plant molecular farming: opportunities and challenges

Critical Reviews in Biotechnology, 28:153–172, 2008Copyright c© Informa UK Ltd.ISSN: 0738-8551 print / 1549-7801 onlineDOI: 10.1080/07388550802046624

Plant Molecular Farming: Opportunities and Challenges

Pervin Basaran and Emilio Rodrıguez-CerezoEuropean Commission, Joint Research Center (JRC), Institute for Prospective Technological Studies(IPTS), Edificio Expo, Calle Inca Garcilaso s/n, E 41092 Seville, Spain

Production of foreign molecules in transgenic plants is anticipated to be an alternative to alreadyestablished, microbial or animal expression systems with lower production costs. This articlereviews the different technologies and approaches currently used to produce economically in-teresting molecules in plants or plant cell cultures, to evaluate their technical feasibility andeconomic implications, and to assess the potential socioeconomic and environmental impactsderiving from the adoption of molecular farming products.

Keywords transgenic plants, genetically modified organisms (GMOs)

SCIENTIFIC STATE-OF-THE-ART AND CURRENTCOMMERCIAL APPLICATIONS

The recombinant production of pharmaceuticals, functionalproteins, industrial enzymes and functional secondary metabo-lites in plants is referred as plant molecular farming (PMF). Theterms molecular farming, biofarming, molecular pharming, phy-tomanufacturing, recombinant or plant-made industrials, planta-pharma, plant bioreactors, plant biofactories, pharmaceuticalgardening, and phytomanufacturing are used interchangeably(Basaran and Rodrıguez-Cerezo, 2008). Molecular farming inplants is expected by some to challenge already established pro-duction technologies for pharmaceuticals that currently use bac-teria, yeast, and cultured mammalian cells because plants lackhuman pathogens, oncogenic DNA sequences, prions, and en-dotoxins (Commandeur, Twyman, and Fischer, 2003). As re-search in using plants as manufacturing platforms becomes morewidespread, the commercial success will rest on the efficiencyof the technology, solving current drawbacks in the existingplant expression and production systems, safety of final prod-ucts, health and environmental testing, economic considerations,the readiness of the regulatory environment, intellectual prop-erty regimes, ethical issues, public acceptance, and overcomingof related social and policy challenges (Drossard, 2004).

Delivery and expression of heterologous genes in plants mayinvolve several strategies such as nuclear transformation, plas-tid (e.g., chloroplast) transformation, transient expression, viral

Disclaimer: The views expressed in this study are purely those ofthe authors and may not in any circumstance be regarded as stating anofficial position of the European Commission.

Address correspondence to Pervin Basaran, University, Depart-ment of Food Engineering, 32260 Cunur Isparta, Turkey. E-mail:[email protected] or [email protected]

transfection, and agroinfilitration (Bock, 2001; Gleba, Klimyuk,and Marillonnet, 2005). The major disadvantage of expressionin nuclear system is that the technology is unpredictable at itscurrent technology. Unnecessary DNA insertions, deletions orre-arrangement of inserted genes within the chromosomes mayoccur, the expression level of each gene is variable and diffi-cult to control, and there are valid regulatory concerns over thecontainment of nuclear transformed plants of which changesare inherited to new generations (Bogorad, 2000; Streatfield,2006). Another developing technology is the synthesis of plant-derived proteins in cellular plastids, which allow extremely highlevels of recombinant protein expression (Bock, 2007; Daniell,2002). Because, chloroplasts are transferred to progeny throughthe maternal inheritance, using chloroplast transformation tech-nology offers a natural containment with preventing transgenetransmit to non-genetically modified (GM) crops and wild rela-tives (Bock, 2007; Bogorad, 2000; Maliga, 2004). Furthermore,chloroplast transformation allows precise targeting of insertedgenes and accumulation of foreign proteins in an enclosed en-vironment (Bock, 2007). The major restriction of using chloro-plasts is the limited number of suitable hosts (e.g., tobacco)(Bock, 2007; Tregoning et al., 2003). When marker-free engi-neering within the plastids is desirable, there are several contem-porary applications available, including transient cointegration(Lutz and Maliga, 2007). Production of pharmaceuticals usingplant viral vectors is also a promising application (Pogue et al.,2002). These viruses are not infectious in humans or animalsand can accumulate large quantities of heterologous proteins inthe plants. Two major strategies are ‘full virus,’ which allows ex-pression of large fusion proteins in the coat, and a developing ap-proach of ‘deconstructed virus,’ which relies on Agrobacteriumas a vector to deliver DNA of one or more viral RNA repliconsto plant cells (Gleba, Klimyuk, and Marillonnet, 2007). Because

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154 P. BASARAN AND E. RODRIGUEZ-CEREZO

the gene is incorporated into the plant genome, it does not forma heritable trait and thus remains contained. Virus approach islimited primarily by the number and type of proteins that can beexpressed (Gleba, Klimyuk, and Maillonnet, 2007). Agroinfil-tration uses recombinant Agrobacterium tumefaciens infiltratedinto plant tissue and the T-DNA is transferred to the nucleus. Theshortcoming in the method of agroinfiltration is that the amountof culture that infiltrated the leaf and, ultimately the number ofcells into which the Agrobacterium delivered are variable be-yond control (Usharani, Periasamy, and Malathi, 2006)

Although in its initial stages, in recent years the plant molec-ular farming industry has demonstrated considerable growth inresearch and development activity around the world. In this studywe have identified more than one hundred and twenty smallcompanies, universities, and research institutes that are activein molecular farming in plants (Table 1). Nearly half of the re-search and development activities originate in North America(United States and Canada), and more than one third of theorganizations are based in Europe. A recent analysis revealedthat the total number of patents has increased more than threetimes between 2002 and 2006 timeframe, and by nationality,US inventors were followed by Germany, Denmark and Japan(Basaran and Rodrıguez-Cerezo, 2008). The patent ownershipdistribution between the private sector and academic institu-tions represented a trend towards the private sector (Basaranand Rodrıguez-Cerezo, 2008). The study here indicated that themajority of private entities are small companies with the capac-ity to produce small amounts of proteins with their host plants.The reasons stated for the lack of interest among research-basedlarge companies are concerns about investment returns, the lackof mature and detailed regulatory frameworks, and lack of com-mercially successful examples to date (Bischoff, 2004).

PRODUCTION OF PHARMACEUTICAL COMPOUNDSAND THEIR PROSPECTIVE APPLICATIONS

No plant-made pharmaceutical products are currently on themarket, but to date eighteen plant-derived pharmaceuticals havebeen submitted for clinical trials (Table 2).

Production of Plant-Derived AntibodiesThe most advanced work for human use of recombinant plant

proteins involves monoclonal antibodies, which are large, multi-meric glycoproteins that bind specifically to their cognate anti-gens and are exploited to treat diseases (Frigerio, 2000). Theincreasing market demand causes pressure for cost-effective al-ternatives for bulk manufacturing of antibodies. The amenablelarge scale production of recombinant antibodies is a challeng-ing task, because there are two distinct cell types (plasma andepithelial cells) involved in the production, and the final prod-uct is a complex molecule of almost 400 kDa, displaying nu-merous posttranslational modifications (intra- and inter-chaindisulfide bonds and glycosylation) and assembly requirements(proteolysis) (Chargelegue et al., 2005; Wieland et al., 2006).

The first successful assembly and expression of the secretoryimmunoglobulin A was reported in transgenic tobacco in 1998(Ma et al., 1995, 1998). At present, six different plant-derivedmonoclonal antibodies are being tested in clinical trials, namelyCaroRx (Phase II) for the prevention of dental caries (made intransgenic tobacco, designed to block adherence of the bacteriathat cause cavities), various single-chain Fv antibody fragmentsproduced by viral vectors in tobacco against non-Hodgkin’s Dis-ease (Phase I), IgG (ICAM1) for the prevention of commoncold (Phase I), an antibody against cancer (Phase II), and RhinoRX for the treatment of respiratory syncytial disease (Phase I).Recently, an anti-hepatitis B surface-antigen antibody receivedregulatory approval in Cuba for large-scale plant-made antibodyproduction (Pujol et al., 2005).

Plant-Derived Human and Animal Vaccinesfor Immunotherapy

Recombinant plant systems may be used as an economic al-ternative to produce animal and human vaccines. The primaryaim of work with plant-derived-vaccines is to prove that theyare comparable to the existing conventional vaccine productionmethods (Center for Infectious Diseases and Vaccinology, 2005;Paez et al., 2005). Surely, the main advantage is the potentialfor very large-scale production, particularly if open field-grownplants can be used. Furthermore, for biologically active vac-cines, immune responses against multiple antigens and toxinsmay be prerequisite, and in this manner, the expression of mul-tiple genes is possible in the same plant (Koprowski, 2005). InJanuary 2006, Dow Agro Science has received the world’s firstregulatory approval from U.S. Agriculture Department’s Centerfor Veterinary Biologics, and also met the stringent requirementsof FDA for a plant-made veterinary vaccine that protects poultryfrom Newcastle disease (www.thepoultrysite.com). The tech-nology uses plant a cell culture, instead of whole reproducingplants in an attempt to overcome existing environmental, agro-nomic and public concerns. The company indicated that it wasnot planning to commercialize the Newcastle vaccine (becausethere is apparently no compelling business case), but to use thesame technology to develop other vaccines for pets, horses, andanimals used for food production (www.thepoultrysite.com).

Plant systems could offer oral delivery option, overcomingthe cost and inconvenience of purification and injection of vac-cines (Yusibov et al., 2002). In the beginning, consumption ofantigen-bearing fruit and vegetables was proposed for this pur-pose. Considerations and hesitation about the regulatory require-ments such as consistency between lots and uniformity of dosagehave resulted in a refinement of the concept of “immunizationby eating” or ‘’edible vaccines.” Furthermore, achieving ade-quate immunogenic response with orally delivered vaccines re-quires an understanding of mucosal immunization; that is, manyorally delivered antigens induce a state of immunological un-responsiveness, known as oral tolerance (Tacket et al., 2000).Currently, orally administered vaccines are expected to be de-veloped more as soluble dry powders of highly purified antigens

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PLANT MOLECULAR FARMING 155

TABLE 1Partial listing of worldwide institutions and organizations actively involved in plant molecular farming research and development∗

Institution Country of origin Products or other indications

Abbott Laboratories USA Polyunsaturated fatty acids (PUFAs)AIST Japan PhytoremediationAjinomoto Co., Inc. Japan EnzymesAgouron Pharmaceuticals, Inc. USA AntibodiesAgrisoma Biosciences, Inc. Canada TherapeuticsApplied Biotechnology Institute USA Enzymes, vaccines, expression toolsAresa Denmark Land mine detectionArizona State University USA VaccinesAthena Bioproduction APS Denmark AntibodiesAubern University USA Expression toolsBASF Plant Sci. GMBH Germany PUFAs, modified starchesBayer Crop Science, BioScience Germany Vaccines, antibodies, enzymes, starchBiolex USA Locteron, antibodies, interferonsBIOPRO Baden-Wurttemberg Germany BioplasticsBiotechnology Foundation, Inc. USA AntibodiesBioTec-GEN Italy AntibodiesBoyce Thompson Inst. Plant Res. USA Edible vaccines, immunocontraceptionBrasilian Biotechnology Institute Brazil Vaccine production, antibodiesCarnegie Institution of Washington USA PUFAsCenter for Genetic Engineering and Biotechnology Cuba Antigens from hepatitis virusCentocor, Inc. USA AntibodiesChinese Academy of Agriculture Science China Animal vaccinesChlorogen USA Antibodies, cancer therapyChonnam National University Korea Enzymes, expression systemsChristian-Albrechts Universitat zu Kiel Germany Silk, biodegradable plasticClemson University USA Expression toolsCobento Biotech Denmark Human intrinsic factorCommonwealth Sci. & Ind. Res. Org. Australia PUFAsConopco, Inc. UK AntibodiesCornell University Research Found. Inc. USA Fagopyritol synthase, insulin mediatorCropTech Corporation USA Expression tools, enzymesDelta Biotechnology Ltd. USA Fusion proteinsDokuritsu Gyosei Hojin Nogro Seibutsu Sh. Japan NutraceuticalsDow Agrosciences USA Glycoprotein, vaccinesENEA-BIOTEC Sezione Genetica e Genomica Vegetale Italy VaccinesEpicyte Pharm Inc. USA Immunoglobulins, antibodiesERA Plantech Spain CalcitoninErasmus University Medical Center The Netherlands VaccinesEvogene Israel ToolsFarmacula BioIndustries Australia Bioplastics, vaccinesFlanders Interuniversity Institute for Biotechnology Belgium GlycosylationFraunhofer IME Germany Antibodies, vaccines, enzymesFreiberg University Germany Expression toolsHorticulture & Food Res. Inst. New Zealand Fragrance additiveGenesis Res. & Dev. Cor. Ltd. New Zealand Expression toolsGenset Corporation France AntibodiesGhent University Belgium AntibodiesGlycart Biotechnology AG Switzerland AntibodiesGreenovation Biotech GMBH Germany Glycosylation, antibodies

