Transgenic Animals, biotechnologia UP Wrocław losowe pierdoły, Biotechnologia zwierząt i roślin, ...
[ Pobierz całość w formacie PDF ]//-->CH A P T E RFOURTransgenic AnimalsWalking Bioreactorsby S. Anne Montgomeryork on transgenicexpression systemsusing animals beganin the early 1980s,primarily as a way ofimproving the genetic characteristicsof livestock. Transgenic animalsacquire genetic material (sometimesfrom another species) throughhuman intervention rather thanthrough normal sexual reproduction.The hope was to accomplish withmicroinjection of functional genesinto ova what would otherwise takeyears with traditional breedingprograms: mosquitoes incapable ofcarrying malaria, for example, orproduction of leaner meat by beefcattle. In an early experiment, a“Super Mouse” was created when arat gene for growth hormone wasinjected into and expressed by thegenome of a parent mouse (1).Aside from applications designedto improve characteristics of aparticular species or to create“specialized” research animals(expressing green fluorescentprotein in zebrafish embryos, forexample, as a marker for geneticstudies) (2), transgenic technology isalso achieving increasing success asan alternative to producing proteinsin cell culture and microbialsystems. The goal of such work is toproduce large quantities ofrecombinant proteins in the milk orplasma of transgenic mammals or inthe eggs of transgenic hens. Manyof these efforts are progressingthrough clinical trials, and a few40BioProcess InternationalJUNE2004WGTC BIOTHERAPEUTICS(WWW.GTC-BIO.COM)companies appear to be close toachieving market approval. In fact,one company, GTC Biotherapeutics,Inc. (Framingham, MA; www.gtc-bio.com), is undergoing review formarket authorization in Europe forATryn, its recombinant form ofhuman antithrombin expressed inthe milk of transgenic goats.In the first successful case oftransgenic production of atherapeutic protein, mouse embryoswere injected with a DNA construct.It was made by inserting thepromoter and upstream regulatorysequence from the mouse wheyacidic protein gene (murine milkcontains distinctively high levels ofthis whey protein) into the genecoding for human tissue plasminogenactivator (tPA). The resultanttransgenic offspring producedbiologically active tPA in their milk: aheterologous protein of tremendoustherapeutic potential (3).Several companies are alreadyselling research-grade products(including “customized” mice)produced transgenically for use inmodeling human diseases inpreclinical studies or generatingantibody candidates for furtherdevelopment. Transgenic companieswith transgenic platform technologiesto produce clinical materials maypartner with other pharmaceuticaland biotechnology companies toproduce their products transgenicallyin addition to developing an in-housepipeline of products. Some of thesecompanies are also capable of thedownstream processing developmentand manufacturing of transgenicproducts, at least to clinical scale,whereas others rely on themanufacturing capabilities of partners.Transgenic production offerscertain advantages compared totraditional mammalian cellproduction systems. One sourcecompares the average generation of0.2–1.0 g/L of recombinant proteinin highly optimized cell cultures topossible expression levels of2–10 g/L of milk in transgenicSUPPLEMENTlivestock (4). Another mentions thatone sheep can produce 2–3 L ofmilk per day. If a recombinantprotein is expressed at a level of1 g/L, a single sheep could produceup to 20 g of product per week (3).Similar estimates are offered bymany other companies. Proponentsof transgenic technology also notethat scaling up transgenicproduction involves increasing thepopulation of a herd rather thanbuilding a mammalian cellproduction facility that costs tens,even hundreds of millions of dollars.The capital costs of building andmaintaining a farm are also small incomparison with building andmaintaining a typical biotech facility.Other companies are leveraging thecapabilities of transgenic productionto develop recombinant forms ofproteins, such as blood proteins,that can be difficult to express usingbioreactor based methods.Collecting source material from a“living bioreactor” also uses a well-established method: either milkingthe animals or gathering the eggs,depending on the species involved.Dairy farming already incorporateshygienic practices, and thecomposition of milk, even as it variesfrom species to species, is well known.