Risk Assessment and Regulation of Molecular Farming - a Comparison Between Europe and US

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Risk Assessment and Regulation of Molecular Farming - A Comparison between Europe and US

Penelope Sparrow1*, Inge Broer2, Elizabeth E. Hood3, Kellye Eversole4, Frank Hartung5 and Joachim Schie- mann5

1John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK; 2University of Rostock, Justus von Liebig-Weg 8, D-18059 Rostock, Germany; 3Arkansas State University College of Agriculture, PO Box 639, State University, AR 72467, United States; 4Eversole Associates, 5207 Wyoming Road, Bethesda, Maryland, 20816, United States; 5Julius Kühn-Institut, Federal Research Cen- tre for Cultivated Plants (JKI), Institute for Biosafety in Plant Biotechnology, Erwin-Baur-Str. 27, D-06484 Quedlinburg, Germany

Abstract: In this article, the general principles of genetically modified (GM) plant risk assessment and the regulatory framework for contained use and open field production of plant-made pharmaceuticals/plant-made industrials (PMP/PMI) are described. While significant progress has been made for the containment grown (plant cell culture) production of PMPs, with the first regulatory approval made by the FDA in 2012, the commercialization of medicinal or industrial products produced in the field has yet to emerge in either Europe or the US. In the current paper, we discribe the regulatory environment in Europe and the US surrounding GM crops, and provide case studies for experimental field releases of PMP and PMI producing plants in both regions. Suggestions for reducing the regulatory burden for GM plants will be discussed, also in light of the emerging new technologies to modify the genetics of plants. Since regulations surrounding the commercialization of GM crops are very costly and not appropriate for most of the PMP/PMI applications in Europe, we propose that amendments to the EU Directive 2001/18/EC are necessary to allow for the commercialization of products from GM plants without the need of an ‘authorization’. To fully acknowledge the overall outcome of adopting plants to produce PMP/PMI, the conclusion is that broader and more balanced legislative oversight is needed in Europe; while specific legislation is not needed in the US. Keywords: Plant molecular farming (PMF), plant made pharmaceuticals (PMP), plant made industrials (PMI), genetically modified plants, GM plant regulation, GM plant risk assessment.

INTRODUCTION move ahead to the field or production arena. As with all GM plants, Plant genetic modification (GM), developed over the past those intended for molecular farming must go through a thorough twenty five-years, has been applied to input traits, to improve plant risk assessment before they can be used in the field. In that respect, production in the form of herbicide and pest resistant crops, as well whereas existing guidance on the environmental risk assessment of as to output traits in the form of nutritional improvement (such as GM plants for food/feed uses is considered adequate to also cover Golden Rice), industrial proteins (e.g. cellulase, and trypsin) and GM plants for non-food/feed uses [3], PMF raises novel questions pharmaceuticals and vaccines (Plant Molecular Farming - PMF). that might trigger a need for specific biosafety considerations due to The benefits of input and output traits are now extensively docu- the nature of the proteins to be expressed. mented, yet the full exploitation of this technology remains hin- Commercialization has many hurdles including not only the dered by the long lead times associated with such emerging R&D science and associated intellectual property to protect the inven- technology combined with the timescales involved in introducing tions, but also market realities and government regulations. As each these new traits into local adapted varieties. One of the biggest GM plant moves from the laboratory to scaled-up greenhouse pro- barriers to the adoption of this technology into the commercial sec- duction or to the field, issues emerge that trigger additional over- tor is the intense scrutiny of these new plant varieties by regulatory sight for environmental release or ultimate use by humans. In this agencies and the public, and the costs associated with regulatory review, we outline the current state of play regarding the risk as- approval. The average cost for having GMOs approved in Europe sessment and regulations surrounding GM plants and how the cur- has been estimated at 7-10 million Euros per event [1]. These rent regulatory systems are adapting to accommodate PMF. costs mainly accrue from the large number of studies which the applicant companies have to present to the European Food Safety 2. GENERAL PRINCIPLES OF GM PLANT RISK ASSESS- Authority (EFSA). The 30 approvals (including for imports) having MENT been granted by April 2011 represent total costs to companies of 2.1. Interplay of Risk Assessment, Risk Management and Risk between 210 and 300 million [2]. Communication PMF consists of using plants as production platforms for the In different countries across the world (termed hereafter as “ju- synthesis of compounds for pharmaceutical or industrial purposes. risdictions”), GM plants and their derived food and feed products PMF has been presented as a convenient way to produce molecules are subject to a risk analysis before they can be used for experimen- of interest on a large scale at low costs. Other benefits associated tal or commercial uses. The case-by-case risk analysis encompasses with the use of plants are; rapid scaling up, convenient storage of all components of risk, and consists of assessment, management, raw material (in the form of seed) and less concern over human and risk communication. In risk assessment, potential adverse im- pathogen contamination. However, in order for any impact to be pacts associated with a specific activity are scientifically character- realized from this research, these laboratory experiments must ized on a case-by-case basis. Risks are identified from plausible sets of circumstances that may result in harm to human and animal *Address correspondence to this author at the John Innes Centre, Norwich health or the environment, and subsequently the level of risk is Research Park, Norwich, NR4 7UH, UK; Tel: +44 (0) 1603 450547; estimated on the basis of the seriousness and chance of harm. Risk Fax: +44 (0) 1603 450045; E-mail: [email protected] management evaluates, selects, and implements plans or actions to

