In Scotland, a transgenic ewe named Tracy produced high levels ofhuman alpha-1-antitrypsin (an emphysema therapeutic) in her milk.In Holland, a Holstein bull named Herman is siring female calves thatgrow up to yield milk containing antibacterial human lactoferrin.And transgenic goats in Massachusetts are secreting up to 7 grams ofantithrombin III per liter in their milk.Getting these dairy animals to express high-value, high-volume humanproteins in their mammary glands involves microinjection of a fewhundred molecules of the foreign DNA into the pronucleus of afertilized egg, reimplanting it in the uterus of a surrogate mother,selecting progeny in which the transfection took, rearing males tobreeding age, and their transgenic female offspring to lactatingmaturity.Dairy and animal scientists at the University of Wisconsin report thatthey have found a potentially better way, using somatic gene therapyinstead of germline transgenic techniques.In the July 19 issue of the Proceedings of the National Academy ofSciences (PNAS), dairy scientist Robert Bremel and his co-authorsreport: 'Human growth hormone (hGH) secretion in milk of goats afterdirect transfer of the hGH gene into the mammary gland by usingreplication-defective retrovirus vectors.'By implanting the hGH expression package directly into the milk-making cells of the goats' mammary glands, Bremel told BioWorldToday, 'we're looking at a couple of weeks vs. a couple of years' forraising protein-expressing ruminants.To turn on the milk glands of these animals in the first place, Bremel'steam administered a two-week course of estrogen and progesteroneinjections. 'It's a standard animal veterinary management practice,' heexplained. 'Vets will do this in a cow, for example, which for whateverreason has stopped giving milk.'To carry the hGH gene sequence into the epithelial cells of the udder,they recruited the standard gene-therapy non-replicating xenotropicnon-species-specific retrovirus 'with as high a titer as we could get,'Bremel said.Such viruses, he added, 'insert their DNA into cells during mitosis. Sowe needed dividing cells, which we got by inducing lactation.'On alternate days during this fortnight of hormonal treatment, Bremel'steam infused the viral vector containing the hGH gene into the rightudder of each animal, by syringe. The left udder, as a control, receivedonly culture medium.Principle ProvedIn this initial proof-of-principle experiment, all four goats producedtotal levels of hGH in their milk, ranging from 0.3 to 2 micrograms perday during the first 10 days of lactation. This output was measured byradio-immune assay, rather than protein recovery.The human cell lines that produced the viruses, Bremel added, weretransfected by biolistic bombardment with gene guns, at Agracetus Middleton, Wis.The Wisconsin somatic-cell transfection approach is protected by U.S.Patent No. 5,215,904, 'Method for Producing a Recombinant Mammalin Vivo,' issued June 1, 1993, to Bremel's co-author, oncologistMichael Gould.Since submitting its paper to PNAS late last year, the Wisconsin grouphas been working on methods to optimize the viral titer. 'This is themajor limitation on expressing human protein in the ruminants milk,'Bremel said. 'The udder of an animal has somewhere around 1010 (10billion) cells, and we're at least a thousandfold below that.'His group is now collaborating with molecular biologist Jane Burns atthe University of California-San Diego. She has found titers as high as1010 in vesicular stomatitis virus, another candidate vector.Bremel doesn't predict that his viral-vector ploy will render all thosetransgenic sheep, cows and goats obsolete - not just yet. Initially, hesees his system as providing a method of evaluating the viability of agiven human protein before transgenic host ruminants are created.'It's a big gamble,' he pointed out, 'to go ahead and make a transgenicanimal, take all of that time and money, only to find out that thedesired protein isn't stable at the pH of milk.''In the long run, if we could get a factor of 100 in the number ofmammary epithelial cells that are targeted, I guess our gene-therapysystem could be competitive with the transgenics. It would certainly befaster, because you can do it in a couple of weeks' time.'Transgenic Company Line-upMeanwhile, Bremel has talked to all of the different [transgenic]companies, presumably:- Pharmaceutical Proteins Ltd. (PPL), Edinburgh, Scotland, whosetransgenic sheep, Tracy and her ilk, were the bellwethers in efforts tocommercialize human protein production in the milk of transgenicruminants. PPL is now approaching clinical trials of its first product,alpha-1-antitrypsin, to treat emphysema.- GenPharm Intl., Mountain View, Calif., with Gene Pharming Europe,at the Dutch city of Leiden, for whom Herman the bull's harem isbeing milked to recover human lactoferrin, a protective additive toinfants' formula.- Genzyme Transgenics Corp., Framingham, Mass., which with nearbyTufts University is raising nanny goats that secrete antithrombin-III. Itforesees clinical trials in 1995.- TransPharm Inc., Blacksburg, Va., (PPL's U.S. arm), is developinghuman protein C, an anticoagulant, in pigs' milk, and a nutritionalhuman protein in cows' milk.Reproductive physiologist Will Eyestone directs TransPharm's bovinetransgenic program. He told BioWorld Today, anent Bremel's PNASpaper, 'It's the first report of this kind of approach to appear in thescientific literature. That in and of itself indicates that the technology isin its infancy, as is to a large degree, the pure transgenic approach. Itremains to be seen if the somatic-cell transfection approach really takeshold.'Eyestone (who previously worked in Leiden, helping to constructHerman) added, 'In that context, the approach has tremendouspotential value, as an adjunct to the development of systems forproducing heterologous proteins by transgenic animals. There's noquestion that if the somatic technology were to develop and besuccessful, it may have much use for evaluating ultimately what couldbe produced in a transgenic animal.' Eyestone added, 'Whether itcould become a substitute remains to be seen.'Asked what response the transgenic companies had made to theWisconsin inquiries, Bremel replied, 'They expressed interest. Let'sput it that way. We'll have to see whether their interest goes to the nextlevel.'He is now attempting to set up a consortium in Wisconsin of interestedcompanies 'to do some of the basic biology that has longer-term timelines to evaluate than most venture capital companies can afford.'Most basic of all, at least at present, is the prospect of transfectingruminants via embryonic stem cells rather than retroviruses.Embryologist Neil First of Wisconsin's meat and animal sciencedepartment, reported last month in PNAS (Vol. 90, p. 6143) theproduction of calves by using bovine embryonic stem cells.TransPharm's Eyestone welcomes this feat. 'Certainly, our aim, andthe hope of everyone working in the transgenic livestock field, is tohave stem cells at some point. What they will allow us to do is thingslike homologous recombination, so that we can do gene substitutionsand knockouts, etc. It will make our genetic modifications a lot moreprecise and varied than what we now do with microinjection.' n

-- David N. Leff Science Editor

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