(Continued on next page)

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TABLE 1Partial listing of worldwide institutions and organizations actively involved in plant molecular farming research and

development∗ (Continued)


GREENTECH SA France Collagen and gelatinGTC Biotherapeutics, Inc. USA Polypeptides, antibodies, fusion proteinsGuardian Biotech Canada VaccinesHuman Genome Sciences, Inc. USA Fusion proteinsHungarian Academy of Sciences Hungary PhytoremediationIcon Genetics GmbH Germany Interferon, antibodies, enzymes, albumin,

insulinIRE-IFO Italy AntibodiesInsight Biopharmaceuticals Ltd. Israel EnzymesInstitut de Biologie Moleculaire des Plantes France VaccinesInstitut fur Pflanzengenetik und

KulturpflanzenforschungGermany Nutraceuticals, silk, antibodies

Institut National de la Recherche Agronomique France Expression toolsInstitute for Applied Science and Technology Portugal EnzymesInstitute Immunology Pla. China VaccinesInstitute of Bioorganic Chemistry Poland Swine vaccinesInstituto de Tecnologia Quimica e Biologica Portugal Production systemsInstituto de Virologia Argentina VaccinesInternational Centre for Genetic Engineering

and BiotechnologyItaly/India Chloroplast engineering

Israel Min. Agric. Rural Dev. Israel EnzymesIstituto Biosintesi Vegetali Italy AntibodiesJapan Chemical Research Co. Ltd. Japan Human interferonJohn Innes Centre UK Antibodies, expression toolsJohn P. Roberts Research Institute Canada Plant bioreactorsJulius-Maximilians Universitat Germany CytochromeKentucky Tobacco Research and Development

CenterUSA Expression tools

Korea Res. Inst. Bioscience & Biotechn. Korea VaccinesKumho Life & Environmental Science Lab. Korea Biodegradable plasticKyowa Hakko Kogyo KK Japan Heparin-binding polypeptide, insulin,

antibodiesLeiden University The Netherlands AlkaloidsLemna Gene France, USA Tools, enzymes, antibodiesLexicon Genetics, Inc. USA Human transferase, promoter moleculesLinnaeus Plant Sciences, Inc. Canada Vegetable-based motor oilLondon Health Science Center Canada BioreactorsMaltagen Forschung GmbH Germany Vaccines, lactoferrinMapp Biopharmaceutical, Inc. USA AntibodiesMassachusetts Institute of Technology USA SilkMax-Planck-Institut fur Molekulare

PflanzenphysiologieGermany Expression tools

Maxygen Denmark Human antitissue antibodies, immunoglobulins,PUFAs

Medicago, Inc. Sweden/Canada Therapeutic molecules, vaccines, antibodiesMeristem Therapeutics France Gastric lipase, albumin, human collagen, human

lactoferrin, antibodies, plasma proteinsMetabolix USA Enzymes, biodegradable plastic


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Millennium Pharmaceuticals Inc. USA Small target proteinsMonsanto Co. USA Expression toolsMonsanto Protein Technologies USA Expression of eukaryotic peptides, enzymesMPB Cologne GmbH Germany Transgenic peptides and proteinsNara Institute of Science and Technology Japan Expression systemsNational Botanical Research Institute India Rabies vaccineNational University of Ireland Ireland PharmaceuticalsNeose Technologies, Inc. USA Albumin fusion proteins, urokinasesNexgen Biotechnologies, Inc. Korea Vaccines, polypeptides,New York University USA AntibodiesNovartis USA AntibodiesNovo Nordisk As Denmark Coagulation factorsNovoplant GmbH Germany AntibodiesNovozymes Biotech. USA Recombinant polypeptidesNovozymes A/S Denmark Serine protease, antimicrobial polypeptides,

cellobiaseNucycle Therapy, Inc. USA VaccineOhio University USA GlycoproteinsORF Genetics Iceland Growth factors, cytokinesPeking Union Medical College China Soluble receptorsPeking University China PhytoremediationPhycotransgenics LLC USA AntibodiesPhytomedics USA PharmaceuticalsPhyton Biotech. Germany, USA Growth hormones, pharmaceuticalsPhytovation B.V. The Netherlands VaccinesPioneer Hi-Bred Int. Inc. USA Gums, expression tools, biodegradable plasticsPlant Bio Products Spain Biodegradable plasticsPlant Research International The Netherlands GlycoproteinsPlanet Biotech. USA AntibodiesPlantechno SRL Italy Enzymes, phytoremediationPlantigen Canada AntibodiesPlanton Germany Antimicrobial compoundsProdiGene, Inc. USA Antigens, enzymesPromega Corporation USA AntibodiesProtalix Biotherap. Israel Glucocerebrosidase, high mannose proteinsPurdue University USA IsoflavonesRubber Institute of Malaysia Malaysia AlbuminRutgers University USA Transformation, vaccinesSaponin, Inc. Canada Antimicrobial peptidesSchering-Plough Ltd. Switzerland TherapeuticsSemBioSys Genetics Inc. Canada Thioredoxin, insulin, multimeric proteinsSerono Genetics Institute S.A. France Expression toolsShanxi Agricultural University China Expression toolsSouth African Medical Res. Council South Africa HIV polypeptideSouth Carolina Center for Biotechnology USA Edible vaccineSouthern Crop Prot. & Food Res. Center Canada Therapeutic proteinsSt. Georges Hospital Medical School UK Vaccines


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SunGene GmbH & Co. KGaA Germany Lactoferrin, enzymes, immune molecules,transformation methods

Sunol Molecular Corporation USA Antibodies, pharmaceuticalsSuntory Ltd. Japan Glycoprotein expressionSyngenta participations Ag. USA Antibodies, nutraceuticalsQueens University Canada Plant bioreactorsTexas A&M University USA Expression in sugar caneThe Fraunhofer Institute Germany BioreactorsThe Macfarlane Burnet Institute for Medical

Research and Public HealthAustralia Vaccines

The Scripps Research Institute USA Antibodies, human protein expression methodsThe State University of New Jersey USA Expression systemsThe University of Queensland Australia Human cytochromesThomas Jefferson University USA Vaccines, antibodiesToray Ind., Inc. Japan Human interferonTufts University USA SilkUniCrop Ltd. Finland Antibodies, enzymesUnilever UK Feed additivesUniversidad Publica de Navarra Spain Expression vectorsUniversidade Federal do Rio de Janeiro Brasil PharmaceuticalsUniversita di Verona Italy VaccinesUniversity of Aachen Germany Expression tools, vaccinesUniversity of Agricultural Sciences Austria Expression toolsUniversity of Bristol UK PUFAsUniversity of Calgary Canada Expression hostsUniversity of California USA Vaccines, insulin, phytoremediation,

interferon, expression toolsUniversity of Cambridge UK Expression toolsUniversity of Cape Town South Africa VaccinesUniversity of Central Florida USA Expression tools, vaccinesUniversity of Central Florida USA Insulin, vaccines, phytoremediation, interferonUniversity of College Cork Ireland Expression systemsUniversity of Dublin Ireland Expression hostsUniversity of Freiburg Germany BioreactorsUniversity Georgia Res. Found., Inc. USA PectinsUniversity of Guelph Canada Recombinant protein productionUniversity of Hohenheim Germany VaccinesUniversity of Hong Kong China Expression vectors, vaccinesUniversity of Illinois USA VaccinesUniversity of Kentucky USA Tobacco-based expressionUniversity Kyoto Japan PharmaceuticalsUniversite Laval Canada Expression toolsUniversity of Malaya Malaysia Expression toolsUniversity of Maryland USA VaccinesUniversity of Melbourne Australia AntibodiesUniversity of Munster Germany Expression toolsUniversity of Naples Italy TransglutaminaseUniversity of Natural Res.& App. Life Sci. Austria Production systems


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University of Northern Iowa USA Expression toolsUniversity of Nottingham UK Expression toolsUniversity of Oxford UK AntibodiesUniversity of Rochester USA VaccinesUniversity of Rouen France VaccinesUniversity of Salzburg Austria Expression toolsUniversity of Vienna Austria Antibodies, glycosylationUniversity of Warwick UK Expression tools, vaccinesUniversity of Wisconsin USA VaccinesUniversity of Wyoming USA Spider-silk proteinUniversity of York UK BioremediationUniversity of Zhejiang China Animal vaccinesUniversity of Zhengzhou China BioremediationVentria Biosc. USA Lysozyme, lactoferrin, vaccines, milk proteinsVerdia Inc. USA Transformation methods, vaccinesVirginia Tech. USA Expression toolsWageningen University The Netherlands Antibodies, vaccinesWashington State University USA Enzymes

∗Based on patent applications, publications, and Web site information.

prepared through a combination of membrane recovery, purifi-cation, concentration, and drying, and delivered in capsule form(Daniell, Carmona-Sanchez, and Burns, 2004).

No phase III trial has yet been carried out with plant-derivedhuman vaccines (Pezzotti et al., 2005). To date, the results ofeleven clinical trials involving orally delivered plant-derivedvaccines have been published (Table 2). Escherichia coli andVibrio cholerae are two well-studied, heat-labile enterotoxigenicmicroorganisms that cause bacterial diarrhea. The β-subunits ofthese two toxins expressed in edible plants such as potatoes(Arizona State University), and corn seeds (Prodigene) showedpromising results in Phase I clinical trials (Tacket et al., 2005).Transgenic potato tubers and transgenic tomatoes carrying agene for Norwalk virus capsid protein have undergone clinicaltrials where potato-delivered proteins (The Boyce ThompsonInstitute for Plant Research) were found to be less effective thantomato-derived ones (The Boyce Thompson Institute for PlantResearch), probably due to different digestibility and absorbancein the gastrointestinal tract (Tacket et al., 2000). Clinical trials,on tomato powder in gelatine capsules, have been started at thesame institute (Khalsa, Mason, and Arntzen, 2004). There aretwo different reports of clinical trials of Hepatitis B virus pro-teins, a leading vaccine candidate, expressed in potato (RoswellPark Cancer Institute) and lettuce (Thomas Jefferson Univer-sity). The potato HBsAg vaccine was reported to boost serumIgG in half of the volunteers in the trials (Kapusta et al., 1999).A vaccine made of a Rabies glycoprotein produced in spinachby using viral vectors is also in Phase I trials (Thomas Jefferson

University). There are two reports of Human Papilloma Virus(HPV) (Khalsa, Mason, and Arnzten, 2004). More recently, alettuce-made measles vaccine has been tested for immunizationof mice (Webster et al., 2006), and HIV-1 Tat protein expressedin spinach plants was explored as a potential vaccine candidate inmice (Karasev et al., 2005). An unidentified viral vaccine againstunspecified disease of horses, dogs and birds is being tested inPhase I clinical trials (Dow Agro, USA), and a poultry vaccineagainst Coccidiosis infection has entereted to Phase II clinicaltrials (Guardian Biosciences, Canada). In contrast to edible vac-cines, there is little or no information on plant-derived injectablevaccines under development. In more recent reports, guinea pigswere immunized intramuscularly against foot-and-mouth dis-ease with transgenic tomato plants expressing two recombinantviral proteins (Pan et al., 2008), and mice intraperitoneally in-jected with recombinant alfalfa mosaic virus particles harbor-ing anthraxt vaccine, and elicited an immune response (Brodziket al., 2005).