1381-6128/13 $58.00+.00 © 2013 Bentham Science Publishers 2 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al. ensure risks are appropriately managed. Thereby, policy alterna- with the abiotic environment; and (13) the scientific quality of the tives to accept, minimize, or reduce the characterized risks are proposed post-market (environmental) monitoring plan [8, 9, 10]. weighed and, if needed, appropriate prevention and control options Moreover, an assessment of direct and indirect, as well as immedi- are selected. Because risk managers and regulators rely on risk ate and delayed, including cumulative long-term effects is required assessments to make an informed decision on whether or not to in several jurisdictions. Now it could be argued that a lot of the approve a certain use of a GM plant, it should explain clearly what criteria set out above, would not be applicable to PMF as these assumptions have been made during the risk assessment, and what crops are not designed to enter the food chain. However, if they are is the nature and magnitude of scientific uncertainties associated engineered into a food crop then these assessments will be neces- with the characterized risks. The decision whether a certain risk is sary, as part of the precautionary approach – i.e. what if they did acceptable and/or tolerable under a particular set of conditions is become mixed in the food chain. EFSA have produced guidance not part of the risk assessment itself, but part of the wider risk notes on the Risk Assessment for GM crops for non-food/feed uses analysis, as this choice is not only based on scientific criteria, but to address some of these differences [3]. also involves political, social, cultural and economic considerations Even though there are considerable differences in regulatory - for example in the case of PMF it will be critical that these crops requirements for GM plants and derived products, priorities in should not become mingled with commodity crops. While this is terms of protection goals, as well as risk terminology among juris- also the case for agricultural GM crops, the perceived public back- dictions globally, most risk assessments: (1) follow sequential lash is likely to be greater for a pharmaceutical crop. Risk manage- steps; (2) are science-based, where quantitative information is ment is also functionally and temporally separate from risk assess- available, and use qualitative information in the form of expert ment in order to reduce any conflict of interest and to protect the judgment; (3) use a comparative approach whereby the level of risk scientific integrity of risk assessment [4]. Risk communication is is estimated through comparison with a non-GM counterpart; (4) defined as an interactive exchange of information and opinions on are case-specific; (5) are iterative and, in a transparent manner, risk throughout risk analysis, between risk assessors, risk managers examine previous conclusions in light of new information; and (6) and other interested parties. It includes the explanation of risk as- follow a tiered approach. For a more detailed account of GM risk sessment findings and of the basis upon which risk management assessment we refer you to [11]. decisions are made [5]. Harm refers to adverse effects to the human and animal health 3. NEW BREEDING TECHNIQUES IN MOLECULAR or the environment, while risk is recognized as a function of the FARMING AND IMPLICATIONS FOR THE CURRENT EU probability of an adverse effect occurring to human and animal REGULATORY SYSTEM health or the environment and its seriousness, following exposure to Several new breeding techniques (NBT) are currently under a hazard due to an activity i.e. the possible intended use(s) of a GM way which differ substantially from the transgenic (GM) techniques plant. Without hazard (i.e., the potential of an agent or situation to that have been used in the last two decades. For some of them the cause harm), there is no harm and thus no risk. It is therefore crucial regulatory framework in the EU may no longer be feasible, result- to distinguish risk from hazard in risk assessment, as both terms ing in a legislative/regulatory uncertainty concerning these tech- have different meanings. Various studies, assessing the impact of niques. In 2007, due to a mandate of the EU commission a new GM plants, have postulated dire risks when all they have done is techniques working group (NTWG) was established in which ex- characterized either a hazard associated with the use of GM plants, perts from competent authorities of several European countries or an exposure to the GM plant without demonstrating whether this evaluated the following eight NBTs: Oligo-directed mutagenesis exposure is hazardous (reviewed by [4]). Such studies rarely link (ODM); Zinc finger nuclease technology (ZFN); Cisgene- hazard to exposure, and in effect confuse hazard or exposure with sis/intragenesis; RNA-dependent DNA methylation (RdDM); Graft- risk. ing (on GM rootstock); Reverse breeding; Agro-infiltration; and Synthetic genomics. In 2012, the working group finished the 2.2. Risk Assessment Principles and Concepts evaluation and circulated its opinion for comments. In 2010, a Regulatory requirements involve the consideration of a range of working group led by the Joint Research Centre of the EU commis- issues (areas of risk) relevant to the overall risk assessment, in order sion started to develop a study concerning the same eight tech- to determine the impact of the GM plant on human and animal niques. The final report of this study was published last year [12]. health or the environment relative to the comparable non-GM crop, In 2011 on request of DG SANCO, the EFSA also set up a working and thus its relative safety [6, 7, 8, 9]. These include amongst others group to determine whether the existing guidance on risk assess- (and differently required in different jurisdictions): (1) molecular ment is appropriate concerning NBTs. The EFSA working group characterization of the GM plant, which provides information on will be providing its opinion one by one, starting with cisgenesis the structure and expression of the insert(s) and on the stability of and intragenesis [13]. the intended trait(s); (2) the comparative analysis of compositional, Cisgenesis/intragenesis technique refers to the integration of phenotypic and agronomic characteristics to identify intended and genes into a plant genome: a transgenic (GM) approach, but re- unintended changes in the GM plant; (3) the toxicological assess- stricting the source of the genes to sexually compatible crossing ment of the genetic modification, which addresses the impact on partners of the recipient plant. By this approach only cisgenesis human and animal health of biologically relevant change(s) in the produces progeny which can arise by chance by a conventional GM plant and/or derived food and feed resulting from the genetic breeding process (CBP), whereas intragenesis always produces modification; (4) the assessment of the allergenic potential of the progeny which cannot be achieved by CBP. As the process itself novel protein(s) (in the case of PMF, this assesment would deter- introduces foreign DNA which is capable of replication and inherit- mine whether approval would be granted for a field release); (5) the able, the technique might be considered to be regulated by GMO nutritional assessment, which aims to demonstrate that the food and legislation. If a cisgene without a selection marker or other foreign feed derived from a GM plant is not nutritionally disadvantageous DNA-sequences is transferred using nuclease technique (see ZFN- to humans and/or animals; (6) potential changes in the persistence 3), the resulting plant is nearly indistinguishable from the one de- (weediness) and invasiveness ability of the GM plant; (7) potential rived through a CBP and meets the criteria of self-cloning. In this for gene transfer; (8) interactions between the GM plant and target case, which is described in Annex II, part A of Directive organisms; (9) interactions between the GM plant and non-target 2009/41/EC it might be falling outside the scope of the GMO legis- organisms; (10) potential adverse effects on biogeochemical proc- lation. esses; (11) impacts of altered farm management practices associated with the cultivation of the GM plant; (12) potential interactions Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 3