“Known composition,” however,means that the milk must undergosome intermediate processing toremove much of its componentsbefore fluid is introduced as startingmaterial to downstream purificationby chromatography.Among the problems still to beworked out are efficiency and thespeed with which a commercialproduct can be produced in largeanimals. The current methods ofproducing transgenic animals have alow rate of live births: The typicalsuccess rate is 10–20%, and with theuse of microinjection techniques, thesuccessful expression of transgenes inoffspring runs at much less than 50%.The next challenge is to identifyand screen founder animals thatproduce high levels of protein. Afterthose founders are identified, it cantake months or years to breed andestablish a production herd,SUPPLEMENTdepending on the species and theirage to sexual maturity. Therefore, lowsuccessful expression of transgenes intransgenic offspring is not a problemwhen people are working with mice,but it is costly when developingtransgenic livestock. Recently, nucleartransfer technology has been shownto significantly reduce the timerequired for production ofrecombinant proteins and to morereliably establish “founders” to abreeding herd in which all offspringborn are transgenic.Each mammalian system willintroduce its own posttranslationalmodifications, especiallyglycosylation patterns (3).Mammary tissue can carry out abroad range of such modifications,but whether those modifications areimmunogenic to humans dependson the protein of interest and thespecies being used.Another concern is “leakage” of atarget protein into the circulation byway of the mammary epithelial cells— and as measured by increasedplasma levels of the proteindesigned to be expressed only in theanimal’s milk. Therefore, unless thetransgene construct integrates in anappropriate way in the genome,certain highly active hormones andcytokines could have detrimentaleffects on the host animal and maynot be possible transgenically.CREATING ATRANSGENICANIMALJE Smith, author ofBiotechnology,provides a useful sequential list ofsteps toward creating a transgenicanimal, which are generallyapplicable regardless of species:• Identification and constructionof the foreign gene and anypromoter sequences (geneticengineering)• Microinjection of DNA directlyinto the pronucleus of a singlefertilized egg (or introductionthrough nuclear transfer, a viralvector, or other means, as touchedon below)• Implantation of theseengineered cells into surrogatemothers• Bringing the developingembryo to termA milking parlorPurification skid and operatorPart of the purification process(GTC BIOTHERAPEUTICS)• Proving that the foreign DNAhas been stably and heritablyincorporated into the DNA of atleast some of the newborn offspring.• Demonstrating that the gene isregulated well enough to functionin its new environment (1).Figure 1 illustrates onecompany’s procedure.Construction of the Foreign Gene:Genetic engineering for a protein ofinterest has already been discussed inprevious chapters. The focus oftransgenic production, however, isthe construction of atransgene:agene foreign to the animal species inwhich it will be expressed. Arecombinant DNA construct isformed by combining a cloned targetJUNE2004BioProcess International41Figure 1:Making a transgenic product (SCHEMATICCOURTESY OFGTC THERAPEUTICS)protein gene with a regulatorysequence (promoter) of a milk-specific gene that will direct itsexpression to the mammary glandduring lactation (3, 5). Transgenicproduction of proteins in bloodplasma/serum, urine, and semen hasalso been investigated and may provefeasible for some unique products(e.g., see www.hematech.com forproduction of human polyclonalantibodies in transgenic bovineplasma, and www.polyclonals.com forproduction of humanized polyclonalantibodies in rabbits). Somecompanies are working on transgenichens, but milk appears to be theprimary choice for production ofrecombinant proteins. Companieshave developed proprietary mammarypromoters, some of which containadditional regulatory sequences tofurther direct expression forspecialized applications — such as tosecrete a protein that would normallybe membrane-bound.Why milk?Major milk-specificproteins are caseins and wheyproteins, most of which have beencloned and are well characterized.According to one publication, themammary gland — with a celldensity of up to 1000 times that ofa mammalian cell-culture bioreactor— can produce greater than 10grams of recombinant protein perliter of milk per day. (5).Although major differences existin milk composition from species tospecies, generally “milk isapproximately 85–90% water, the42BioProcess