Transgenic Mammals Likely To Transform Drug-Making

Transgenic mice have become the stock-in-trade of basic biomedical research since their creation in the early 1980s. The techniques used to insert human genes into non-human species that were refined in rodents are now being applied to a menagerie of other mammals. These chimeric goats, pigs, sheep, cows, and rabbits are essentially living bioreactors: When a gene inserted into their DNA is specifically activated in mammary gland cells, the desired human protein is secreted into the animal's milk. The protein is then isolated and purified. Academic, government, and industry researchers in the United States and Europe have been developing transgenic animals in hopes of producing safer and cheaper pharmaceuticals. BUNNIES AND BULLS: Netherlands-based Pharming BV uses cattle and rabbits for its transgenic products, notes Gerard van Beynum. While no transgenic products are on the market or licensed, two are in clinical trials and more are expected to go into trials within the next year, researchers report. For example, Framingham, Mass.-based Genzyme Transgenics Corp. started clinical trials with antithrombin III-an anticoagulant-last year. This was the first use of a transgenic, pharmaceutical protein in human trials. Another area being pursued by researchers is the mass production of hemophiliac blood factors and other blood proteins. The American Red Cross set up an R&D transfusion biology lab about 10 years ago to help contain the growing problem of HIV contamination in the U.S. blood supply. "We were looking at what the future would be in terms of trying to replace some of the unsafe proteins," says Bill Drohan, senior director of plasma development at the American Red Cross's Holland Laboratory, the organization's research arm based in Rockville, Md. "We took two tacks: One [involved] ways of inactivating viruses in plasma and the other was trying to produce the proteins by recombinant technology." Drohan notes that blood proteins had been made this way in tissue culture, but this was more expensive than isolating them from plasma. "So we looked at other ways of making it, and that's what brought us into the production of proteins in the milk of transgenic animals." Researchers are looking toward the day when herds of transgenic livestock will supplant the now laborious and costly production of human proteins. Currently, proteins and other molecules are purified from human blood or grown in cell culture in enormous metal vats. The purchase of blood-clotting factors like Factor VIII produced by these methods can cost a hemophiliac tens of thousands of dollars annually. The efficiency of recovery of a protein from livestock milk varies with the type of protein "plus or minus 10 [percent] to 50 percent," says Drohan. "Fifty percent of [a blood factor] found in the milk can be recovered in a useful form to help patients. That's about the same amount that we can recover from plasma." William H. Velander, a professor of chemical engineering at the Virginia Polytechnic Institute and State University in Blacksburg, Va., and a collaborator with the Red Cross, says that the Virginia Tech-Red Cross team picked pigs for "a good reason." One pig can produce 300 to 500 liters of milk. Velander predicts that about 100 transgenic pigs could produce the entire U.S. supply of Factor IX, another hemophilia-related blood protein. A few years ago, Drohan, Velander, and colleagues produced Genie, the first transgenic pig. The gene for protein C, a clotting agent found in human blood, was inserted into her genome. HOG WILD: William Velander, left, and William Drohan, creators of transgenic pigs (right) The procedure for making a transgenic animal is similar for most species, although nuances in cellular and reproductive biology differ by species and must be taken into account. The gene for the desired protein is cloned and put into the single cell of a fertilized embryo. "Then that egg is put into a female that is hormonally induced," explains Drohan. "She brings those eggs to term, and the pig-lets are born [nearly four months] later. When they're about three weeks old, we snip a piece of tail and sample DNA from it for the presence of the human gene. If it's there, we assume that every cell has the human gene, and we call the pig transgenic." The genes are inserted into the receiving animal's genome in such a way that the expression of the protein is controlled by a promoter sequence that is introduced at the same time as the gene for the target protein. The promoter encourages expression of a gene only in the mammary gland. The protein is then secreted into the milk and collected. The livestock mammals take up the