Oligo-directed mutagenesis is a technique using short oli- integration by homologous recombination, long DNA sequences gonucleotides as a tool to induce targeted alterations in the genome (500-1000 nt) with homology nearby the DSB site have to be pre- ranging from one to a few adjacent nucleotides. The alterations are sent on the targeting construct. caused by mispairing of nucleotides and the subsequent DNA repair Two other nuclease techniques have been developed to achieve by the host cell. The ODM technique can be used, e.g., for induc- the same task by slightly differing means, meganucleases (MN) and tion of herbicide resistance in tobacco due to point mutations [14]. transcription activator like effector nucleases (TALENs) [17, 18]. However, because the ODM technique does not introduce recombi- In general all three types of site directed nucleases (SDN) perform nant DNA into the plant which is capable of replication, it is dis- two functions: i) specific DNA recognition and binding and ii) in- cussed by the NTWG to be not regulated by the GMO legislation or troduction of a DSB in or nearby the bound sequence. This DSB to be excluded from Directive 2001/18/EC (Annex IA, Part 1). greatly enhances the chance for homologous recombination and The aim of RNA-dependent DNA methylation is the induc- thereby offers the opportunity to integrate any DNA sequence spe- tion of gene silencing via promoter methylation of target genes. cifically at a targeted locus instead of elsewhere in the genome [19, Depending on the delivery method of the DNA which encodes for 20]. Therefore, SDN technique allows choosing or designing a ge- the silencing RNAs, the expression can be transient thus only in- nomic locus which possesses the best performance for stable and ducing epigenetic changes of the genome by the RNA silencing high expression of, e.g., PMP genes. pathway. If the DNA coding for the silencing RNA is stably inte- The nuclease techniques described under ZFN-1 and -2 used for grated into a plant genome it is regulated by current GMO legisla- gene alteration with only point mutations or a few nucleotide tion. If only transient expression occurs, it should not be in the changes, might be considered to be not regulated by GMO legisla- scope of the GMO legislation because methylation of nucleotides is tion as far as no foreign DNA is integrated in the plant cell. The not considered to be an alteration of genomic material. outcome of ZFN-1 and -2 is indistinguishable from natural muta- Grafting with GM scions or rootstocks can be used for the tions. ZFN-3 technique used for gene integration is covered by the transient expression of proteins or other effectors like RNAs to existing guidance for risk assessment and GMO legislation as it induce, e.g., enhanced pathogen resistance. This technique can be does not differ in its mode of action from the integration of genes performed using a non-GM scion onto a GM rootstock or vice by transgenesis. However, nuclease technique in general greatly versa. In both cases the resulting plant is a chimeric plant which enhances the accuracy of gene integration in a preselected target falls within the scope of the GMO legislation. The fruits, seeds or locus, thereby minimizing unattended side effects which might offspring of a GM scion are also regulated by GMO legislation, result in an easier risk assessment. whereas the fruits, seeds or offspring derived from a non-GM scion The term Agro-infiltration (or Agro-inoculation) means that a grafted onto a GM rootstock might not fall under the scope of Di- plant or plant tissue is infiltrated with a liquid Agrobacterium sp. rective 2001/18/EC. suspension. The Agrobacterium delivers T-DNA from which ge- The Reverse breeding technique is able to reconstitute the netic material is expressed locally and transiently at high levels in parental lines starting with an elite F1 hybrid whose genetic mate- the plant to produce a specific protein. Agro-infiltration has been rial is unknown [15, 16]. Reverse breeding combines several other used in research for, e.g., the transient expression of resistance techniques, i) RNAi to suppress meiotic recombination; ii) tissue genes from wild grape in susceptible grapevine cultivars or for the culture to regenerate plants from cells; and iii) the double haploidi- expression of artificial micro RNAs conferring resistance to potato zation technique to create double haploid plants which are used as virus X and Y [21]. Concerning the production of pharmaceutical the respective parental lines to produce new elite F1 hybrids. Dur- proteins in plants, e.g., Artemisinin precursors as well as IgG or ing this procedure a transgenic step can be included but the final IgM antibodies have been produced in Nicotiana benthamiana re- plant lines are non-transgenic and therefore might be considered as sulting in yields of 40 to about 600 mg/kg of fresh weight [22-25]. falling outside the scope of the GMO legislation. As the technique described does not aim for progeny of the Synthetic genomics aims to synthesize large DNA molecules inoculated plant tissue, the only GMO involved in the process is the followed by transformation in a recipient structure. As the applica- Agrobacterium. Therefore, Agro-infiltration is covered by the cur- tion of this technique to genetically modify plants is in a very early rently existing legislation and risk assessment as GMM in plants. stage, possible future implications have been evaluated. Only if the Usually the plants or the plant tissue are destroyed after the experi- synthesized DNA is assembled to result in an entity which is a mi- ment. Progeny derived from this plant material in which absence of croorganism will it fall under the scope of Directives 2009/41/EC stably integrated DNA is proven might not be considered a GMO (Annex I Part A) and 2001/18/EC (Annex 1A Part 1). and therefore might not fall under the scope of the GMO legisla- Several of the NBTs are already in use or planned to be used for tion. Agro-infiltration approaches for PMP production, of contain- general plant breeding in the near future. The following two NBTs ment grown plants, are beginning to see investment. Two compa- especially, may have great benefits for molecular farming purposes: nies leading in this area are Kentucky Bioprocessing Zinc finger nuclease technology and Agro-infiltration. (www.kbpllc.com/) and Medicago (www.medicago.com/), the later now being in scale up for phase II clinical trials of their avian flu Zinc finger nuclease technique was the original term describ- vaccine, produced using transient expression systems. ing a new technique which uses an unspecific nuclease (usually FokI) coupled to a sequence specific DNA binding element, the 4. CONDITIONS FOR MOLECULAR FARMING IN zinc finger. The restriction activity is directed to a certain locus in EUROPE the genome of an organism by the zinc finger binding activity. Af- 4.1. Regulatory Framework for Contained and Open Field use ter induction of a double strand break (DSB), the repair is done of GM Plants completely by the host cell. The working group of new techniques discriminated between three different ways how ZFN can be used. The European Union (EU) is considered to have the toughest ZFN-1: Without any additional DNA homology, the DSB will be and most stringent legislation on GM products and commodities in repaired in almost all cases by the non-homologous end joining the world. The enforcement of such legislation has three main ob- pathway of the cell. This will result in point mutations or small jectives: to protect human and animal health and the environment; InDels at the DSB site. ZFN-2: Adding short DNA sequences to guarantee consumers’ freedom of choice without misleading which are differing only in a few nucleotides to the genomic se- consumers/users; and to create an internal market allowing the free quence around the DSB can result in a repair which copies these movement of safe and healthy GM products within the EU, thus differences in the genomic sequence. ZFN-3: To enable targeted 4 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al. avoiding unequal competition and trade impediments between and - All waste material generated in containment is steam within EU Member States. treated/autoclaved or incinerated (soil, plant debris, pots and The EU regulatory approach is precautionary, process-related, disposable protective clothing like gloves, hair nets, etc.); and includes mandatory labelling and traceability requirements for - Personnel wear protective clothing and hand washing facilities food and feed products derived from GM plants, unprocessed or are used where appropriate, again to minimize or prevent pol- processed. Only non-food GM products (unseeded), such as textile len transfer and insect movement into and out of the facility; or other industrial products are not subject to any requirements for - Live tissue leaving the premises (e.g. leaf tissue, or seeds) is labelling. moved in double contained vessels, with an appropriate risk EU legislation is adopted through a system of interactions be- assessment, to ensure that no unintended release into the envi- tween the three main EU institutions: the European Parliament, the ronment occurs. Council of the European Union (i.e., representatives of all the EU It should be noted that these plants in general will not pose any Member States at the ministerial level), and the European Commis- health or environmental risks, but EU legislation follows a precau- sion. In most cases, the European Commission initiates legislative tionary principle and all ‘what if’ scenarios need to be addressed. proposals that are decided jointly by the Council and the European Implementation of these regulations is very straightforward, and Parliament; the most common legislative measures are Regulations indeed have become common practice for all EU universi- (acts that are binding in their entirety and are immediately applica- ties/institutes and organizations. For PMPs and PMIs these meas- ble throughout the EU) and Directives (acts that require the modifi- urements would also ensure that plants are as clean as technically cation or establishment of national measures, generally for har- possible (for cultivation in a non-sterile environment) before they monization purposes). can be processed prior to entering cGMP facilities (see Fischer et In the early 1990s, two European Directives for the use of al., this issue). GMOs were adopted: Directive 90/219/EEC (which has since been When GM plants are used to produce therapeutic or industrial amended by Directive 98/81/EEC, and more recently 2009/41/EC), proteins in plant suspension cell systems, containment in enclosed which regulated the contained use of GM micro-organisms; and sealed bioreactors would in some cases simplify the risk assessment Directive 90/220/EEC (which has since been replaced by Directive and biosafety issues. These types of culture systems are likely to 2001/18/EC) which regulated the deliberate release of GMOs into also be more familiar to the current pharmaceutical industry (cf., the environment, covering both the release for research purposes use of mammalian and bacterial cell culture systems). In 2006, the (part B) and for commercial use as or in products (part C). This first PMP, a vaccine for poultry (against Newcastle disease) gained triad reflects the stepwise process GM plants go through, beginning regulatory approval from the US Department of Agriculture. Al- with experiments under contained use (e.g., laboratory, green- though this poultry vaccine remained a proof-of-concept and was house), through experimental release, up to the placing on the mar- never sold, it paved the way for future PMF. Indeed, the first plant- ket. According to the step-by-step principle, the containment of made biologic, for human use, to gain [US] regulatory approval was GMOs can be reduced and the scale of release increased gradually, produced in a carrot cell system (for the production of Elelyso, an if assessment of earlier steps indicated that the next step can be enzyme to treat type 1 Gaucher’s disease) by the Israel-based Pro- taken. Thus broadly speaking there are two categories of authoriza- talix BioTherapeutics, which gained FDA regulatory approval in tion: (1) for the contained use (i.e., those grown in laboratories, May 2012 (http://www.protalix.com/product- glasshouses, or fully contained facilities such as cell culture sys- development/elelyso.asp). tems); and (2) for the ‘deliberate release into the environment’, either for experimental purposes or for commercial cultivation. If Meanwhile, in Europe an EU-funded project that ran from the GMO is intended for food and/or feed uses, its potential uses are 2004-2011 (www.pharma-planta.net) saw the first regulatory ap- regulated under the scope of GM food and feed Regulation (EC) proval for a PMF product to enter phase 1 clinical trial in humans. 1829/2003. The product was an anti-HIV monoclonal antibody; using whole tobacco plants grown in containment as the platform. The choice of When the end product of a molecular farming activity is a host plant and growing environment was somewhat determined as pharmaceutical, the safety or toxicological aspects of the product the EU program developed, in order to meet the least regulatory and its uses fall under Regulation (EC) 726/2004 on medicinal resistance, and is further discussed later. Although plants have been products. shown to be potentially useful for the production of therapeutic 4.1.1. Contained use of GM Plants proteins, they are at a relatively early stage of development and Within Europe the contained use of all GMOs (which includes more needs to be done so that they can be used to produce proteins GM bacterial, mammalian and plant cultures) as previously men- in a consistent manner. Another EU funded project, tioned, is covered by Directive 2009/41/EC. Contained use covers (www.comofarm.org), was established to address these issues, and the initial production of a GMO in the laboratory, as well as the use, to look at a range of containment platforms (hydroponic plants, root storage, transport and destruction of the GMO. Any organization culture, cultivated moss and cell suspension culture) across a range wanting to produce or handle GMOs must notify the relevant com- of plant species. Ultimately, the results from the CoMoFarm (con- petent authorities of their intention to use their premises for contained molecular farming) project, which concludes at the end of tained use activities for the first time. It then becomes the responsi- 2012, aims to help reduce the costs involved in the production of bility of the organization to ensure that the appropriate risk assess- pharmaceutical and industrial proteins and ensure that pharmaceuti- ments are in place, and the appropriate level of containment is se- cals from plants are produced to the highest possible standards. lected to minimize any potential risks to negligible or low. In the However, to fully exploit the claims of using plants as produc- case of growing a GM plant in glass house containment, for exam- tion vehicles for cheap production of therapeutic and industrial ple, measures would be put in place: proteins (especially the lower value proteins) it may be desirable for - To reduce the likelihood of, or prevent insects or rodents from plants to be cultivated on a larger scale than containment-grown entering the glasshouse - thereby minimizing any potential approaches. For example, it has been estimated that one acre of transfer of material, i.e., pollen or seed out of the glasshouse safflower engineered to produce insulin by the Canadian company and reducing any potential unintended effects resulting in ex- SemBioSys, could produce more than one kilogram of insulin, posure of these groups to the plants; enough to treat 2,500 diabetic patients for one year. That means just 16,000 acres of safflower could meet the world’s total demand each year. Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 5