Currently available plant-derived vaccine research resultsclearly indicate the need for providing consistent levels of target-ing and effectiveness with the intended delivery method (immu-nization through oral, aerosol, nasal, or skin patches). An assess-ment of the efficacy of plant-derived vaccines is still awaitingthe results of phase III clinical trials, only at the end of phase IIItrials (nearly year 9), effectiveness of the purification, the in vivosafety and stability, and the ideal dose be known; and the issueof any risk factors related to unintentional exposure recognized(Bischoff, 2004). Eventually, end product quality, stability, and

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TABLE 2Plant-derived pharmaceutical proteins for potential medical use that have reached clinical development

Product Treatment Recombinant source Clinical trial status Company

AntibodiesAvicidin Colorectal cancer Transgenic corn Withdraw from

Ph IIMonsanto Protein

Technology (USA)CaroRX Dental caries Transgenic tobacco Ph II Planet Biotech (USA)IgG (ICAM1) Prevention of common

coldTransgenic tobacco Ph I Planet Biotech (USA)

Fv antibodies Non-Hodgkin’slymphoma

Modified viral vectors intobacco

Ph I Large Scale Bio. Corp.(USA)

Rhino RX Respiratory syncytialdisease

Transgenic tobacco Ph I Planet Biotech (USA)

Antibody Cancer Transgenic corn Ph II NeoRX/Monsanto(USA)

VaccinesEsherichia coli

heat-labile toxinDiarrhea Transgenic corn

Transgenic potatoPh IPh I

ProdiGene (USA)Arizona StateUniversity (USA)

Vibrio cholerae Cholerae Transgenic potato Ph I Arizona State University(USA)

HBsAg Hepatitis B Transgenic lettuce

Transgenic potato

PhIPh II

Thomas JeffersonUniversity (USA)

Arizona State University(USA)

Lt-B vaccine Traveler’s diarrhea Transgenic corn Ph I ProdiGene (USA)TGE vaccine Piglet gastroenteritis Transgenic corn Ph I ProdiGene (USA)Norwalk virus capsid Diarrhea Transgenic potato

Transgenic tomatoPh I Arizona State University

(USA)Newcastle vaccine Newcastle viral

infection PoultryCultured transgene cells Approved (USDA)

commercialDow Agro (USA)

Viral vaccine mixture Diseases of horses,dogs, and birds

Tobacco transgene cells Ph I Dow Agro (USA)

Cancer vaccine Non-Hodgkin’slymphoma

Transgenic tobacco Ph II Large Scale Biotech(USA)

Viral vaccine Feline parvovirus Transgenic tobacco Ph I Large Scale Biotech(USA)

Poultry vaccine Coccidiosis infection Transgenic canola Ph II Guardian Biosciences(Canada)

Therapeutic proteinsGastric lipase Cystic fibrosis Transgenic corn

Transgenic tobaccoPh II Meristem Therapeutics

(EU)Human intrinsic

factorVitamin B12 deficiency Transgenic Arabidopsis Ph II Cobento Biotech AS

(EU)α-Galactosidase Fabry disease Transgenic tobacco Ph I Planet Biotech (USA)α-Interferon Hepatitis B&C Transgenic duckweed Ph I Biolex (USA)Lactoferrin Gastrointestinal

disordersTransgenic corn

Transgenic rice

Ph I

Commercial

MeristemTherapeutics (EU)Ventria Biosciences

(USA)


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TABLE 2Plant-derived pharmaceutical proteins for potential medical use that have reached clinical development (Continued)

Product Treatment Recombinant source Clinical trial status Company

Interleukin Crohn’s disease Transgenic tobacco Field trials Southern Crop Protectionand Food ResearchCentre (Canada)

Fibrinolytic drug(thrombolytic drug)

Blood clot dissolver Transgenic duckweed Ph I Biolex (USA)

Glucocerebrosidase Gaucher’s disease Plant cell Ph III Protalix(Israel)Insulin Diabetes Transgenic safflower Ph I Sembiosys (Canada)Apolipoprotein AI Cardiovascular Transgenic safflower Ph I (expected

in 2008)Sembiosys (Canada)

∗This product has been commercialized through Sigma Chemical Co., which specializes in products for research and diagnostic purposes.

safety must be consistently assured in order to obtain the regu-latory approval necessary to commercialize any pharmaceuticalproduct, regardless of the production source whether plant orelse.

Pharmaceutical and Nutraceutical Protein ProductionThe first reported pharmaceutical protein made in plants was

tobacco callus expressing human growth hormone (Barta et al.,1986). Tobacco plants, genetically modified to produce inter-leukin, an enzyme used in treatments for disease (e.g, Crohn’sdisease), are being tested in field trials in Canada by the South-ern Crop Protection and Food Research Centre (Einsiedel andMedlock, 2005). Gastric lipase, an enzyme produced from trans-genic corn and used to treat cystic fibrosis, is in Phase II clini-cal trials (Meristem Therapeutics, France). Human intrinsic fac-tor, to be used against vitamin B12 deficiency and producedin transgenic Arabidopsis thaliana plants has started Phase IIclinical trials (Cobento Biotech, Denmark) (Horn, Woodard,and Howard, 2004). Lactoferrin (a functional food ingredient),produced from transgenic corn and used as a dietary supple-ment (Meristem Therapeutics, France), and α-galactosidase forthe treatment of Fabry disease produced in transgenic tobacco(Planet Biotech, USA) have reached the phase I trial stage. Re-garding other pharmaceutical proteins, enterotoxin B producedin transgenic corn (ProdiGene, USA) and potato (Arizona StateUniversity, USA), and aprotinin have reached the phase I trialstage. The companies working on other human products includeSembiosys (Canada), which is developing a method of produc-ing human insulin in plants; Ventria (United States), which isworking with rice to develop treatments for childhood dysentery;and Chlorogen (United States), which is experimenting with to-bacco to produce pharmaceutical products in plants for ovariancancer therapy (www.plantpharma.org). More recently, a bloodclot dissolver produced in duckweed (Biolex, United States) hasentered phase I clinical trials, and an apolipoprotein expressedin safflower (Sembiosys, Canada) to be used against cardio-vascular diseases is expected to enter clinical trials in 2008.Based on the scientific data available in 2005 and 2006, poten-

tial products expected to be in clinical trials in the short term thathave been patented as potential molecular farming products in-clude synthesis of long-chain polyunsaturated fatty acids, humanmilk proteins in transgenic plants, and microbicide productionin transgenic plants.

Industrial Enzymes and Other Molecules for Industrial useDevelopment of non-pharmaceutical products from plant

molecular farming has made them faster to the market. Elevenplant-derived proteins (avidin, tyrpsin, β-glucuronidase, apro-tinin, lactoferrin, lysozyme, thyroid-stimulating hormone re-ceptor, Hantaan and Puumala viral antigens, peroxidase, lac-case and cellulase) were developed and marketed (Table 3).In 2004, TrypZean, the industry’s the first commercial proteinproduct from transgenic plant technologies recombinant bovine-sequence trypsin, was expressed in corn (ProdiGene) (Horn,Woodard, and Howard, 2004). Trypsin has several commercialapplications, including the manufacture vaccines and it is alsoused in large quantities in the detergent and leather industries(Howard and Hood, 1998). Historically, this protein has beenisolated and purified from bovine or porcine pancreas; how-ever, this practice has come under increasing scrutiny due toconcerns about bovine spongiform encephalopathy (Woodardet al., 2003). Peroxidase and laccase are fungal enzymes largelyconsumed in paper industry and the encooding genes have beenexpressed in maize (Applied Biotechnology Institute, UnitedStates). With increasing oil prices, biofuel production fromplants has become important. The cellulose composed of longchains of glucose molecules, is the most abundant biopolymer inplant cell walls. When hydrolyzed by the cellulase enzyme, thesechains are broken down to glucose monomers which can even-tually be fermented to ethanol by microorganisms. Recently,genes expressing fungal cellulase has been expressed in corn toproduce large quantity of this enyzme (Applied BiotechnologyInstitute, United States).

Biodegradable plastics are environmentally friendly alter-native to petrochemical polymers. The first plastic-like com-pound synthesized in transgenic plants was polyhydroxybutyrate

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TABLE 3Commercial industrial proteins from plant molecular farming

Source Commercial CompanyProduct Trade name Pharma crop of genes purpose producing

Avidin Recombinant Transgenic corn Chicken Research & diagnosis ProdiGene (USA)avidin

Aprotinin AproliZean Transgenic corn Cow Research & diagnosis ProdiGene (USA)Aprotinin AproliZean Modified viral vectors

in tobaccoCow Research & diagnosis Large Scale Biol.

(USA)β-Glucuronidase N/A Transgenic corn Bacteria Research & diagnosis ProdiGene (USA)Trypsin TypZean Transgenic corn Cow Research & diagnosis ProdiGene (USALactoferrin N/A Transgenic rice Human Research Ventria Biosc. (USA)Lysozyme N/A Transgenic rice Human Research Ventria Biosc. (USA)Thyroid-stimulating

hormone receptorN/A Transgenic oriental

melonHuman Diagnosis Nexgen (Korea)

Hantaan and Puumalaviral antigens

N/A Transgenic orientalmelon

Virus Diagnosis Nexgen (Korea)

Peroxidase N/A Transgenic Corn Fungus Paper bleaching Applied BiotechnologyInstitute (USA)

Laccase N/A Transgenic Corn Fungus Paper bleaching Applied BiotechnologyInstitute (USA)

Cellulase N/A Transgenic Corn Fungus Ethanol production Applied BiotechnologyInstitute (USA)

(PHB), reported by Poirier and his colleagues (1992). In thecase of biodegradable plastic-like compounds, high expressionof PHB has been achieved, although in most cases a negativecorrelation between PHB accumulation and plant growth hasbeen reported. Spider-silk proteins, elastins and collagen havebeen expressed in transgenic plants (Scheller and Conrad, 2004).In the case of spider-silk proteins, only the major ampullatespidroins I and II or their artificial derivatives have been pro-duced in plants (Scheller et al., 2004). Collagens have also beenproduced and processed in transgenic plants, in which the intro-duction of a second gene was reported as necessary for success-ful protein processing (Christou, Stoger, and Twyman, 2004).

TECHNOLOGICAL CONSTRAINTS, SAFETY ASPECTS,CURRENT LIMITATIONS AND FUTURE POTENTIALSOF THE TECHNOLOGY

Efficacy and Reliability of Recombinant Plant ProductsSeveral alternative plant production systems are being devel-

oped. Each system has its strengths and weaknesses in termsof the economics of production, the location of the expression,options for alternative routes of administration, authenticity ofproducts, and ease with which the agricultural production systemcan be controlled (Chong et al., 1997; Maliga, 2002; Mayfield,Franklin, and Lerner, 2003). The choice of the plant to be useddepends on a number of factors, including its cultivation meth-ods, transformability, growing cost, production and processingof the target tissue, existence of wild relatives, and degree of

outcross with wild relatives (Howard and Hood, 2005). Thetype of tissue that is used for protein accumulation is oftenchosen on the basis of the type of plant used and the levelof expression. Fischer and Stoger (2004) gave an overview ofthe emerging plant platforms, cultured cells, and their advan-tages and disadvantages. A large number of species have beentested. Open to molecular farming are following: model plants(tobacco, Arabidopsis), cereal crops (wheat, rice, maize, bar-ley), legumes (pea, soybean, alfalfa, peanut, pigeon pea), fruitcrops (tomato, banana, carrot), solanaceous species (potato),and oil crops (oilseed rape, safflower, camelina) (Hood, 2002;Twyman, 2004). Recently, there have been significant develop-ments in the use of more diverse plant species such as green algae(Chlamydomonas reinhardtii), duckweed (Lemna), and moss(Phscomitrella patens), which can be propagated and trans-formed to produce the recombinant proteins (Gasdaska, Spencer,and Dickey, 2003; Mayfield, Franklin, and Lerner, 2003). Theuse of nonfood or nonfeed crop plants would obviously eliminatethe possibility of unintentional and unwanted contact and con-tamination of the food or feed supply, yet may increase the en-vironmental and agronomic concerns if the species chosen haveknown weedy or invasive characteristics (Howard and Hood,2005).