human DNA as their own and pass the new trait on to subsequent generations. Individual animals, however, differ in the amount of protein that they produce. The U.S. Department of Agriculture's Agricultural Research Service (ARS) in Beltsville, Md., has been peripherally involved in the development of transgenic livestock like Genie. "We do think that livestock animals make good models, but it hasn't been real clear that we should take off down that path," says Caird Rexroad, research leader of ARS's gene evaluation and mapping laboratory. "We've sort of purposely avoided that because it's not clearly within our mandate. What we have done is help other people interested in this area find out how to go about doing this kind of work." About a year ago, Vernon Pursel, an ARC research physiologist, and researchers at the National Institutes of Health tried to produce the enzyme glucose cerebrosidase in pigs, with hopes of using it to treat Gaucher's disease. People with this disorder are missing this enzyme. However, as Pursel puts it, the research didn't "pan out." The enzyme wasn't expressed in the pig's mammary glands. One government-industry alliance to create transgenic animals made a media splash earlier this year. The research collaboration between PPL Therapeutics PLC-an Edinburgh, Scotland-based biotech firm-and the Roslin Institute-a United Kingdom government-sponsored laboratory-created Dolly, the cloned ewe (K.Y. Kreeger, The Scientist, April 14, 1997, page 1). One of their goals was to someday make herds of sheep clones that produce a desired protein in high amounts. GOT MILK? Genzyme Transgenics is conducting human clinical trials with antithrombin III isolated from the milk of transgenic goats. "Certainly with cows that would be desirable," says Alan Colman, PPL's research director. "But that isn't our vision of the use of the technology. That is an adjunct, a peripheral." PPL's transgenic sheep are currently being made by what Colman terms "conventional routes," much the same way as Velander and Drohan's pigs or Genzyme's goats. Now, the goal is to make many animals in one generation-"a production flock instantly by using the cloning technology. What gave rise to Dolly could give rise to 100 sheep expressing a particular human gene," says Drohan. If this could be achieved, it would bypass the need to clone an adult with a desired trait. "We could do everything at the cellular level in the lab and then make animals from these identical cells," he explains. "We see that as a way of bringing these products to the market a lot more quickly. We cut out this quite extensive breeding program that we use at the moment. We could actually make what I call an 'instant flock.'" While some have grand ideas of streamlining the production of transgenic animals, most of the research and development efforts lie with producing pharmaceutical products. Observers predict that many such pharmaceuticals will be on the market within the next few years. The three main biotech firms working with transgenic animals-Genzyme, PPL, and Pharming BV, based in Leiden, the Netherlands-have proteins and other substances in various stages of development. "The total number of proteins that we've expressed is now around 25 in either mice, goats, or rabbits," reports Suzanne Groet, Genzyme's vice president of business development. "Six or seven have been scaled up to the goat system." These include human monoclonal antibodies, tissue plasminogen activator, and alpha-1-antitrypsin, along with the antithrombin III in clinical trials. Antithrombin III is an anticoagulant that could be used in bypass graft procedures to decrease the amount of donor blood required. Patient enrollment for the Phase II trials for antithrombin III has just been completed. Genzyme also collaborates with New York-based Bristol-Myers Squibb Co. to produce monoclonal antibodies in the milk of goats. The pharmaceutical company is developing a cancer therapy called BR96-doxorubicin, a combination of the monoclonal antibody BR96 and the drug doxorubicin. Monoclonal antibodies are proteins that seek out and latch onto distinct cell types. American Chemical Society 1155 16th St., N.W. Washington, D.C. 20036 (202) 872-4600 http://www.chemcenter.org American Red Cross http://www.redcross.org Genzyme Transgenics Corp. http://www.genzyme.com/transgenics Roslin Institute http://www.ri.bbsrc.ac.uk Virginia Polytechnic Institute and State University College of Engineering http://www.eng.vt.edu/ For more information on transgenic pigs, see W.H. Verlander, H. Lubon, W.N. Drohan, Scientific American, 276(1):70-4, 1997. Pharming BV is betting the farm, so to speak, on cows and rabbits for its transgenic products. The company