4.1.2. Open field use of GM Plants tion information format). The SNIF document is then made public, Growing GM plants in the field for experimental purposes con- which in turn supports the transparency (openness) of the activities stitutes a ‘deliberate environmental release’ and as such requires proposed – although this openness of precise field locations may prior notification under the Directive 2001/18/EC to the local com- then render field trials vulnerable to abuse by potential protestors. petent authority of the EU Member State in which the proposed Information about all experimental releases authorized by the EU field release was intended. Such notifications must include details member states is available to all and can be accessed at the follow- about the host plant, the introduced trait, the purpose of the release ing URL: http://gmoinfo.jrc.ec.europa.eu/. According to Article 6.5 and details of the exact location and size of the trial, an environ- of Directive 2001/18/EC, the local (national and/or regional) mental risk assessment and details of the risk management strate- authority must respond to the notification within 90 days by ap- gies [26]. Once the notification has been sent to the competent na- proving or rejecting it, if no additional information is requested tional authority it is passed on to a scientific advisory committee, from the notifier. Field trials are then subject to [annual] inspection who will look at the risk assessments and potential environmental throughout the life time of the trial by the GM Inspectorate; and for impacts and assess if the trial would pose a threat to health or the a set period of time post trial to ensure compliance. environment. Although the regulations cover the whole of the EU, Currently many EU countries have a negative position on GM the power to approve or reject a notification rests solely with the plants and do not allow experimental field trials in their territory. national competent authority, in some cases with additional input Other countries are more open to the evaluation of this technology from national scientific advisory bodies, other member states and and while they allow field trials they impose strict compliance the European Commission. It is also mandatory for the notifier to measures to avoid potential risks to the environment or human and advertise the proposed release (usually in a national paper) within animal health. Usually, specific conditions are imposed under 10 days of submission, and for the national authority to provide the which the field trial may go ahead, and these include: isolation European Commission with a summary communicating the most distances and buffer strips to minimize outcrossing with the con- important information in the application (SNIF, summary notifica- ventional counterpart or hybridization with sexually cross-

Table 1. Overview of field trials in Europe (1995-2012) with GM plants used as production platforms for pharmaceutical or industrial products

Crop Trait / Molecule of Interest No. of Trials

Barley Thaumatin (sweetner) 1 Barley Human serum albium 1 Barley Growth factor 1 Flax Biodegradable plastic 1 Maize Rabies virus G glycoprotein 1 Maize Dog gastric lipase 6 Maize Human collagen 2 Maize Human lactoferrin 2 Maize Human albumin 1 Maize Antibodies 1

Rape Dog gastric lipase 2 Rice human enzyme acid -glucosidase (GCase) 1 Pea Alpha-amylase 1 Feed pea Alpha-amylase 1 Potato Spider silk 1 Potato Non-plant carbohydrates 1 Potato Spider silk-elastin fusion protein 1 Potato Expression of Cyanophycin synthetase 5 Potato Expression of the viral coat protein VP60 and choleratoxin B 6 Tobacco Human alpha-1 anti-trypsin 1 Tobacco Dog gastric lipase 7 Tobacco Rabies virus G glycoprotein 1 Tobacco Technical enzymes 1 Tobacco Human collagen 3 Tobacco Human glucocerebrosidase protein 1 Tobacco Taxane diterpenoid 1