There are practical issues related to the large-scale cultiva-tion of certain plant species that previously were not suited orproposed to conventional agriculture (Datar, Cartwright, andRosen, 1993). Expression stability and the biochemical activ-ity of the product are important for plant-based production

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systems. Molecular farming requires that DNA encoding theprotein of choice be introduced into the plant of choice. Multi-ple methods of human-directed foreign DNA transfer of nucleiinto plants have been developed: Agrobacterium-mediated trans-formation, micro projectile, bombardment, electroporation, sil-icon carbide fibers, electrophoresis, and microinjection (Hoodand Jilka, 1999). Thus far, it has been difficult to evaluate therelative performance of different crops for industrial or pharma-ceutical molecular farming because this requires the productionof the same protein in a range of hosts, using a ‘standardized’ ex-pression construct. The performance of an expression constructacross species is itself difficult to judge, because the same pro-moter (or the regulatory sequence) may have a different intrinsiclevel of activity and specificity in different genetic backgrounds(Inaba et al., 2007). Production of recombinant proteins in plantsrefers to the growing, harvesting, transport, storage, and tissueprocessing of the crop after harvest, as well as extraction andpurification of the molecule of interest (Wilde et al., 2002). Ex-pression of foreign molecules in plants is the most importantfactor for most recombinant proteins and can dictate the eco-nomics of the product as well as regulatory issues (Wilde et al.,2002).

The amount of the protein as a percentage can range from 1%to over 40% depending on the plant and tissue source (Howardand Hood, 2005). The major factors in this decision are biomassyield, ease of transformation and in vitro manipulation, and con-venience of scale-up. Choice of host species and productionsystem is also affected by setup costs, scale-up and maintenancecost, length of production cycle, biomass yield, costs of pro-cessing and edibility, costs of storage and distribution, and costsof contamination clean up (Schillberg et al., 2005). As seedscontain a less complex mixture of proteins and lipids, this ap-pears to offer an advantage for purification (Lamphear et al.,2002). Fiedler et al. (1997) reported a 50% loss of functionalantibodies after eighteen months in storage. In addition to theneed for high-level protein expression to provide good yieldsin plant-based production systems, the efficient recovery of re-combinant proteins from plant tissues must also be evaluatedin care and eventually optimized. At first, protein expressionin edible fruit and vegetable crops has been considered advan-tageous, because edible organs can be consumed as uncooked,unprocessed or partially-processed material, making them idealfor the production of recombinant subunit vaccines, nutriceu-ticals and antibodies designed for topical application (Yu andLangridge, 2003). However, the term “edible vaccines” is nowbeing avoided by researchers in this field so as to avoid givingthe mistaken impression of specialty foods.

Tobacco has been adopted as a platform by several biotech-nology companies, including the only two companies that haveplant-derived pharmaceuticals undergoing phase II clinical trials(Table 1). The popularity of tobacco reflects its status as a well-established expression host for controlled transgene expression(Fischer et al., 2004). Furthermore, it is also a nonfood, nonfeedcrop that carries a reduced risk of transgenic material contami-

nating feed and human food chains (Stoger et al., 2005). There isalso an emerging tobacco transient-expression technology basedon plant virus expression vectors, one of which is undergoingphase I clinical trials (McCormick et al., 1999, 2003). The ma-jor disadvantages of tobacco are its high content of nicotine andother alkaloids, which have to be removed completely duringdownstream processing after harvest, and the fact that proteinsexpressed in the leaves tend to be unstable and unprotected fromproteolytic degradation, which means that the harvested materialhas to be processed immediately (Fischer et al., 2004).

The major technological bottleneck to be addressed by re-searchers is to ensure that the structure and functionality isequivalent to that of the native form (Ammann, 2004). Plantsare preferred over prokaryotic hosts, such as E. coli, from theexpression of recombinant proteins because most C- and post-translational protein maturation events, including signal peptidecleavage, protein folding, disulfide-bond formation, and glyco-sylation (sugar attachment), are similar in mammals and plants(Frigerio et al., 2000). However, there are some crucial vari-ations in posttranslational modifications, specifically covalentlinkage of sugar chains in the formation of mature proteinsin plants and animals (Saint-Jore-Dupas, Faye, and Gomord,2007). These posttranslational maturations can have dramaticeffects on the accumulation of a recombinant protein; if pro-teins are not properly cleaved or conformed, proproteins willaccumulate instead of mature proteins (Bakker et al., 2001).Gomord and Faye (2004) discussed the similarities in and dif-ferences between posttranslational protein modification in plantand mammalian hosts that are relevant to the posttranslationalmodification of therapeutic proteins in plants. Differences inthe glycosylation patterns of produced proteins in plants andhumans have raised concerns regarding the potential immuno-genicity of plant-specific complex N-glycans, which are presentin the heavy chain of plant-derived proteins (Chargelegue et al.,2000). Also, there is the question of whether recombinant pro-teins bearing plant N-glycans are immunogenic in humans(Gomord et al., 2004, 2005). Several tools and strategies forproducing human proteins are under development (Bardor et al.,2006). Two major strategies tested are (1) in vitro attachmentof glycan structures to recombinant proteins by using modi-fied bacterial galactosyltransferase enzymes (Bernatchez et al.,2007), and (2) alternatively co-expression of human β-1,4-glycosyltransferase together the gene of interest in transgenicplants to produce recombinant antibodies extended with galac-tose glycans (Bakker et al., 2001).

Scale-Up Considerations for Downstream Processingand Recovery

The yield of the recombinant proteins produced in a plantsystem depends on three factors: the intrinsic limitations of theproduction host and the expression system, limitations imposedby the level of transgene expression, and limitations imposed bythe stability of the recombinant protein (Schillberg et al., 2005).Most studies indicate that efficacy could be further improved

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through higher yield in plants. Stability and degradation of theproduct are the major issues in choosing the production sys-tems, and industry would prefer plant production systems thatoffer the possibility of long term storage and easy transport fromharvest to downstream processing facility. This is difficult if thetransgenes are expressed in fragile leaves due to faster proteindegradation (Ma et al., 1994). Despite the seed-based produc-tion, not all target proteins can be expressed in seeds (Boschet al., 1994). Nevertheless, seed propagation makes it neces-sary to monitor the stability of the transgene expression afterreamplification from the master seed collection (Miele, 1997),for example, in a study, E. coli Lt-B protein was not found tobe uniformly distributed throughout the defatted maize germ(Lamphear et al., 2002).

Techniques have been developed for the expression of pro-teins in stable transformed plants (where the gene is incorporatedinto the plant genome) or in transiently transformed plant tis-sues (Schillberg et al., 2005). Transient gene expression is rapidcompared to stable plant transformation and gives results in days(Cramer et al., 1999; Mercer et al., 2001). However, transientgene expression is limited in scale and is generally preferredto test the new constructs used for protein expression before astable transformation is performed (Schillberg et al., 2005). Inaddition, transient expression systems involving viral vectors oragro infiltration are effective ways of obtaining moderate quan-tities of recombinant product within a very short time frame(Roggero et al., 2001). The overall yield can be higher with theviral vector, which can spread to all cells. This production wasapplied by Large Scale Corp. to develop personalized vaccineagainst B-cell non-Hodgkins lymphoma (Bischoff, 2004). Viralvectors that produce high levels of protein in leaves have beenshown to be efficient in the case of hepatitis B virus core antigenusing tobacco mosaic virus vector and mice immunized mucos-ally (oral and intranasal) with the extract (Huang et al., 2006).When sufficiently high levels of expression and protein accumu-lation are achieved, efficient downstream processing protocolsmust be developed to ensure product quality and the economicfeasibility of production. For instance, tobacco has a long andpromising history in molecular farming, but due to its high con-tent of nicotine and other alkaloids it has drawbacks in certainproduction processes. Maintaining the expression levels of re-combinant proteins within a defined range is also a prerequisitefor edible vaccines (Wang et al., 2008). The ideal crops shouldalso be amenable to transformation and regeneration. In termsof speed and fidelity, crops that can be reproduced by vegetativepropagation might be preferred.

Bio- and Environmental Safety Aspects of MolecularFarming in Plants

In a case study conducted in Canada, participants raised majorquestions and concerns about the impact that molecular farm-ing would have on water supply, quality of the soil, and theexisting ecosystem (Meurer, 2004). Specific bio-safety aspectsof the cultivation of molecular farming crops fall into two major

groups: transgene spread and unintended exposure. The trans-gene spread can result from cross-pollination of the molecularfarming crop and conventional crops or weed relatives (Jiangand Sung, 2002). Unintended exposure can originate from thecommingling of molecular farming harvests with conventionalor organic harvests further down in the food chain.

Risk assessments of those transgenic plants destined for thepharmaceutical and industrial production vary according to thetype of confinement and containment measures. Confinementmeasures include agronomic and genetic measures to keep reg-ulated products in an open space, such as in a field, whilecontainment requires a closed physically isolated environmentfor production (Menassa et al., 2001). Agronomic confinementmeasures the geography and physical parameters of the field, un-cultivated ground and surroundings, physical isolation from sex-ually compatible plants, crop destruction and postplanting sea-son, use of dedicated nonfood/nonfeed equipment and facilities,planting at a different period from other sexually-compatibleplants in the area to ensure that the fertility periods will inter-sect (Brandle, 2004; Spok, 2007). The use of extensively grownfood or feed commodity crops for molecular farming purposesincreases the possibility of unintentional mixing with food orfeed supplies, if the molecular farming is conducted under non-confined conditions (Jiang and Sun, 2002). The containment canbe physical and it is proposed that transgenic plants can be main-tained in greenhouses, in artificially irrigated desert plots, or inunderground caves, and specific treatment in the field such asconcealing of flowers and fruits should be applied (Tackaberryet al., 2003). Some suggest production in a closed-loop produc-tion as a fully confined and contained production system. Bio-logical containment measures such as self-pollinating species,chloroplast transformation, or crops with no sexually compati-ble wild relatives may provide additional natural barriers (Bock,2001; Commandeur, Twyman, and Fischer, 2003). Alternativesinclude growing plants in glasshouses or other types of con-tained environments to ensure isolation from food crops. Themajor containment practices currently available are greenhouseproduction, the use of hydroponics, indoor growing facilitiesor laboratories, and use of sterile males, etc. (Commandeur,Twyman, and Fischer, 2003; Fu et al., 2000). The use of plantcultures that can be maintained in a precisely controlled envi-ronment of stainless steel tanks and not whole plants sproutingin open fields as practiced in the case of Dow Agro vaccineproduction.

Adaptation of current agronomic practices for manufacturingof pharmaceuticals in plants can be essential. The recombinantproteins with medicinal properties may present the risk of illeffects on nontarget organisms, particularly insects, birds andsoil microorganisms, by simple exposure to plants in the field(Lee et al., 2003). The element of danger posed by accidentalingestion or exposure to plant made pharmaceuticals also needsto be addressed. These proteins might have a direct toxic effecton bystanders and the consumption of dead decaying transgenicplant material by saprophytes and other insects is also a cause

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of major concern. Although antibiotic resistance genes are themain focus because of their generally selective advantage in hu-man pathogens, horizontal transgene transfer from plants to mi-crobes is considered to be extremely small because no gene flowto bacterial strains has been demonstrated to date. Furthermore,all natural plants are already covered with bacteria naturally car-rying antibiotic-resistant genes (Nielsen et al., 1998; Schluter,Futterer, and Potrykus, 1995). Persistence of plant molecularfarmed by-products in the soil is reported as another concern(Freese, 2002). Even though the biopharmaceuticals are proteinsand therefore generally expected to break down more rapidlythan synthetic drugs, several plant-grown insecticidal and drugproteins have been shown to have surprising stability (Freese,2002). Methodologies for purified industrial pharmaceutical for-mulations require development of quality assurance and qualitycontrol processes, and defining detailed after-processes such assterilization, ecologically sound waste disposal is a requirement(Daniell, Khan, and Allison, 2002).

Non-food plants could be grown in containment or iso-lated locations. However, if food crops must be used, it is rec-ommended to apply risk-based containment and confinement;phenotypic variation (obvious, traceable visual changes, ifpossible); exclusive facilities for guidance; strict monitoring andenforcement regulations; liability issues; insurability of industryas security; supply-chain training and certification; training offarmers, workers, and auditors; and development of guidelines,and finally procedures for robust identification and traceabil-ity of plants and their derived products (Spok, 2007). Some ofthese have already been implemented by the industry, includingmonitoring (Spok, 2007; Nap et al., 2003).