InternationalJUNE2004pH is 6.5–6.7 and as high as pH 6.8in ewe’s milk” (7). A target proteinexpressed in milk is usually found insolution with a colloidal mixture offats and proteins in which aresuspended casein micelles, somaticcells, and bacteria from thelymphatic ducts of the udder (7).Although purification methodsdiffer from company to company andare still being developed andoptimized, generally the raw milk isfiltered to remove fat, casein, cells,and other particulates, yielding a clearamber-colored fluid. That fluid thenundergoes a capture chromatographystep specific for the therapeuticprotein, followed by additionalchromatography steps to achieveclinical grade purity (7). So once thecapture is performed, downstreamprocessing is indistinguishable for theproducts of transgenic animals andcell culture or fermentation. Becausetransgenics dramatically lowers thecost of bulk production, theChromosomes from a transgenicanimal after fluorescence in situhybridization (FISH); the red and greendots in the upper left show integrationof the transgene.(GTC BIOTHERAPEUTICS)processing and purification stagestend to be the most expensive part ofthe manufacturing process in bothlabor and materials. The overall costof purification of transgenicallyproduced material is about the sameas that for bioreactor-producedmaterial.Lowering the Cost of Processing:Asan example of work being done tofurther improve the downstreamprocessing of transgenic proteinscontained in milk, BioSantePharmaceuticals, Inc. (Lincolnshire,Il; www.biosantepharma.com) haspatented calcium phosphatenanoparticle (CAP) technology forrecovering more than 90% of drugprotein from milk, requiring less(costly) downstream processing andperhaps resulting in higher yields.The scalable technique separates(dissolves) clusters of milk caseins,which make up 70–80% of total milkprotein, in initial processing steps;caseins tend to aggregate, trappingthe therapeutic proteins (8, 9).Speaking of costs, whereas milkcontains fewer proteins thantraditional fermentation broths,chicken eggs contain only 12 totalproteins — one of those beingovalbumin, which may be useful inprocessing or formulation down theline. A number of companies arepredicting successful production oftherapeutics in chicken eggs fromchimeric hens. So far they’reclaiming high, if variable, expressionlevels and the potential forsimplified purification. We focus ontransgenic mammals here onlybecause they are further along indevelopment as an expressionsystem. (For the same reason, we donot discuss investigations intotransgenic expression in blood,urine, and semen.)Microinjection:Pronuclearmicroinjection, although not theonly method under development,was the first method used. In thismethod, the fertilized eggs used tocreate the transgenes are flushedfrom the oviducts of “superovulateddonor females”: females that havebeen mated with fertile males andthat, depending on the species, maySUPPLEMENThave received pregnant mare serumgonadotropin, fluorogestone acetate,or prostoglandin (hormonallystimulating them to produce lots ofeggs at once instead of the one ortwo common in large animals). Thecritical step is then to develop thetransgene and get it into embryo andthe embryo into the host female. Inthis process, the transgene is injectedinto the pronucleus of a fertilizedegg. The technician uses a speciallydesigned micromanipulation pipetteand works under extrememagnification. It is tedious work, andnot all injections are successful.Nuclear Transfer:Some companiesare no longer using microinjectionand have developed methods totransfer nuclei isolated fromembryo-derived cells into oocyteswith their nuclei removed. Theadvantage of nuclear transfer is thatits success rate replaces the time-consuming process of cullingnontransgenic offspring from thebreeding program, and therebyaccelerates formation of thetransgenic herd.The process is explainedsuccinctly on the Geron web site:In this process, the nucleuscontaining all of the chromosomalDNA is removed from an egg celland replaced with the nucleuscontaining all of the chromosomalDNA from a donor somatic ornonreproductive cell. Fusionbetween the resulting egg cell andthe donor somatic nucleus resultsin a new cell which gains acomplete set of chromosomesderived entirely from the donornucleus. Mitochondrial DNA,providing some of the genes forenergy production, resides outsidethe nucleus and is provided by theegg. After a brief culture period,the resulting embryo is implantedinto the uterus of a female animal,where it can develop and