(Source: Adapted from Breyer et al. (2009). Joint Research Centre of the European Commission, 2012: http://gmoinfo.jrc.it/) 6 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al. compatible wild relatives; confinement measures to avoid the oc- rounding master and working cell banks, compliance with GMP currence of volunteer plants in subsequent years originating from and particularly issues regarding batch-to-batch consistency, stan- spilled seeds and/or vegetative plant parts during the trial and mix- dard operating procedures for different production systems and ing of plant material in machinery during sowing, harvest and/or downstream processing requirements need to be addressed. The post-harvest operations [27]; post-release follow-up to control po- European Medicines Agency (EMEA) that oversees the assessment tential volunteer plants occurring in subsequent years; and crop of biopharmaceuticals and vaccines, published guidance notes in destruction. Plant material from these trials cannot enter the food 2006 on the ‘quality of biological active substances produced by and feed chain and has to be destroyed at the end of the trial [28, stable transgene expression in higher plants’ [34], which looks at 29, 30]. such issues. As with all emerging technologies, it will be the early In the EU, 51 field trials with GM plants serving as production and first products to enter the approval/regulatory systems that will platform for pharmaceutical or industrial products have been noti- ultimately help shape and evolve the regulation surrounding PMPs. fied since 1995 (see Table 1). However, Europe has yet to witness When considering host plants in the context of biosafety and the commercial application of PMP technology, although some risk assessment, the better scientific option (e.g., plants with the pharmaceutical proteins (gastric lipase, lactoferrin) have entered highest yield, best protein stability, and better downstream process- phase I and II clinical trials [31, 32]. ing ability) may not be the best choice of host from a biosafety and regulatory perspective. 4.2. Regulatory Requirements for PMP in Europe The choice of host crop falls into three main categories: non- There is currently no ‘natural’ home for the start to finish regu- food crops, food crops and non-cultivated crops (reviewed in [35]. lation of PMP/PMIs. Their regulation in Europe therefore falls to a Desirable crops from a biosafety perspective could include male number of regulatory bodies (Table 2). PMPs will need to adhere to sterile plants (such as male sterile lines of potato), crops with no the regulations that cover the contained use of GMOs wild relatives within the growing region (such as maize, tobacco or (2009/41/EC), for the early laboratory R&D phase, as well as cov- potato), or plants with heavy pollen (such as maize, barley and rice, ering GM material used in bioreactors and the growth of whole again in countries with no wild relatives). A wealth of knowledge plants for PMF in contained facilities such as glass-houses. For surrounds the cultivation of food crops and transformation proce- some PMP products, where a therapeutic protein is considered dures are well defined for a number of major crop species. How- ‘high risk’ because it could result in adverse effects if inadvertently ever, the regulators may view such crops as less desirable because eaten, regulatory approval would only be granted if crops were they represent major food crops, and therefore the risk of PMPs grown under containment conditions. PMP producing plants grown entering the food chain (even if only a perceived risk) would be under containment conditions are likely to face fewer regulatory higher than it would be when using non-food crops such as tobacco hurdles than field-grown PMP producing plants because the risk to or non-cultivated crops, such as duckweed, that could be grown in the environment is lower. containment. Nevertheless, the lack of toxic ingredients is an im- Regulatory oversight for PMF in the field is covered by Direc- portant advantage of food crops which should be taken into active 2001/18/EC, which regulates the deliberate release into the count. environment of GMOs. Part B of this Directive regulates the ex- The choice of expression site within the plant can also have perimental field release (for which Europe has seen a number of biosafety implications. Targeting expression to the seed reduces the notifications for PMF experimental release (see Table 1), although potential exposure of herbivores and other non-target organisms to far behind the numbers for US). Part C of this Directive would pharmaceutical products expressed in the leaves as well as leaching regulate the import, cultivation, processing and marketing for of recombinant proteins into the environment from roots. However, commercial purposes. The EFSA, who oversees the assessment of one advantage of harvesting leaf tissue, rather than seeds or fruit, is GM plants to be deliberately released into the environment, pub- that the plants do not need to flower, thereby reducing the risk of lished guidance notes in 2009 on the risk assessment of genetically pollen and seed dispersal. modified plants used for non-food or non-feed purposes [3] includ- Some plants have already been recommended as not suitable for ing GM plants as production platform for PMI/PMP. field production by regulatory bodies such as the US Animal and Europe has yet to see any Part C application for a commercial Plant Health Inspectorate (APHIS) and the Canadian regulatory scale field release of a GM crop for PMF. Indeed, Europe in general authorities, these included oilseed rape (canola) and alfalfa. These falls far behind the rest of the world in approvals of GM crops for plants are considered to be unsuitable because they are bee- cultivation, with only two products being approved in the last 14 pollinated, sexually compatible with local weed species and have years for cultivation (insect resistant maize (MON810) and a modi- multiple year seed dormancy. These traits therefore increase the fied starch potato (Amflora)). The maize MON810, and derivatives risk of transgene spread and volunteer plants in subsequent crops. thereof (i.e., subsequent introduction of the insect resistant trait into However, such crops may be considered as beneficial hosts if maize breeding programs) are the only GM crops currently being grown in containment. cultivated in Europe. However, across Europe the political view point of cultivating GM crops is far from harmonious, with a num- 4.3. Case Studies for Experimental Field Releases of PMP and ber of Member States banning such cultivation PMI Producing Plants (www.greenbiotech.org). Given the state of play surrounding the In October 2008, the University of Rostock applied for the cultivation of agricultural GM crops, it is unlikely that we will see a permission to release potatoes each carrying one of three different pharma crop grown commercially in Europe any time soon. transgenes in a field trial planned from 2009 to 2011 at two sites When the plant cells/plants are harvested they then need to (Thulendorf in Mecklenburg Pomerania and Üplingen in Saxony enter downstream processing in order to isolate the active sub- Anhalt). For each transgene, different events in different cultivars stance, which can then enter cGMP facilities (discussed in detail in should be released in order to identify effects caused by somaclonal Schillberg et al., this issue). It is under cGMP that the formulation variation or cultivar, instead of the specific transgene (Table 3). The and assembly of a finished product, to enter clinical assessment, first transgene (35SctxBSEK) codes for the non-toxic B subunit will take place and this will be in line with Regulation (EC) (CTB) of Cholera toxin (CTA-CTB). CTB is one of the most potent 726/2004 on ‘Biopharmaceuticals and vaccines for human and vet- natural adjuvants which functions as both mucosal and parenteral erinary use’. This Directive was originally set up for cell lines (e.g. adjuvant [36, 37]. The natural CTB of Vibrio cholerae contains a CHO and bacterial cells), and it is argued that it needs to be refined specific signal peptide at its amino terminal end [38] that facilitates to be more specific for plants [32, 33]. In particular, issues sur- transport into the periplasmatic space, where the CTB monomers Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 7

Table 2. Overview of EU Regulations Currently Surrounding PMF

Directive / Regulation Covers Relevance to Pharma - covers Authority

Directive 2009/41/EC Contained use of GMOs • Early laboratory R&D phase Member States National compe- • GMOs grown in enclosed cell culture systems tent authorities (Bioreactors) • GM plants grown in containment, i.e. glasshouses or controlled environment rooms

Directive Deliberate release to the environ- Part B of the directive covering experimental field Member States National compe- 2001/18/EC ment release tent authorities Part C covering cultivation at the commercial level Member States National competent authorities, European Com- mission, EFSA

Regulation (EC) Regulation on GM food/feed Applies only if GM plant parts are to enter the European Commission, EFSA, 1829/2003 food or feed chain (highly unlikely for Pharma) or Member States National compe- may also apply if a ‘food’ crop is used for produc- tent authorities tion even if for non-food/feed purposes

Directive 2001/10/EC Safety and efficacy testing of drugs Clinical trials Overseen by the Member States National competent authorities, at the R&D stage and by EMEA at the marketing stage