Loss of confinement of modified plant products could re-sult in the adventitious presence of unregulated products, andhave an adverse impact on food safety (illness, injury, distress),potentially leading to trend name exposure, recalls, and result-ing domestic and international market disruption (Commandeur,Twyman, and Fischer, 2003; Daniell, 1999; Mascia and Flavell,2004). Cropping systems and climate may need to be modeledfor cross pollination (Argevin et al., 2008), accordingly guide-lines would be prepared for the design and operation of con-tainment spaces. From the safety viewpoint, it is expected thata safeguard of exceptional proportions compared to other agri-cultural activities would need to be put in place, for instance, inrespect to threshold levels required for organic farming labels.Currently, there are no enforced tolerance levels for pharmaceu-ticals and industrial products produced in transgenic plants oranimals, in the regulatory frameworks of USA, EU, Canada andelsewhere. The point is made that there is a lack of appropriatetools to manage a betrayal of confinement, such as safety assess-ments; detection procedures for rapid qualitative evaluation ofexposure; executing preestablished corrective measures-actions,including recall policies, transgenic biomass destruction, foodand feed recalls in case of human errors and contamination; andeventually preestablished corrective measures (contingencies),etc. (Commandeur, Twyman, and Fischer, 2003). This increases

hesitation of the general public on prospects of plant molecularfarming. Production of plant cell-organ cultures in bioreactorsis more costly than conventional open field production, but of-fers the advantage of a high level of containment and controlledproduction with good manufacturing practices (Shadwick andDoran, 2004; Sharp and Doran, 2001).

The possible transgene escape through pollen or seed disper-sal and the potential for recombinant molecules to enter the foodchain are two major public concerns about coexistence (Easthamand Sweet, 2002). The risk of transgene spread can be definedas the potential for transgene DNA sequences to spread outsidethe intended host plants and production site. This is primar-ily associated with nuclear transformation rather than chloro-plast transformation or use of viral vectors, and, furthermore, acase-by-case risk assessment of each particular crop-moleculecombination would be needed for final conclusions. Experiencegained from co-existence studies demonstrates that the technicalfeasibility studies of production at different thresholds is neces-sary (Gressel, 2000; Smith, Kilpatrick, and Whitelam, 2001). Anumber of potential solutions to the problem of transgene pol-lution are based on prevention of the spread of transgene pollen,by either physical or genetic containment. The requirements forproducing nonfood products in transgenic plants are expected tobe considerably different from those for producing food prod-ucts. They include physical isolation, agronomic support (e.g.,delayed planting times for food crops), dedicated equipmentand frequent monitoring of contamination. Seed dispersals fromtransgenic crops also result in colonization of natural ecosystem,and this is more prevalent if the crop is dormant (Gruber et al.,2001). Gene flow from transgenic to non-transgenic relative re-cipient populations of the same crop occurs if two populationsare close enough for cross-contamination, as documented in twostudies. Kling (1996) reported 50% of wild strawberries grow-ing near a field where cultivated transgenic strawberries con-tained marker genes. Similarly, herbicide resistance genes haveintrogressed from transgenic oilseed rape (Brassica napus) intoits weedy cousin B. campestris by hybridization (Mikkelsen,Jensen, and Jorgensen, 1996). Yet, there are however examplesof spontaneous hybridisation in the field between oilseed rapeand a number of its wild relatives (e.g., wild turnip [Brassicarapa]). Another group of crops are those that have relatives withwhich they are more or less fully compatible. Sugar beet (Betavulgaris ssp. vulgaris) which has been the subject of many GMfield trials, can hybridise readily with wild sea beet (Beta vul-garis ssp. maritima), but the removal of flower spikes beforeflowering and efficient segregation measures manages the risk(Smyth and Philips, 2002). There are already similar segregationregulations applied for high erucic acid rape, whose industrialproduct erucic acid is toxic, and so the production protocols aretightly defined (Smyth and Philips, 2002).

The risk of unintended exposure can be defined as the po-tential for nontarget organisms (including humans and farm an-imals) to come in contact with recombinant protein producedby a transgenic plant. Horizontal gene flow, which also includes

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horizontal gene transfer by asexual means, is not the same aspollen dispersal. The different mechanisms are outlined, includ-ing herbivory and parasitism; exposure of pollinating insectsto transgenic pollens; the exposure of microbes’ rhizosphere toroot exudates; the exposure of non-target microbes and animals(e.g., gastrointestinal tract of farm animals) to proteins secretedin the leaf guttation fluid; and the release of recombinant pro-teins by dead and decaying transgenic plant material (Comman-deur, Twyman, and Fischer, 2003; Eastham and Sweet, 2002).Although these risks apply to all field-grown transgenic crops,those planted for molecular farming deserve special attention be-cause of pharmaceutical or consequently toxic properties, andthere are apparent informational gaps in the exposure and riskassessments that need to be analyzed and evaluated qualitatively.

ECONOMIC EVALUATION OF RECOMBINANTPRODUCTION TECHNOLOGY IN PLANTS

Plants can be altered in ways that increase their value asagricultural trade commodities. Molecular farming is seen asa new subset of agrobiotechnology, within the estimated $550billion global pharmaceutical market in 2006. Several analystspredict that recombinant biopharmaceuticals (animal or plant)may make up 35% of the market by 2010 (Arcand and Arnison,2004). One of the driving forces behind molecular farming ofpharmaceuticals in plants from the industrial perspective wouldbe the low initial investment needed when compared with mam-malian cell culture production (Bischoff, 2004; Maliga andGraham, 2004). It has been estimated that proteins can be pro-duced in plants at 2% to 10% of the cost of microbial fermenta-tion systems, and at 0.1% of the cost of mammalian cell culturesor transgenic animals, as long as adequate yields can be achieved(Hood, Woodard, and Horn, 2002). The cost of producing anyrecombinant protein in cell fermenters is estimated to be in ex-cess of $300 per g of protein that is approximately 3,000 timeshigher than estimates for recombinant plants (Hood, Woodard,and Horn, 2002; Walsh, 2003). The cost of antibody produc-tion in plants is expected to be half that of transgenic animalsand 20 times lower than in mammalian cell cultures (Daniell,Streatfield, and Wycoff, 2001). One bushel of corn-producingrecombinant avidin at 20% of total soluble seed protein is es-timated to yield the same as a thousand kilos of chicken eggs(which is the common source of avidin) (Twyman et al., 2003).It is predicted that enough hepatitis B-antigen to vaccinate allthe babies in the world each year could be grown on roughly200 acres of land, and all the HBV vaccine required annuallyin China could be produced on 40 acres (Center for InfectiousDiseases and Vaccinology, 2006).

Costs related to plant-made products may comprise devel-opment costs of the new transgenic product, the costs requiredobtaining authorization for the new transgenic product, the costof industrial production, the cost of the final purified and pro-cessed product, and lastly the additional costs required to over-come negative public view, if any (Sedjo, 2005). Although thedirect oral delivery of plant extracts might also be considered

possible for medicinal use; for the efficiency, safety and stabilityof the transgenic product, a high level of protein purification-as it is the case for all pharmaceuticals produced today- wouldbe essential. Therefore, the production cost in the agriculturalfield would constitute only a fraction of the cost of a plant-derived pharmaceutical. The greatest cost is that of downstreamprocessing, isolation and purification of the product, in partic-ular if the molecule produced is a clinical product for humans.Even with the nonclinical proteins, downstream processing ofrecombinant proteins (extraction, purification, and characteri-zation) can account for >80% of overall production (Kusnadiet al., 1998). When choosing a plant expression system, down-stream processing (good manufacturing practice requirements,purification, quality control for approval) should also be takeninto consideration as it makes up a substantial proportion ofthe total cost of the final product. Gleba, Klimyuk, and Maril-lonnet (2005) estimated around $0.01 per one 0.1 mg vaccinedose if Agrobacterium is used as an infective systemic agent thatdelivers viral replicons. Gleba, Klimyuk, and Marillonnet alsocalculated the capital cost of a production facility with a capacityof 100 kg purified recombinant protein per year, approximately$15 million if it is built in the United States.

Many of the biopharmaceutical candidate drugs are beingdeveloped as if the production costs were not an issue. Conven-tional pharmaceutical development has been estimated to costroughly between $100 and $800 million per product and takesover 12 years on average. In general, the first phases of drugdevelopment cost approximately US $25 million that is for tech-nical proof of concept, including initial platform developmentand preclinical trials. An additional US $100 million is neededfor development to phase III clinical trials (Bischoff, 2004). Theactual decision for the capital investment comes when thera-peutic molecules enter phase III of clinical trial, because theproduct to be used in the phase III trial has to be producedidentical to that envisaged for the future industrial production(Bischoff, 2004), but the major bottleneck is that nearly 50%of the phase III clinical trials are reported to be failures. TheFrench company, Meristem Therapeutics, estimates the cost ofa 100-kg/year facility manufacturing gastric lipase in plants foranticipated phase III clinical tests, and eventual commercial-ization, at US$ 35 million; and Dow Chemical estimates thecost of a 400 kg/year facility at US$ 95 million for plant-basedproduction, compared to US$ 300 million for mammalian cellculture-based production (Arcand and Arnison, 2004).

Financial data in regards to plant production platforms are notavailable, because companies tend to conceal budget-productioncost information, and the shortage of existing data makes it diffi-cult to predict about the commercial viability or success of plantmolecular farming. Several observers have stated three reasonsfor believing that molecular farming will be unprofitable: (1)the concentration of the therapeutic protein must be at least 1%of the total protein to be commercially viable (Daniell, Streat-field, and Wycoff, 2001); (2) the extraction and purification ofplant biomolecules will be too costly; and (3) massive sums may

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have to be invested to confinement and containment measures(e.g., prevent cross-pollination), and liability insurance for con-tamination of conventional crops (Huot, 2003). Even further,each application is different and numerous variables have tobe evaluated individually, including agronomic practices, effec-tiveness of gene expression, isolation requirements, health andenvironment safety testing of the compound and plant, and easeof extraction and purification of the product (Chu and Robinson;2001; Taticek, Lee, and Shukler, 1994; Walsh, 2003).

Given the high prices of proprietary drugs, in many caseslower-cost production may not be expected as an issue. Never-theless, at present, none of the major pharmaceutical companiesis directing funding towards the development of plant-derivedmedicines. Drug companies are uncertain about how easy it willbe to gain regulatory approval for such plant-made pharmaceuti-cals since no examples yet to exist (Khalsa, Mason, and Arntzen,2004; Guruatma, Mason, and Arntzen, 2004). Other factors thatadd to the uncertainty are limited human clinical data and on-going public debate on the potential transformation of the tech-nology (Khalsa, Mason, and Arntzen, 2004). Expert opinion onthe issue is diverse and even conflicting. Some experts predictthat products reaching the commercialization stage may not beconvincing the pharmaceutical industry and instead the phar-maceutical industry may expand current production systems tomeet the market needs; in contrast, some predict that consumerdemand will intensify the call for the production of plant-derivedbiopharmaceuticals to treat chronic diseases (Drossard, 2004).Although it is not the expected case, the potential for unac-ceptance by the public might discourage entrepreneur interestfrom exploring this technology. The expectation of nonprof-itable, poor return in investing on pharmaceuticals developedto be used largely in resource-limited locations and conditions,are considered not so encouraging to private sector participa-tion in research and development (Drossard, 2004). Intellectualproperty (IP) is crucially important due to the increased costsof compulsory regulatory requirements for constructing properproduction facilities and for providing final quality and safetycontrols. In the area of molecular farming (plant or animal), threeprincipal challenges to IP management are outlined as shapingliberty to operate; securing a new IP as it is developed; anddeploying IP to collaborate with other private companies (Maet al., 2005).

