Plant Molecular Farming: Opportunities and Challenges

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  • Critical Reviews in Biotechnology, 28:153172, 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 Rodrguez-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 Rodrguez-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:pervinb@ziraat.sdu.edu.tr or pb27@cornell.edu

    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 Rodrguez-Cerezo, 2008). The patent ownershipdistribution between the private sector and academic institu-tions represented a trend towards the private sector (Basaranand Rodrguez-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-Hodgkins 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 worlds firstregulatory approval from U.S. Agriculture Departments 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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  • 156 P. BASARAN AND E. RODRIGUEZ-CEREZO

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

    development (Continued)Institution Country of origin Products or other indications

    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, album...

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