Regulation (EC) ‘laying down Community proce- Would follow the existing regulations developed for EMEA (assessment only), Euro- 726/2004 dures for the authorization and su- mammalian/ bacterial systems and draw guidance pean Commission, Member pervision of medicinal products for from EMEA guidance notes published on the ‘qual- States National competent human and veterinary use and estab- ity of biological active substances produced by authorities lishing a European Medicines stable transgene expression in higher plants’ [34] (Market authorization) Agency

EFSA = European Food Safety Authority; EMEA = European Medicines Agency oligomerize [39]. In plants, both secretory and chloroplast-derived cyanophycin synthetase, encoded by cphA, is necessary to catalyze CTB expression have been demonstrated [40, 41]. Plant-derived the ATP dependent elongation of the cyanophycin primer by the fusion proteins of CTB expressing different antigens via the secre- consecutive addition of aspartic acid and arginine. All transgenic tory pathway have been successfully used to induce specific im- events carried the nptII gene as selectable marker. As controls, a mune responses and therapeutic effects [42-48]. No harmful effects transgenic line transformed with the empty vector carrying only the could be detected when CTB was applied to mice [36, 40, 49, 50]. nptII gene was selected. In addition the cultivar ‘Albatros’ was used A very similar protein, Lt-B, was applied to humans without any as near-isogenic variant (niv) and ‘Desiree’ as second potato culti- deleterious effect [51]. The second transgene (35Svp60SEK) codes var. for an antigen supposed to lead to immunogenicity against the rab- The aim of the release was to identify potential effects of the bit haemorrhagic disease (RHD) virus. RHD is an acute and highly three transgenes on the environment, to analyse the variability of infectious viral disease of the European rabbit (Oryctolagus cunicu- transgene expression in comparison to greenhouse trials and to lus). Its capsid protein VP60, which forms a 27-40 nm virion with a produce material for feeding studies. The number of tubers released structured surface of regularly arranged cup-shaped depressions is given in (Table 3). [42], was chosen as antigen. The third transgene (pPsbY-cphA) codes for a cyanophycin synthetase. Cyanophycin is a cyanobacte- In October 2008 the application was sent to the German Federal rial reserve polymer composed of a poly--aspartic acid backbone Office of Consumer Protection and Food Safety (BVL) which is with arginine residues linked via their -amino group to the - responsible for the approval of the trial. The application was a fol- carboxyl group of each aspartate residue [52-54]. Mild hydrolysis low-up of a release that was carried out in 2005 to 2008, where of cyanophycin [55] results in homo- and copolymers of polyaspar- transgenic potatoes carrying the same transgenes have already been tate and additionally L-arginine as products. The latter one has a released at the same sites. No signs of any detrimental effect have broad range of technical and medical applications, amongst others been observed. In December, the application was declared to be as a stimulator of the immune system [56-60], growth increase by complete by BVL and this was announced in the federal bulletin as feeding [61, 62], and as an inhibitor of tumour cell growth [63]. well as in local newspapers at the sites of release. In addition, the Polyaspartate is a soluble, non-toxic and biodegradable polycar- complete application was displayed for one month to the public at boxylate [64], which could replace the non-biodegradable polyacry- both locations and in Berlin in order to give everybody the chance lates in many of its industrial, agricultural, and medical applications to be informed. The whole process is expected to be finished in 90 [55, 65, 66]. Cyanophycin is synthesized via non-ribosomal poly- days after the submission of the application, the time needed for peptide synthesis in many cyanobacteria and some other non photo- corrections by the applicant and the month of public display is synthetic bacteria [67, 68]. For cyanophycin synthesis only the added. The approval was expected at least at the beginning of March 2009 in order to allow a timely release of the potatoes. 8 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.

Table 3. Events and number of tubers predicted to be released in the field trial

Vector Cultivar Event No. of tubers (in year)

2009 2010-2012, each

35Svp60SEK Desiree 6 458 458

35Svp60SEK Fasan 201 38 38

35Svp60SEK Albatros 204 143 437

35SctxBSEK Albatros 204 38 38

pPsbY-cphA Albatros 12 458 458

pPsbY-cphA Albatros 23 38 38

pPsbY-cphA Albatros 9 38 38

pPsbY-cphA Albatros 10 38 38

pPsbY-cphA Albatros 24 38 38

pPsbY-cphA Albatros 31 38 38

pPsbY-cphA Albatros 35 38 38

35S (nptII) Desiree 6 458 458

35S (nptII) Fasan 203 38 38

35S (nptII) Albatros 205 458 458

near-isogenic variant (niv) Desiree niv 458 458

near-isogenic variant (niv) Fasan niv 38 38

near-isogenic variant (niv) Albatros niv 547 547

Amazingly, only a partial permission was given in May 2009. A 5. CONDITIONS FOR MOLECULAR FARMING IN THE US prerequisite for this permission was the agreement to postpone the release of the 76 CTB tubers to 2010. Hence the application for the 5.1. Regulatory Framework for Contained and Open Field use permission to release the CTB event 35SctxBsek 204 was repeated of Genetically Engineered (GE) Plants in 2009. The approval was provided in time to add these tubers to In the US, laboratory practices using recombinant DNA the field experiment in 2010. No additional information confirming (rDNA) must comply with National Institutes of Health (NIH) the safety of the release of these potatoes was necessary. The loca- guidelines for recombinant DNA and these guidelines are adminis- tion of the release had to be announced in the German ‘Standortreg- tered by the Institutional Biosafety Committees of whatever entity ister’ (http://apps2.bvl.bund.de/stareg_web/showflaechen.do) in is performing the research. However, in order for any impact to be detail that it was easy to identify the place of release. realized from this research, these laboratory experiments must Twelve additional measures were ordered by the BVL including move ahead to the field or production arena. Commercialization has that 1) during the release and the monitoring period, a 20 m wide many hurdles including not only the science and associated intellec- area surrounding the trial had to be controlled every month for po- tual property to protect the inventions, but also market realities and tential transgenic progeny; 2) after the release the area had to be government regulations. As each engineered plant moves from the loosened up 15 to 20 cm deep. Plugging was not permitted; 3) the laboratory to scaled-up greenhouse production or to the field, issues area had to be monitored once a month in the following year in emerge that trigger additional oversight for environmental release order to identify volunteers. If any transgenic volunteer were found or ultimate use by humans. in the area where the trial occurred, the monitoring period had to be The regulatory framework for genetically engineered plants in prolonged for a second year. The tubers were planted by hand to- the United States has been discussed in many articles from several gether with the controls and the area was surrounded by a 2 m high perspectives [69-71]. In contrast to the development of regulations fence buried 30 cm deep into the soil. Watchmen were employed to in Europe, no specific legislation was enacted in the United States protect the site, increasing the cost for the field trial drastically. that provided guidance or directives relating to the regulation of Nevertheless, the field was attacked twice in 2009 and again in genetically engineered plants. Rather, it was concluded in the 1980s 2011. Fortunately, the destroyers were only able to tear some of the that existing statutes provided sufficient general authority for the plants out of the soil, and in some cases it was possible to identify implementation of genetically engineered crop regulations. The the plants so they could be replaced into the soil and the trial could White House Office of Science and Technology Policy (OSTP) be evaluated, at least partially. established the Biotechnology Science Coordinating Committee (BSCC, 1984), with representatives from 18 agencies including the Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 9