CURRENT INTERNATIONAL REGULATIONS,LEGISLATION AND POLICIES AND POTENTIALREGULATORY CHALLENGES

In the European Union, when placing genetically modifiedorganisms (GMOs) on the market, the request for European au-thorization should be submitted to the competent environmentalrisk assessment authorities in one of the member states. Thereare four directives, and regulations governing the development,production, release, import/export, and marketing of GMOs. Theplacing on the market of GMOs, (e.g., for cultivation, release,export-import, and processing into industrial products) is sub-

ject to Directive 2001/18/EC. The individual member state isthen responsible for preparing the environmental risk assess-ment report and proposing the marketing measures to be taken.Contrary to the procedure for GMO products for food or feed,assessment of medicinal products for human and veterinary useobtained through GMOs is not the responsibility of any sin-gle member state, but is coordinated by the European Agencyfor the Evaluation of Medicinal Products (EMEA). The EMEAconstitutes a special regulatory case by their registration beinggoverned by reciprocal provisions in the Directive 2001/18/ECand Regulation 2004/726/EC. Applications are made directly tothe EMEA and are evaluated by one of the scientific committeesconsidering the quality, safety, and efficacy of the medicines.For the Authorization of a GMO-derived medicinal product,an applicant submits a dossier to the EMEA, which includesall the necessary administrative, quality, non-clinical and clin-ical data for medicinal product as well as environmental im-pact and risk assessment documentation of the GMO source, asdescribed in the legislation. The EMEA has published guide-lines for applicants preparing to market medicinal products ob-tained from plant molecular farming, both on the quality aspects(EMEA/CPMP/726/2002) and environmental risk assessment(EMEA/CHMP/135148/2004). The European Food Safety Au-thority (EFSA) Panel on GMOs deals with questions as definedin Directive 2001/18/EC, relating to deliberate release of ge-netically modified food/feed and their derived products into theenvironment. The EFSA, which is an independent agency, hasa role with respect to the European legislative framework forGMOs to carry out scientific risk assessment and providing ad-vice on GMOs.

In the United States, the production of pharmaceuticals intransgenic plants is regulated by two agencies, the U.S. De-partment of Agriculture’s (USDA’s) Animal and Plant HealthInspection Service (APHIS) focuses on containment of recom-binant plants and the Food and Drug Administration (FDA) con-centrates on the production system as it relates to manufactureof the drug or vaccine. The key principle is that genetically en-gineered products should continue to be regulated according totheir characteristics and unique features, and not according totheir production method. Current regulations for the productionof plant-derived pharmaceuticals mainly concentrate to preventrecombinant proteins from entering the food chain or from per-sisting in the environment, and to guard against recombinantnucleic acid sequences entering genomes of food or feed crops,or wild species. The APHIS is the first agency that applicants de-veloping bioengineered plants must work with before initiatingreviews with FDA. APHIS conducts reviews of permit applica-tions in which it evaluates the probable environmental impactof the release and prepares an environmental assessment. Thepermit application process requires the developer to disclose in-formation about the transgenic plant, the gene, and control mea-sures to be in place during transport and field tests (trial site, thetrial start and end dates, the manner in which the crop will beused and destroyed after trial). If the agency reaches a “finding

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of no significant impact” (FONSI), a permit, which might in-clude inspection of laboratories, greenhouses and other fieldsites, is subsequently issued. APHIS authorization must also beobtained prior to import, interstate movement, or environmentalrelease. In 2005, APHIS and the FDA have published a draftguidance for industy entitled “Drugs, Biologics, and MedicalDevices Derived from Bioengineered Plants for Use in humansand Animals.”

Canada’s Plant Bio-Safety Authority requires that the devel-oper must provide detailed and complete molecular and biolog-ical information when requesting authorization to proceed forthe field trial. The required data relates to the actual geneticmodification as well as the fertility, genetic variability, allelopa-thy, dormancy, pollen, seed, vegetative reproduction, floweringand fructification patterns, habitat, pollination and seed disper-sal mechanisms, weediness potential, toxicity, allergenicity, andfinally, protocols for the use of the site during and after trial.

There have been common calls for tighter regulations andconsistency in legal process, strict and controllable agriculturalpractices, better labelling, and clear regulatory frameworks cor-responding to expectations need to be defined. Due to the reg-ulatory requirements in developed countries, particularly forconstructing production facilities and final quality control inrecent years, a new strategy adopted by the research and devel-opment institutions is to develop plant-derived pharmaceuticalsfirst in a developing country. For instance, GlaxoSmithKlinehas recently licensed a rotavirus vaccine in Mexico. In 2005,The World Health Organization (WHO) organized a meetingin Geneva that considered the development of regulations forplant-derived vaccines, and an informal consultation documenton a scientific basis for regulatory evaluation of candidate hu-man vaccines derived from plants was published (van der Laanet al., 2006).

Most of the remaining challenges to commercialization ofplant-made molecules are associated with public perceptionsand acceptance. Various exercises have been conducted in recentyears to estimate attitudes, awareness and acceptance amongthe general public toward plant-made molecules. In an earlystudy, Nevitt et al. (2003) evaluated the perception of risksand benefits for a pharmaceutical expressed in transgenic to-bacco. Overall, more respondents expressed concerns associ-ated with GMO rice intended for the food chain as compared topharmaceutical-producing transgenic tobacco. In a more recentstudy, Einsidel and Medlock (2005) questioned perceptions ofsix different plant-made pharmaceuticals: 52% of respondentsdescribed plant-made pharmaceuticals as “more acceptable,”27% of respondents described this application as “unacceptable”or “less acceptable.” When two more questions were included onthe production of interleukin in plants, 80% of respondents sup-ported the application, and 78% supported the use of technologyin the production of biodegradable plastics (Einsiedel and Med-lock, 2005). A survey comprising multiple-choice questions wasconducted with over 700 respondents with the major objectiveof evaluating market preference of vaccines among the general

population. More than 40% of respondents chose the oral option(Kirk and McIntosh, 2005). A professional survey conducted inthe USA and Canada which put specific questions to representa-tive populations revealed that three out of four respondents havea positive attitude towards the use of plant-based, even includ-ing food plants (http://www.inspection.gc.ca). When membersof anti-GM campaigns were asked how they perceive the risksand benefits of genetically modified technology, respondentswere not convinced that there is an inverse relationship betweenperceived risk and perceived benefit (Hall and Moran, 2006).It is difficult to draw any specific and universal conclusions,however, about acceptance of plant-made pharmaceuticals orother molecules by the general public. Public perceptions ofany new technology are complex and cannot be generalized asthese separate studies, and individual surveys designed with dif-ferent methodologies could not be compared. The subject ofplant made products is expected to acquire more public debateand present new policy challenges to all.

Future studies are expected to overcome current technologi-cal drawbacks emphasized by the researchers and the commer-cial bodies, to evaluate the most likely scenarios and impactof certain initiatives for commercial development, to estimatescale-up investment costs depending on the market demand, toassess the cost effectiveness and financial returns of large-scaleproduction, and, last but least, to analyze social acceptance andsocio-economic impacts of molecular farmed products in plants.

REFERENCESAmmann, K. 2004. The role of science and discourse in the application

of the precautionary approach. In: Molecular Farming: Plant-MadePharmaceuticals and Technical Proteins, 291–301. Fischer, R., andSchillberg, S., eds., Wiley-VCH, Weinheim, Germany.

Arcand, F., and Arnison, P. 2004, April. Development ofNovel Protein-Production Systems and Economic Opportu-nities and Regulatory Challenges for Canada. Available at:www.plantpharma.org/documents/NPPS 040412.pdf. AccessedMarch 31, 2008.

Argevin, F., Klein, E. K., Choimet, C., Gauffreteau, A., Lavigne, C.,Messean, A., and Meynard, J. M. 2008. Modelling impacts of crop-ping systems and climate on maize cross-pollination in agriculturallandscapes: The MAPOD model. Eur. J. Agronomy 28: 471–484.

Bakker, H., Bardor, M., Molthoff, J. W., Gomord, V., Elbers, I., Stevens,L.H., Jordi, W., Lommen, A., Faye, L., and Lerouge, P. 2001.Galactose-extended glycans of antibodies produced by transgenicplants. Proc. Natl. Acad. Sci. USA 98: 2899–2904.

Bardor, M., Cabrera, G., Rudd, P. M., Dwek, R. A., Cremata, J. A., andLerouge, P. 2006. Analytical strategies to investigate plant N-glycanprofiles in the context of plant-made pharmaceuticals. Curr. Opin.Struct. Biol. 16: 576–583.

Barta, A., Sommengruber, K., Thompson, D., Hartmuth, K., Matzke,M., and Matzke, A. 1986. The expression of a napoline synthasehuman growth hormone chimeric gene in transformed tobacco andsunflower callus tissue. Plant Mol. Biol. 6: 347–357.

Basaran, P., and Rodrıguez-Cerezo, E. 2008. An assessment of emerg-ing molecular farming activities based on patent analysis (2002–2006). Biotech. Bioprocess Engineer. (In press)

Cri

tical

Rev

iew

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Bio

tech

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ownl

oade

d fr

om in

form

ahea

lthca

re.c

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nive

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of

Not

re D

ame

Aus

tral

ia o

n 05

/05/

13Fo

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al u

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nly.


Bernatchez, S., Gilbert, M., Blanchard, M. C., Karwaski, M. F., Li,L., DeFrees, S., and Wakarchuk, W. W. 2007. Variants of the ß1,3-Galactosyltransferase CgtB from the bacterium Campylobacter je-juni have distinct acceptor specificities. Glycobiology 17: 1333–1343.

Bischoff, F., 2004. A top-down view of molecular farming from thepharmaceutical industry: requirements ad expectations. In: Molecu-lar Farming: Plant-Made Pharmaceuticals and Technical Proteins,267–287. Fischer, R., and Schillberg, S., eds., Wiley-VCH, Wein-heim, Germany.

Bock, R. 2001. Transgenic chloroplasts in basic research and plantbiotechnology. J. Mol. Biol. 312: 425–438.

Bock, R. 2007. Plastid biotechnology: prospects for herbicide and in-sect resistance, metabolic engineering and molecular farming. Curr.Opin. Biotechnol. 18: 100–106.

Bogorad, L. 2000. Engineering chloroplasts: an alternative site for for-eign genes, proteins, reactions and products. Tibtech. 18: 257–263.

Bosch, D., Small, J., and Krebbers, E. 1994. A trout growth hormone isexpressed, correctly folded and partially glycosylated in the leavesbut not the seeds of transgenic plants. Transgenic Res. 3: 304–310.

Brandle, J. 2004. The field evaluated of transgenic crops engineered toproduce recombinant proteins. In Molecular Farming: Plant-MadePharmaceuticals and Technical Proteins, 69–76. Fischer, R., andSchillberg, S., eds., Wiley-VCH, Weinheim, Germany.

Brodzik, R., Bandurska, K., Deka, D., Golovkin, M., and Koprowski,H. 2005. Advances in alfalfa mosaic virus-mediated expression ofanthrax antigen in planta. Biochem. Biophys. Res. Commun. 338:717–722.

Center for Infectious Diseases and Vaccinology. 2006. ProVacs. Avail-able at: www.biodesign.asu.edu/centers/idv/projects/provacs. Ac-cessed March 31, 2008.

Chargelegue, N. D., Drake, P. M. W., Obregon, P., and Ma, J. C. K.2005. Production of secretory IgA in transgenic plants. In Molecu-lar Farming: Plant-Made Pharmaceuticals and Technical Proteins,159–169. Fischer, R., and Schillberg, S., eds., Wiley-VCH, Wein-heim, Germany.

Chargelegue, N. D., van Dolleweerd, C. J., Drake, P. M., and Ma, J. C. K.2000. A murine monoclonal antibody produced in transgenic plantswith plant-specific glycans is not immunogenic in mice. TrangenicRes. 9: 187–194.

Chong, D. K. X., Roberts, W., Arakawa, T., Illes, K., Bagi, G., Slattery,C. W., and Langridge, W. H. R. 1997. Expression of the human milkprotein beta-casein in transgenic potato plants. Transgenic Res. 6:289–296.

Christou, P., Stoger, E., and Twyman, R. M. 2004. Monocot expressionsystems for Molecular Farming. In Molecular Farming: Plant-MadePharmaceuticals and Technical Proteins, 55–67. Fischer, R., andSchillberg, S., eds., Wiley-VCH, Weinheim, Germany.

Chu, L., and Robinson, D. K. 2001. Industrial choices for protein pro-duction by large-scale cell culture. Curr. Opin. Biotechnol. 12: 180–187.

Commandeur, U.,Twyman, R. M., and Fischer, R.2003. The biosafetyof molecular farming in plants. AgBiotechNet 30: 109–112.