was the first to produce transgenic cattle, reports Gerard van Beynum, Pharming's vice president of product development. Pharming BV has two main products, one made in cows and another in rabbits. Human lectoferrin, a protein that works as a natural antibiotic, is made in the milk of female offspring of Herman the bull, one of the first animals used in Pharming's transgenic program. "In fact, it's found in human milk," notes van Beynum. One potential application, he says, is as a neutraceutical, an infant formula additive to be used for immune-depressed babies or perhaps a product for older patients on chemo- or radiotherapy. Another use could be as a pharmaceutical during cardiac surgery to decrease operation-related infections. Preclinical trials have started for this product. At the end of this year, says van Beynum, Pharming expects to start clinical trials in Europe using human alpha glucosidase produced in rabbits to treat Pompe's disease. Patients with this lethal and incurable muscle disorder lack the enzyme alpha glucosidase. "This is our lead product," remarks van Beynum. Because there's no drug available for treating the disease, van Beynum expects the clinical trials and regulatory review to be hastened. He expects to get the enzyme commercialized by 1999: "We might have the first [transgenic] product on the market." PPL just started Phase II clinical trials with cystic fibrosis patients using alpha-1-antitrypsin, with the first administration to a patient in mid-June, according to Colman. This protease inhibitor is produced in transgenic sheep. Like the American Red Cross, PPL is also interested in blood clotting factors. The company has transgenic sheep in developmental phases for three of these proteins: Factors IX and VII, and protein C. Virginia Tech's Velander notes that Factor IX isolated from pig's milk is now in the process of being commercialized. He predicts that it will be in clinical trials within the next three years and on the market in five. Another clotting substance found in human blood called fibrinogen is attracting the attention of PPL and the Red Cross. They want to develop its use as a tissue glue for use in internal surgical wounds. "It's a biological glue that is more effective than sutures," explains Colman. Calling fibrinogen the Red Cross's most economically important pursuit, Drohan states that "it could be used in bypass surgery grafts to help speed healing of the suture." The Red Cross has finished three preclinical studies with fibrinogen, and it's now in regulatory review at the Food and Drug Administration. Although many potential transgenic pharmaceuticals are in the pipeline, questions regarding efficacy and safety remain. "The downsides are more clinical in terms of the utility of the protein," says Drohan. "Every tissue, including mammary glands, that makes a protein puts its signature on that protein in a process called post-translational modification. So a potential downside would be if the protein made in the mammary gland is not as efficacious as the protein made in its natural human organ." The protein might also be immunogenic, meaning that the patient given the protein might make antibodies to it, Drohan explains. Those antibodies not only would neutralize the transgenic proteins, but also might cross-react with the natural protein and impede a person's ability to make the target protein. So far, this scenario hasn't occurred in the clinical trials with antithrombin III or with alpha-1-antitrypsin. Another potential downside, says Drohan, is that if the signature sugars are added to the protein differently from the way they are in nature, the transgenic protein could circulate longer or shorter than the natural protein. "That could be good or bad, depending in the therapeutic application." One risk that researchers don't like to talk about is the potential for infectious agents in the milk of transgenic animals to jump species into human recipients. "Normally these agents don't jump species," maintains Drohan, but there are possible rare exceptions like bovine spongiform encephalopathy and other prion-like diseases. The question of whether producing human proteins in transgenic farm animals is safer and more efficient than currently used methods remains open. "The honest answer to that is that we don't know whether there is a downside or not to [these technologies]," says PPL's Colman. "These are just the sort of issues being looked at in the human clinical trials."

Transgenic Mammals Likely To Transform Drug-Making

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BUNNIES AND BULLS: Netherlands-based Pharming BV uses cattle and rabbits for its transgenic products, notes Gerard van Beynum.