National Institutes of Health (NIH), the United States Department promoters in genetic engineering, the plants produced from these of Agriculture (USDA), the US Environmental Protection Agency agents are considered plant pests. (EPA), the US Food and Drug Administration (FDA), and the US A GM crop developer must obtain APHIS authorization National Science Foundation [71, 72]. During the Reagan admini- through either the permit process or notification process before a stration in 1986, the Coordinated Framework for Regulation of genetically engineered crop can be field tested or released into the Biotechnology was published in the US Federal Register (51 Fed. environment. Movement and planting restrictions are imposed by Reg. 23302; June 26, 1986). This document included policy state- permits while notification can be used in lieu of a permit when the ments from the agencies involved as well as a proposed USDA plant is not considered a noxious weed [77]. Notifications, i.e., an regulation governing the introduction of organisms and products expedited permitting process, are used primarily for field trial ap- that are plant pests that were developed through genetic engineering proval as well as importation and transport within the US of certain [73]. Of significance and in contrast to European policies, an under- genetically engineered plants. To be eligible for a notification, spe- lying principle of the US Coordinated Framework is that genetically cific criteria as well as designated performance and characteristics engineered organisms are not inherently risky and regulatory re- standards must be met. For example, among other things, the GM quirements should focus on the product instead of the process used plant cannot be considered a noxious weed under USDA regula- to derive the organism. Additionally, the existing agencies and laws tions or a weed in the area of release, the function of the genetic were deemed sufficient to regulate this new technology and the material must be known and its expression must not cause plant agencies agreed to cooperate in the process. The resulting Coordi- disease, the modification should not contain human or animal nated Framework allowed (1) USDA to monitor planting and field pathogenic sequence, and the crop cannot express a plant-made testing as well as to regulate genetically engineered organisms that pharmaceutical or a plant-made industrial product [78]. Permits are might be plant pests, (2) FDA to ensure the safety of most domestic used when the GE plant does not meet the requirements for notifi- and imported foods or feed, including genetically engineered foods, cation. and to enforce pesticide tolerances established for food or feed by the EPA, and (3) the EPA to establish pesticide tolerances for food In 2003, the USDA amended its regulations to require that in- and feed and regulate genetically engineered organisms that contain troductions of plants genetically engineered to produce industrial pesticidal substances and the genetic material necessary for incor- compounds be conducted only under permit, i.e., not eligible for poration of pesticidal substances, new uses of existing pesticides, notification (68 FR 46434-46436, Docket No. 03-038-1, August 6, and novel micro-organisms [71, 74, 75]. Although these divisions 2003). As with the notifications, developers must provide APHIS of responsibility may be clear in theory, in practice the lines are scientists with details about the nature of the GE organism. The often blurred as into whose purview a genetically engineered plant main consideration for field trials is safety and prevention of unin- falls. The US policy decision to use existing laws, in effect, led to tended release in order to prevent spread of a potential plant pest to three different agencies with significant and often duplicative roles the environment. in the regulation of GM crops. Indeed, in most cases, different regu- APHIS regulation 7 CFR Part 340.6 permits a crop developer to latory dossiers and data sets must be submitted to at least two US petition APHIS to evaluate submitted data to determine if a particu- agencies, although these data sets are not mutually exclusive. lar article presents a plant pest risk, and if not, should no longer be Twenty-six years after the implementation of the US Coordinated regulated [71, 77]. Once a developer has sufficient evidence that its Framework (i.e., in early 2012), the USDA, EPA, and FDA entered GM product is no more risky than the equivalent non-GM parent, into a memorandum of understanding (77 Federal Register 5012, the developer may petition APHIS for a determination of non- FRL–9328–7, MOU 225-11-0001) to permit information sharing regulated status to introduce the organism into US agriculture and between the three agencies. This may improve the overall coordina- commerce without APHIS oversight – i.e., to become “deregu- tion of regulation in the US although that remains to be seen. lated”. Petitions must contain extensive data on details about plant 5.1.1. Regulatory Considerations of the US Department of Agri- biology, genetics, the nature and origin of the genetic material, culture – Animal Plant Health Inspection Service (APHIS) expression of the gene product, disease and pest susceptibilities, field test reports and effects on other plants. In addition, data on the The primary regulatory arm of the USDA that is relevant for impact of the GM plant on other organisms including the potential plants and crops is the Animal and Plant Health Inspection Service for gene flow must be determined. Petitions are reviewed by APHIS (APHIS). The focus of APHIS is to protect US agriculture, food, with a focus on the potential for the GM plant to become a weed or and natural resources and it is in this context that it regulates GM plant pest [77, 78]. During the process of evaluation, APHIS at a crops [76], specifically genetically engineered organisms capable of minimum prepares an Environmental Assessment (EA) and a “De- posing risks to plant or animal health. termination of Non-Regulated Status” as well as taking into consid- The US Plant Protection Act (Title IV Public Law 106-224, 114 eration comments from the public. Statute 438, 7 U.S.C. §7701-7772) requires the USDA to prevent While this review is not focused on the legal aspects of regula- the introduction and dissemination of plant pests and weeds into the tions, it warrants mentioning that APHIS has worked through sev- United States and provides specific authority to prohibit or restrict eral lawsuits filed by the Center for Food Safety (CFS) and the the importation, exportation, and interstate movement of plants, International Center for Technology Assessment (ICTA) [70]. plant products, certain biological control organisms, noxious weeds, These organizations alleged, in the early 2000’s, that APHIS did and plant pests [69]. APHIS regulations under 7 CFR §340.1 stipu- not adequately evaluate the environmental risks of pharmaceutical- late that any organism which has been altered or produced through producing GM crops using the directives of the National Environ- genetic engineering is considered a regulated article if the donor mental Policy Act (NEPA) of 1969. An environmental assessment organism, recipient organism, or vector or vector agent meets the prior to issuing permits in some instances would have prevented definition of a plant pest, or is an unclassified organism and/or an litigation over the field-testing of these crops. Current litigation organism whose classification is unknown, or any product which involves the decisions by APHIS to de-regulate glyphosate-tolerant contains such an organism. Additionally, any other organism or (GT) alfalfa and sugar beet. These complex cases allege that APHIS product altered or produced through genetic engineering which the failed to consider outcrossing of GT alfalfa to organic crops or GT Administrator determines is a plant pest or has reason to believe is a sugar beet to seed production sugar beet crops [70]. In each case, plant pest is also regulated [77]. APHIS defines plant pests broadly the de-regulation decision was accompanied by a “Finding of No to include any organisms that can damage or cause injury to plants Significant Impact” or FONSI. In response to these cases, the or plant parts. Since many plant pathogens are used as vectors or USDA prepared Final Environmental Impact Statements (FEIS) for GT alfalfa and sugar beet. USDA published the FEIS in December 10 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.