Cramer, C. L., Boothe, J. G., and Oishi, K. K. 1999.Transgenic plantsfor therapeutic proteins: linking upstream and downstream strategies.Curr. Top. Microbiol. Immunol. 240: 95–118.

Daniell, H. 1999. GM crops: public perception and scientific solutions.Trends Plant Sci. 4: 467–469.

Daniell, H. 2002. Molecular strategies for gene containment in trans-genic crops. Nat. Biotechnol. 20: 581–586.

Daniell, H., Carmona-Sanchez, O., and Burns, B. E. 2004.Chloro-plast derived antibodies, biopharmaceuticals and edible vaccines.In Molecular Farming: Plant-Made Pharmaceuticals and TechnicalProteins.113–133. Fischer, R., and Schillberg, S., eds., Wiley-VCH,Weinheim, Germany.

Daniell, H., Khan, M. S., and Allison, L. 2002. Milestones in chloroplastgenetic engineering: an environmentally friendly era in biotechnol-ogy. Trends Plant Sci. 7: 84–91.

Daniell, H., Streatfield, S. J., and Wycoff, K. 2001. Medical molecularfarming: production of antibodies, biopharmaceuticals and ediblevaccines in plants. Trends Plant Sci. 6: 219–226.

Datar, R. V., Cartwright, T., and Rosen, C. G. 1993. Process eco-nomics of animal cell and bacterial fermentations: a case studyanalysis of tissue plasminogen activator. Bio/technology, 11: 349–357.

Drossard, J. 2004. Downstream processing of plant-derived recom-binant therapeutic proteins. In Molecular Farming: Plant-MadePharmaceuticals and Technical Proteins, 217–230. Fischer, R., andSchillberg, S., eds., Wiley-VCH, Weinheim, Germany.

Eastham, K., and Sweet, J. 2002. Genetically Modified Organisms(GMOs): The Significance of Gene flow Through Pollen Trans-fer. European Environment Agency, Environment Issue ReportNo. 28.

Einsiedel, E. F., and Medlock, J. 2005. A public consultation on plantmolecular farming. AgBioForum 8: 26–32.

Fiedler, U., Phillips, J., Artsaenko, O., and Conrad, U. 1997. Optimiza-tion of scFv antibody production in transgenic plants. Immunotech-nology 3: 205–216.

Fischer, R., and Stoger, E. 2004. Plant-based production of biopharma-ceuticals. Curr. Opin. Plant Biol. 7: 152–158.

Fischer, R., Stoger, E., Schillberg, S., Christou, P., and Twyman, R. M.2004. Plant-based production of biopharmaceuticals. Curr. Opin.Plant Biol. 7: 152–158.

Freese, B. 2002, July. Manufacturing Drugs and Chemical Crops:Biopharming Poses New Threats to Consumers, Farmers, FoodCompanies and the Environment. Available at: www.foe.org/camps/comm/safefood/biopharm/ BIOPHARM REPORT.pdf. Ac-cessed March 31, 2008.

Frigerio, L., Vine, N. D., Pedrazzini, E., Hein, M. B., Wang, F., Ma,J. C. K., and Vitale, A. 2000. Assembly, secretion, and vacuolardelivery of a hybrid immunoglobulin in plants. Plant Physiol. 123:1483–1494.

Fu, X., Duc, L. T., Fontana, S., Bong, B. B., Tinjuangjun, P., Sud-hakar, D., Twyman, R. M., Christou, P., and Kohli, A. 2000. Lineartransgene constructs lacking vector backbone sequences generatelow-copy-number transgenic plants with simple integration patterns.Transgenic Res. 9: 11–19.

Gasdaska, J. R., Spencer, D., and Dickey, L. 2003. Advantages thera-peutic protein production in the aquatic plant Lemma. Bioprocess J.2: 49–56.

Gleba, Y., Klimyuk, V., and Marillonnet, S. 2005. Magnifection-a newplatform for expressing recombinant vaccines in plants. Vaccine, 23:2042–2048.

Gleba, Y., Klimyuk, V., and Marillonnet, S. 2007. Viral vectors for theexpression of proteins in plants . Curr. Opin. Biotechnol. 18: 134–141.

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Not

re D

ame

Aus

tral

ia o

n 05

/05/

13Fo

r pe

rson

al u

se o

nly.


Gomord, V., Chamberlain, P., Jefferis, R., and Faye, L. 2005. Biophar-maceutical production un plants: problems, solutions, and opportu-nities. Trends Biotechnol. 23: 559–565.

Gomord, V., and Faye, L. 2004. Posttranslational modification of ther-apeutic proteins in plants. Curr. Opin. Plant Biol. 7: 171–181.

Gomord V., Fitchette, A. C., and Lerouge, P. 2004. Glycosylation ofplant-made pharmaceuticals. In Molecular Farming: Plant-MadePharmaceuticals and Technical Proteins, 233–250. Fischer, R., andSchillberg, S., eds., Wiley-VCH, Weinheim, Germany.

Gressel, J. 2000. Molecular Biology of weed control. Transgenic Res.9: 355–382.

Guruatma, K., Mason, H. S., and Arntzen, C. J. 2004. Plant-derivedvaccines: progress and constraints. In Molecular Farming: Plant-Made Pharmaceuticals and Technical Proteins, 135–155. Fischer,R., and Schillberg, S., eds., Wiley-VCH, Weinheim, Germany.

Gruber, V., Berna, P., Arnaud, T., Bournat, P., Clement, C., Mison,D., Olagnier, B., Philippe, L., Theisen, M., Baudino, S., Benicourt,C., Cudrey, C., Bloes, C., Duchateau, N., Dufour, S., Gueguen, C.,Jacquet, S., Ollivo, C., Poncetta, C., Zorn, N., Ludevid, D., VanDorsselaer, A., Verger, R., Doherty, A., Merot, B., and Danzin, C.2001. Large-scale production of a therapeutic protein in transgenictobacco plants: effect of subcellular targeting on quality of a recom-binant dog gastric lipase. Mol. Breeding 7: 329–340.

Hall, C., and Moran, D. 2006. Investigating GM risk perceptions: asurvey of anti-GM and environmental campaign group members.J. Rural Studies. 22: 29–37.

Hood, E. E. 2002. From green plants to industrial enzymes. EnzymeMicrob. Technol. 30: 279–283.

Hood, E. E., and Jilka, J. M. 1999. Plant-based production of xenogenicproteins. Curr. Opin. Biotechnol. 10: 382–386.

Hood, E. E., Woodard, S. L., and Horn, M. E. 2002. Antibody manufac-turing in transgenic plants: myths and realities. Curr. Opin. Biotech-nol.13: 630–635.

Horn, M. E., Woodard, S. L., and Howard, J. A. 2004. Plant molec-ular farming: systems and products. Plant Cell Reports 22, 711–720.

Howard J. A., and Hood, E. 2005. Bio-industrial and biopharmaceu-tical products produced in plants. Advances in Agronomy 85: 91–123.

Howard J. A., and Hood, E. E. 1998. Commercial production of pro-teases in plants. US Patent 6,087,558.

Huang, Z., Santi, L., Lepore, K. Kilbourne, J., Arntzen, C. J., andMason, H. S. 2006. Rapid, high-level production of hepatitis B coreantigen in plant leaf and its immunogenicity in mice. Vaccine 24:2506–2513.

Huot, M.-F. 2003, June. Plant Molecular Farming: Issues and Chal-lengesfor Canadian Regulators. Consumers Affairs Office, IndustryCanada. Available at: www.option-consommateurs.org/documents/1130 principal/fr/File/rapports/foods/molecular farming oc0603.pdf. Accessed March 31, 2008.

Inaba, Y., Zhong, W. Q., Zhang, X.-H., and Widholm, J. M. 2007.Specificity of expression of the GUS reporter gene (uidA) driven bythe tobacco ASA2 promoter in soybean plants and tissue cultures.J. Plant Physiol. 164: 824–834.

Jiang, L. W., and Sun, S. S. M. 2002. Membrane anchors for vacuolartargeting: application in plant bioreactors. Trends Biotechnol. 20:99–102.

Kapusta, J., Modelska, A., Figlerowicz, M., Pniewski, T., Letellier,M., Lisowa, O., Yusibov, V., Koprowski, H., Plucienniczak, A., and

Legocki, A. B. 1999. A plant-derived edible vaccine against hepatitisB virus. FASEB Journal 13: 1796–1799.

Karasev, A. V., Foulke, S., Wellens, C., Rich, A., Shon, K. J., Zwierzyn-ski, I., Hone, D. Koprowski, H., and Reitz, M. 2005. Plant basedHIV-1 vaccine candidate: Tat protein produced in spinach. Vaccine23: 1875–1880.

Khalsa, G., Mason, H. S., and Arntzen, C. J. 2004. Plant-derived vac-cines: progress and constrains. In Molecular Farming: Plant-madePharmaceuticals and Technical Proteins, 135–158. Fischer, R., andSchillberg, S., eds., Wiley-VCH, Weinheim, Germany.

Kirk, D. D., and McIntosh, K. 2005. Social acceptance of plant madevaccines: indications from a public survey. AgBioForum 8: 228–234.

Kling, J. 1996. Could transgenic supercrops one day breed superweeds?Science 274: 180–181.

Koprowski, H. 2005. Vaccines and sera through plant biotechnology,Vaccine. 23: 1757–1763.

Kusnadi, A. R., Hood, E. E., Witcher, D. R., Howard, J. A., and Nikolov,Z. L. 1998. Production and purification of two recombinant proteinsfrom transgenic corn. Biotechnol. Prog. 14: 149–155.

Lamphear, B. J., Streatfield, S. J., Jilka, J. M., Brooks, C. A., Barker,D. K., Turner, D. D., Delaney, D. E., Garcia, M., Wiggins, B.,Woodard, S. L., Hood, E. E., Tizard, I. R., Lawhorn, B., and Howard,J. A. 2002. Delivery of subunit vaccines in maize seed. J. Control.Release 85: 169–180.

Lee, R. W. H., Pool, A. N., Ziauddin, A., Lo, R. Y. C., Shewen P. E.,and Strommer, J. N. 2003. Edible vaccine development: stabilityof Mannheimia haemolytica A1 leukotoxin 50 during post-harvestprocessing and storage of field-grown transgenic white clover Mol.Breeding 11: 259–266.

Lutz, K. A., and Maliga, P. 2007. Construction of marker-free transplas-tomic plants. Curr. Opin. Biotechnol. 18: 107–114.

Ma, J. K. C., Chikwamba, R., Sparrow, P., Fischer, R., Manohey, R.,and Twyman, R. M. 2005. Plant-derived pharmaceutical –the roadforward. Trends Plant Sci. 10: 580–585.

Ma, J. K. C., Hiatt, A., Hein, M., Vine, N. D., Wang, F., Stabila, P., vanDolleweerd, C., Mostov, K., and Lehner, T. 1995. Generation andassembly of secretory antibodies in plants. Science, 268: 716–719.

Ma, J. K. C., Hikmat, B. Y., Wycoff, K., Vine, N. D., Chargelegue,D., Yu, L., Hein, M. B., and Lehner, T. 1998. Characterization ofa recombinant plant monoclonal secretory antibody and preventiveimmunotherapy in humans. Nat. Med. 4: 601–606.

Ma, J. K. C., Lehrner, T., Stabila, P., Fux, C. I., and Hiatt, A. 1994.Assembly of monoclonal antibodies with IgG1 and IgA heavy chaindomains in transgenic tobacco plants. Eur. J. Immunol. 24: 131–138.

Maliga, P. 2002. Engineering the plastid genome of higher plants. Curr.Opin. Plant Biol. 5: 164–172.

Maliga, P. 2004. Plastid transformation in higher plants. Annu. Rev.Plant Biol. 55: 289–313.

Maliga, P., and Graham, I. 2004. Molecular farming and metabolicengineering promises a new generation of transgenic crops. Curr.Opin. Plant Biol. 7: 149–151.

Mascia, P. N., and Flavell, R. B. 2004. Safe and acceptable strategiesfor producing foreign molecules in plants. Curr. Opin. Plant Biol. 7:189–195.

