduction of ATryn into theEuropean market demonstrates thecapability of transgenic recombinantdrug production. Other products are sureto follow. Companies that are successfulin the United States must addressacceptable animal health and testingprograms and acceptable animal husbandrypractices. They must provide purity,potency, and efficacy through validatedpurification processes in preclinicaland clinical studies.11
This is no easy task, as evidenced by alack of uniformity of standards and littlein the way of recent regulatory guidancefrom the FDA.
Mr. Sherman is president of ShermanConsulting Services Inc.
For a list of references, send a stamped,self-addressed envelope to: ReferencesDepartment, Attn. A. Rybovic, PharmacyTimes, Ascend Media Healthcare, 103 CollegeRoad East, Princeton, NJ 08540; or send an emailrequest to: arybovic@ascendmedia.com.