producethe live birth of a clonedoffspring. The offspring isessentially a genetic clone of theanimal from which the donornucleus was obtained. (10)Insomatic cell nuclear transfer,also called therapeutic cloning, asomatic cell is fused with aenucleated oocyte. The nucleus ofthe somatic cell provides the geneticinformation, and the oocyteprovides nutrients and other energy-producing materials necessary forthe embryo’s development (11).Use of Viral Vectors:In anothermethod, the helper cell line from agene of interest is “packaged” intoan engineered viral vector: a virus stillencoded to “infect” but with thedisease-causing gene sequenceremoved (replicationdeficient).Thehope is that, if the virus is injectedinto the mammary gland duringhormone-induced mammogenesis,females could begin producing theprotein in milk without having towait through gestation; and theiroffspring would also express thetransgene (5, 12, 13). Productionlevels thus far are lower than desired(5), but in an early success, a gibbonape leukemia virus was used todeliver the structural gene encodingfor human growth hormone to agoat, and the hormone was expressedin her mammary epithelial cells.Implantation:After fertilized eggshave been washed from the oviductof a superovulated female donor andhave received the transgene, they aretransferred to the oviduct or uterusof a “pseudopregnant” recipientanimal and developed to term. Thoserecipients are prepared for embryotransfer by mating with vasectomizedmales. The offspring are eventuallytested through a blood or tissuesample (usually from the ear or tail)for presence of the transgene. Thenthe company must wait for thematurity of the animals to test forproduction of the protein of interest.When microinjection techniquesare used, not all offspring will expressthe transgene, and offspring that domay express it at different levels oreven in different organ systems. Theconsensus indicates that the successrate of germ-line transmission of thetransgene averages 50% or less formicroinjection. The insertion sitemay influence the expression levels oreven result in transgenic animalsshowing no expression at all. Forthese reasons, it is important tocharacterize multiple founders toselect lines with desired phenotypes,and if microinjection is used, severalgenerations may be required before astable transgenic herd is established.The transgene will, however, betransmitted to all offspring in nucleartransfer techniques.ANIMALS ON THEPHARMAlthough a small number ofcompanies are working to developcommercially viable transgenicproduction of protein therapeutics,many are working with multiplespecies and with a number ofpartnering agreements in place atmany different stages. Mosttransgenic species are studied forresearch applications as well aspotential commercial pharmaceuticalproduction.Caveats:Transgenics in general isa rapidly advancing field, andkeeping up to date on work inprogress is far from easy. Therefore,the following examples (presentedalphabetically by species) attemptonly to summarize informationabout work in progress that isreadily available; it is not inclusive,nor can it present the completestory of this segment of thebiotechnology industry. Efforts aremade here to use material no morethan two years old. Any claims ofcost savings and potentialtherapeutic yields are offered toemphasize the potential promise ofthe expression system, but thosediffer from company to companyand as the technology andexpression efficiencies advance (14).Chickens and Eggs:Chickens androosters grow faster than mostmammals, can be raised in closequarters, and can synthesize highlevels of protein in egg whites. Abig advantage in working withchickens is our familiarity with themgained from years of use in vaccineand antibody production. Eggscontain simple and well-characterized proteins (ovalbumin isa specific protein already present).They contain only 12 proteins to befiltered out compared with as manyas 20,000 in traditionalSUPPLEMENT44BioProcess InternationalJUNE2004fermentation. Chickens appear toadd correct sugars to glycosylatedproteins and can be raised at a costof around $20 a year per transgenicchicken (15). One rooster can matewith 10 hens in eight hours and canproduce 100,000 offspring a year.Products in developmentincludevaccines; interferons, commercialcytokines; human serum albumin;HSA, insulin, and MAbs (fromgermline transgenic chickens indevelopment). Additionally,development plans are ongoing (12)for proinsulin produced at$10/gram (in contrast with $1550to $3100 per gram using