Mayfield, S. P., Franklin, S. E., and Lerner, R. A. 2003. Expression andassembly of a fully active antibody in algae. Proc. Natl Aca. Sci. USA100: 438–442.

McCormick, A. A., Kumagai, M. H., Hanley, K., Turpen, T. H.,Hakim, I., Grill, L. K., Tuse, D., Levy, S., and Levy, R. 1999.

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Not

re D

ame

Aus

tral

ia o

n 05

/05/

13Fo

r pe

rson

al u

se o

nly.


Rapid production of specific vaccines for lymphoma by expressionof the tumor-derived single-chain Fv epitopes in tobacco plants. Proc.Natl Acad. Sci. USA 96: 703–708.

McCormick A. A., Reinl, S. J., Cameron, T. I., Vojdani, F., Fronfield, M.,Levy, R., and Tuse, D. I. 2003. Individualized human scFv vaccinesproduced in plants: humoral anti-idiotype responses in vaccinatedmice confirm relevance to the tumor Ig. Immunol Methods 278: 95–104.

Menassa, R., Nguyen, V., Jevnikar, A. N., and Brandle, J. E. 2001. Aself-contained system for the field production of plant recombinanthuman interleukin-10. Mol. Breeding 8: 177–185.

Mercer, D. K., Scott, K. P., Melville, C. M., Glover, L. A., and Flint,H. J. 2001. Transformation of an oral bacterium via chromosomalintegration of free DNA in the presence of human saliva. FEMSMicrobiol. Lett. 200: 163–167.

Meurer, H. 2004, March. Genetic engineering. Alive Maga-zine#257. Available at: www.alive.com/1743a5a2.php?subject breadcramb=635%20(2006). Accessed March 31, 2008.

Miele, L. 1997. Plants as bioreactors for biopharmaceuticals: regulatoryconsiderations. Trends Biotechnol. 15: 45–50.

Mikkelsen, T. R., Jensen, J., and Jorgensen, R. B. 1996. Inheritance ofoilseed rape (Brassica napus) RAPD markers in a backcross progenywith Brassica campestris. Theor. Appl. Genet. 92: 492–497.

Nap, J.-P., Metz, P. L. J., Escaler, M., and Conner, A. J. 2003. Therelease of genetically modified crops into the environment. Part I.Overview of current status and regulations. Plant J. 33: 1–18.

Nevitt, J., Norton, G., Mills, B., Jones, M. E., Ellerbrock, M., andReaves, D. 2003, April. Participatory assessment of social and eco-nomic effects of using transgenic tobacco to produce pharmaceuti-cals. Working Paper No. 2003-1. Virginia Tech, Blacksburg. Avail-able at: www.agecon. vt.edu/biotechimpact/tobacco/WP20031.pdf.Accessed March 31, 2008.

Nielsen, K. M., Bones, A., Smalla, K., and van Elsas J. D. 1998. Hori-zontal gene transfer from transgenic plants to terrestrial bacteria—arare event? FEMS Microbiol. Rev. 22: 79–103.

Paez, R., Cremata, J. A., Enriquez, G., Mendoza, O., Ortega, M., andBorroto, C. 2005. An integral approach towards a practical applica-tion for a plant-made monoclonal antibody in vaccine purification.Vaccine 23: 1833–1837.

Pan, L., Zhang, Y., Wang, Y., Wang, B., Wang, W., Fang, Y., Jiang,S., Lv, J., Wang, W., Sun Y., and Xie, Q. 2008. Foliar extracts fromtransgenic tomato plants expressing the structural polyprotein, P1–2A, and protease, 3C, from foot-and-mouth disease virus elicit aprotective response in guinea pigs. Vet. Immunol. Immunopathol.121: 83–90.

Pezzotti, M., Schillberg, S., Sparrow, P., Stoger, E., and Twyman, R. M.2005. Molecular Farming for new drugs and vaccines. EMBO Re-ports 6: 593–599.

Pogue, G. P., Lindbo, J. A., Garger, S. J., and Fitzmaurice, W. P. 2002.Making an ally from an enemy: plant virology and the new agricul-ture, Annu. Rev. Phytopathol. 40: 45–74.

Poirier, Y., Dennis, D. E., Klomparens, K., and Somerville, C. 1992.Polyhydroxybutyrate, a biodegradable thermoplastic, produced intransgenic plants. Science 256: 520–523.

Pujol, M., Ramırez, N. I., Ayala, M., Gavilondo, J. V., Valdes, R., Ro-driguez M., Brito, J., Padilla, S., Gomez, L., Reyes, B., Peral, R.,Perez, M., Marcelo, J. L., Mila, L., Sanchez, R. F., Rolando, P., Cre-mata, J. A., Enriquez, G., Mendoza, O., Ortega, M, and Borroto,

C. 2005. An integral approach towards a practical application for aplant-made monoclonal antibody in vaccine purification. Vaccine 23:1833–1837.

Roggero, P., Ciuffo, M., Benvenuto, E., and Franconi, R. 2001. Theexpression of a single-chain Fv antibody fragment in different planthosts and tissues by using Potato virus X as a vector. Protein Expr.Purif. 22: 70–74.

Saint-Jore-Dupas, C., Faye L., and Gomord, V. 2007. From planta topharma with glycosylation in the toolbox. Trends Biotechnol. 25:317–323.

Scheller, J., and Conrad, U. 2004. Production of silk proteins in trans-genic tobacco. In Molecular Farming: Plant-Made Pharmaceuticalsand Technical Proteins, 171–181. Fischer, R., and Schillberg, S., eds.,Wiley-VCH, Weinheim, Germany.

Scheller, J., Henggeler, D, Viviani, A., and U. Conrad. 2004. Purifica-tion of spider silk-elastin from transgenic plants and application forhuman chondrocyte proliferation. Transgenic Res. 13:51–57.

Schillberg, S., Twyman, R. M., and Fischer, R. 2005. Opportunities forrecombinant antigen and antibody expression in transgenic plants-technology assessment. Vaccine 23: 1764–1769.

Schluter, K., Futterer, J., and Potrykus, I. 1995. Horizontal gene-transfer from a transgenic potato line to a bacterial pathogen (Er-winiachrysanthem) occurs, if at all, at an extremely low-frequency.Bio/Technology 13: 1094–1098.

Sedjo, R. A. 2005. Will Developing Countries be the Early Adoptersof Genetically Engineered Forests? AgBioForum, 8: 205–212.

Shadwick, F. S., and Doran P. M. 2004. Foreign protein expressionusing plant cell suspension and hairy root cultures. In MolecularFarming: Plant-Made Pharmaceuticals and Technical Proteins, 13–36. Fischer, R., and Schillberg, S., eds., Wiley-VCH, Weinheim,Germany.

Sharp, J. M., and Doran, P. M. 2001. Strategies for enhancing mon-oclonal antibody accumulation in plant cell and organ cultures.Biotechnol. Prog. 17: 979–992.

Smith, N., Kilpatrick, J. B., and Whitelam. G. C. 2001. Superflu-ous transgene integration in plants. Crit. Rev. Plant Sci. 20: 215–249

Smyth, S., and Philips, P. W. B. 2002. Product Differentiation Alterna-tives: Identity Preservation, Segregation, and Traceability. AgBioFo-rum 5: 30–42.

Spok, A. 2007. Molecular Farming on the rise – GMO regulators stillwalking a tightrope. Trends in Biotech. 25: 74–82.

Stoger, E., Ma, J. K. C., Fischer, R., and Christou, P. 2005. Sowing theseeds of success: pharmaceutical proteins from plants. Curr. Opin.Biotechnol. 16: 167–173.

Streatfield, S. J. 2006. Mucosal immunization using recombinant plant-based oral vaccines. Methods 38: 150–157.

Tackaberry, E. S., Prior, F., Bell, M., Tocchi, M., Porter, S., Mehic, L.,Ganz, P. R., Sardana, R., Altosaar, I., and Dudani, A. 2003. Increasedyield of heterologous viral glycoprotein in the seeds of homozygoustransgenic tobacco plants cultivated underground. Genome 46: 521–526.

Tacket, C. O. 2005. Plant-derived vaccines against diarrheal diseases.Vaccine 23: 1866–1869.

Tacket, C. O., Mason, H. S., Losonsky, G., Estes, M. K., Levine, M. M.,and Arntzen, C. J. 2000. Human immune responses to a novel Nor-walk virus vaccine delivered in transgenic potatoes. J. Infect. Dis.182: 302–305.

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Not

re D

ame

Aus

tral

ia o

n 05

/05/

13Fo

r pe

rson

al u

se o

nly.


Taticek, R. A., Lee, C. W. T., and Shukler, M. L. 1994. Large-scaleinsect and plant cell culture. Curr. Opin. Biotechnol. 5: 165–174.

Tregoning, J. S., Nixon, P., Kuroda, H., Svab, Z., Clare, S., Bowe,F., Fairweather, N., Ytterberg, J., van Wijk, K. J., Dougan, G., andMaliga, P. 2003. Expression of tetanus toxin fragment C in tobaccochloroplasts, Nucleic Acids Res. 31: 1174–1179.

Twyman, R. M. 2004. Host plants, systems and expression strategies forMolecular Farming. In Molecular Farming: Plant-made Pharmaceu-ticals and Technical Proteins, 191–216. Fischer, R., and Schillberg,S., eds., Wiley-VCH, Weinheim, Germany.

Twyman, R. M., Stoger, E., Schillberg, S., Chrishou, P., and Fischer.R. 2003. Molecular Farming in plants: host systems and expressiontechnology. Trends Biotechnol. 21: 570–578.

Usharani, K. S., Periasamy, M., and Malathi, V. G. 2006. Studies onthe activity of a bidirectional promoter of Mungbean yellow mosaicIndia virus by agroinfiltration. Virus Res. 119: 154–162.

van der Laan, J. W., Minor, P., Mahoney, R., Arntzen, C., Shin, J.,and Wood, D. 2006. WHO informal consultation on scientific basisfor regulatory evaluation of candidate human vaccines from plants,Geneva, Switzerland, 24–25 January 2005. Vaccine 24: 4271–4278.

Walsh, G. 2003. Biopharmaceutical Benchmarks, Nature Biotechnol.21: 865–870.

Wang, L., Webster D. E., Campbell, A. E., Dry, I. B., Wesselingh, S.L., and Coppel, R. L. 2008. Immunogenicity of Plasmodium yoeliimerozoite surface protein 4/5 produced in transgenic plants. Int. J.Parasitol. 38: 103–110.

Webster, D. E., Smith, S. D., Pickering, R. J., Strugnell, R. A., Dry,I. B., and Wesselingh, S. L. 2006. Measles virus hemagglutininprotein expressed in transgenic lettuce induces neutralising anti-bodies in mice following mucosal vaccination. Vaccine 24: 3538–3544.

Wieland, W. H., Lammers, A., Schots, A., and Orzaez, D. V. 2006. Plantexpression of chicken secretory antibodies derived from combinato-rial libraries. J. Biotechnol. 112, 382–391.

Wilde, C. D., Peeters, K., Jacobs, A., Peck, I., and Depicker, A. 2002.Expression of antibodies and Fab fragments in transgenic potatoplants: A case study for bulk production in crop plants. Mol. Breed.9: 271–282.

Woodard, S. L., Mayor, J. M., Bailey, M. R., Barker, D. K., Love, R.T.,Lane, J. R., Delaney, D. E., McComas-Wagner, J. M., Mallubhotla,H. D., Hood, E. E., Dangott, L. J., Tichy, S. E., and Howard J. A. 2003.Maize (Zea mays)-derived bovine trypsin: characterization of thefirst large-scale, commercial protein product from transgenic plants.Biotechnol. Appl. Biochem. 38: 123–130.

Yu, J., and Langridge, W. 2003. Expression of rotavirus capsid pro-tein VP6 in transgenic potato and its oral immunogenicity in mice.Transgenic Res. 12: 163–169.

Yusibov, V., Hooper, D. C., Spitsin, S. V., Fleysh, N., Kean, R. B.,Mikheeva, T., Deka, D., Karasev, A., Cox, S., Randall, J., andKoprowski, H. 2002. Expression in plants and immunogenicity ofplant virus-based experimental rabies vaccine. Vaccine. 20: 3155–3164.

Cri

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Rev

iew

s in

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plant molecular farming: opportunities and challenges

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