currentproduction methods).Companies, Milestones:Avigenics(Athens, GA, www.avigenics.com)holds a patent on its “WindowingTechnology” for injecting foreigngenetic material through an aperturein an egg shell; TranXenogen(Shrewsbury, MA; www.tranxenogen.com) holds a gene-testes transfectiontechnology and was the first toexpress MAbs in the whites ofchimeric chicken eggs (proof ofprinciple); TransGenRx (Dallas, TX;www.tgrx.com) and Viragen haveproprietary gene transfer vectors.Viragen (Plantation, FL;www.viragen.com) works with avector obtained from OxfordBioMedica plc (San Diego, CA, andOxford, UK; www.oxfordbiomedica.co.uk) with an exclusive license fromthe Roslin Institute (Edinburgh,UK; www.ri.bbsrc.ac.uk). Also ofinterest, GenWay Biotech (SanDiego, CA; www.genwaybio.com) is(among other activities), producinggene-specific IgY (chicken)antibodies.Cows:The prospect of obtainingthe large amounts of milk producedby dairy cows made them earlycandidates for studies into transgenicproduction. Dairy cattle produce 23g of protein/kg of body weightduring peak lactation. A 1997 articleestimated that one transgenic cowcould produce the annual US marketneeds for Factors VIII and IX; twocows could produce enough proteinC, three cows could produceenough antithrombin III, 17 cows46BioProcess InternationalJUNE2004could produce enough fibrinogen,and “35 103” cows could makeenough HSA (16). Thedisadvantages, however, include boththeir size (and therefore the cost oftheir “pharming” habitat) and theseven to eight years required toproduce a milking herd (3).Products in developmentincludeHSA, rHSA, and human milkprotein. A research farm in Alapitkä,Lapinlahti (Finland) is working toproduce lactoferrin for medical use(http://opp.ysao.fi/~pemo/future/breeding.htm).GTC BIOTHERAPEUTICS(WWW.GTC-BIO.COM)Companies:• GTC Biotherapeutics, Inc.(with about a dozen partners) (17)• The Dutch company PharmingBV (Leiden, The Netherlands;www.pharming.com) was the maincompany working with developmentof transgenic cows with the creationof Herman, the bull, designed tobreed progeny that producelactoferrin.• Hematech, LLC (Westport,CT; www.hematech.com) is workingon production of human polyclonalantibodies in transgenic bovineplasma.Goats:Goats are smaller thancattle and also produce a largeamount of milk in a shorter time.Expression though natural lactationtakes 15–18 months, but it can beinduced earlier.Products in developmentincludealpha-1 proteinase inhibitor ; MAbs,Ig fusion proteins, ATryn(recombinant human antithrombinIII); and tPA.Companies, Milestones:GTC’ssubmission of a marketauthorization application to theEMEA for Atryn is the firstapplication submitted in the UnitedStates or Europe for review andapproval of a recombinanttherapeutic protein producedtransgenically. It is also the firsttransgenic recombinant protein tocomplete phase III trials (18).Mice:Mice can be easily raised ina laboratory; gestation takes threeweeks, with sexual maturity reachedin one month, so initial results arepossible in six months or less. Theyare also inexpensive to maintain.Mouse milk has a higherconcentration of acidic whey protein— a desired characteristic for someapplications. Another advantage tousing transgenic mice in research isthat mice lack the cell-surfacemolecule that serves as the receptorfor the polio virus in humans;transgenic mice can express thehuman gene for polio and developsymptoms of the disease.Products in Development:Miceare mostly used in basic research fortransgenesis feasibility studies and asdisease models.Knockoutmicecreated with a nonfunctional geneare tools for studying genefunctions.Mice may yield small amounts ofmilk compared with larger species,but they are still powerful little“bioreactors.” Peptides derived fromantineoplastic urinary protein(ANUP) were shown to reducetumor burden by 70% in nude miceimplanted with human cervicalcancer cells (an avian transgenicplatform is in development forrelated recombinant proteinproduction). Other research withtransgenic mice includes expressionof malaria protein for possiblevaccine; MAbs and Ig fusionproteins; alpha-1 proteinaseinhibitor; antithrombin III;angiogenin; beta interferon; cysticfibrosis transmembrane regulator;Factor X; glutamic aciddecarboxylase; glucocerebrosidase;HGH, HSA, tPA, myelin basicprotein; proinsulin; prolactin; solubleCD4-HIV receptor; and fibrinogen.Companies, Milestones:The firsttransgenic mice were developed in1981. TranXenoGen holds aworldwide license for ANUP;Invitrogen is manufacturing,SUPPLEMENT [ Pobierz całość w formacie PDF ]