2010 for the GT alfalfa lines J101 and J163 (APHIS-2007-0044- pesticides or plant incorporated protectants (PIPs). PIPs, such as the 12532). These lines were determined to be unlikely to pose plant Bacillus thuringiensis (Bt) toxin in corn, are considered pesticides pest risks and thus were no longer considered regulated articles. In under FIFRA because they are introduced into plants with the inten- the FEIS published by the USDA in May 2012, the USDA found tion of preventing, destroying, repelling, or mitigating a pest (FI- that GT sugar beets (H7-1) are not expected to become more inva- FRA §2). Therefore, Plant-Made Pharpaceuticals are not regulated sive in natural environments or have any different effect on critical by EPA. In 2001, EPA published a rule that established the regula- habitat than conventional sugar beets which do not establish or tory framework for PIPs and included (1) an exemption from FI- persist in the environment (APHIS-2010-0047-4658). FRA requirements for those PIPs that are derived entirely through 5.1.1.1. APHIS and New Breeding Technologies conventional breeding between sexually compatible plants and (2) an exemption from the requirement of the Federal Food, Drug, and In recent years, APHIS has taken steps to refine and limit its Cosmetic Act (FFDCA) (21 U.S.C. § 301 et seq.) for the establish- regulatory authority to only plants that are plant pests, were created ment of a residue tolerance level for pesticides [71]. with plant pests, or used a plant pest in the method to create the plant. This has resulted in the exemption from APHIS regulation A developer of a crop that contains a pesticidal property that several new breeding technologies. was not developed entirely through conventional breeding must submit an application to EPA to register a PIP. The EPA evaluates Cibus Global (www.Cibus.com) received a green light from each submission to register a pesticidal substance produced by a a APHIS in 2004 when it learned that its herbicide tolerant canola GM plant on a case-by-case basis, and requires data related to the developed with oligonucleotide-directed mutagenesis was not sub- product characterization which are similar to the data required by ject to regulation. In the March 2004 letter, APHIS indicated that it other agencies regarding the description of the GM plant and the had no authority to regulate products created by mutagenesis tech- details of its modification: i.e., organic source of the pesticidal gene niques. construct (promoter, terminator, enhancer, marker genes and other In May 2006, APHIS exempted from regulation a maize Zinc inserted DNA). The dossier should include information on the biol- Finger Nuclease (ZFN) - deletion technology used by Dow Agro- ogy of the plant, the stability of the genetically engineered DNA, Science (May 26, 2010 letter). The ZFN-12 maize plants with in- and the anti-nutritional substances produced by the plant, among duced deletions due to the use of zinc finger nuclease technology others. were not considered to be articles regulated by APHIS as ZFN-12 Additionally, EPA requires data on mammalian toxicity, the was not a plant pest and did not pose a plant pest risk. effects on non-target organisms, environmental metabolism and an APHIS clarified the scope of its regulatory authority in mid- insect resistance management program for plants specifically ex- 2011 when it informed the public that a genetically engineered, pressing Bt products. Since the EPA focuses on the pesticidal prop- herbicide tolerant Kentucky bluegrass developed by Scotts did not erties, developers should include a full biochemical and amino acid meet the definition of a regulated article (i.e., a plant pest) and thus description of the pesticidal protein. Since most PIPs are proteins, was exempt from regulation by APHIS (APHIS-2011-0080). This the issue of mammalian allergenicity is highly important. Acute conclusion was based on the fact that the Kentucky bluegrass itself digestibility assays and amino acid sequence homology are neces- is not a plant pest and that no organisms or the method used to cre- sary to demonstrate absence of allergenicity concerns. EPA is also ate the variety were plant pests. In a July 2011 Factsheet regarding concerned with gene flow issues as they may result in expanded the action, APHIS made clear that it would exempt from regulation exposure to novel pesticidal substances as well as the environ- other GE organisms that are not a plant pest, are not made using mental fate of PIP substances and the effect on non-target organ- plant pests, and when there is no reason to believe it is a plant pest. isms [71]. While each of these decisions was made relative to a specific In addition to the requirement for a PIP to be registered before case, they indicate that APHIS will not regulate new breeding tech- use unless it is exempted (as noted above), FIFRA section 7 re- nologies in and of themselves. Rather, it appears that APHIS will quires that the producers of pesticides register the facilities where continue to focus its regulatory requirements on plants that are a the pesticide is produced and report the amount produced. In con- plant pest or that pose pest risk, without regard to the technology trast to APHIS requirements, experimental use permits are required, used to create the plant. generally, for larger field trials (i.e., over 10 acres). In an effort to 5.1.2. Regulatory Considerations of the Environmental Protection delay the onset of resistance to pesticides, EPA requires manage- Agency (EPA) ment strategies after commercialization. Farmers cultivating GM crops with PIPs are required to follow the appropriate insect resis- The main role of the US Environmental Protection Agency tance management (IRM) practices that are approved by EPA usu- (EPA) is to ensure that US citizens are protected from significant ally including refuge areas. risks to health and that the environment is protected from invasive species. EPA regulations are designed to make US communities 5.1.2.1. EPA and New Breeding Technologies and ecosystems diverse, sustainable, and economically productive In March 2011, the EPA issued a Notice of Proposed Rule- [76]. The regulatory activities of the EPA are governed by the re- Making (NPRM) that would specifically exempt from registration quirements of specific environmental laws. These laws direct EPA and the need for experimental use permits cisgenic PIPs (EPA-HQ- to regulate many kinds of pollution, including pesticides, with a OPP-2009-0499; FRL-8873-5). Additionally, the proposed rule focus on how they affect different aspects of the environment. Envi- would contain proposed revisions to the data requirements for regis- ronment is generally defined in these statutes as including human tration of PIPs or for an experimental use permit. In the NPRM, the health, plant and animal life, water quality, and air quality [79]. The EPA notified the public that it had sent the draft rule to the USDA courts, the scientific community, and the regulatory community and the FDA. The EPA has yet to finalize the proposed rule and recognize that EPA should consider other factors along with science submit to the Federal Register for comments. when it makes decisions about risk management [80]. 5.1.3. Regulatory Considerations of the US Food and Drug Ad- Under the Federal Insecticide, Fungicide, and Rodenticide Act ministration (FDA) (FIFRA) (7 U.S.C. §135 et seq., 1972), the EPA must determine if a The Food and Drug Administration (FDA) is an agency within new pesticide is able to perform its intended function without un- the US Department of Health and Human Services and is the pri- reasonable adverse effects on human health and the environment mary agency with responsibility for ensuring the safety of food and (FIFRA §3) before it can be registered for use. Within the Coordi- feed, except for meat, poultry, and egg products which are regulated nated Framework, these pesticides include genetically engineered by the USDA. The FDA primarily assures the safety, effectiveness, Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 11 and security of food, animal feed additives, as well as human and Evaluation and Research and the Center for Drug Evaluation and animal drugs under the regulatory authorities of the Federal Food, Research. Drug, and Cosmetic Act (FFDCA) (21 U.S.C. § 301 et seq.) and the Developers are encouraged to submit a summary of the scien- Public Health Service Act (42 U.S.C. § 201 et seq) [77]. The FDA tific and regulatory assessment of the food. The submission should also regulates tobacco products and helps the public obtain accu- include information regarding the genetic stability of the plant, rate, science-based information for their use of medicines and foods nutritional and compositional analyses, allergenicity and toxicology to improve their health (www.fda.org) [76]. Under the FFDCA, studies as well as a complete assessment of the introduced gene food and feed manufacturers must ensure the safety and proper products. In addition to these compositional analyses, the FDA labeling of their products. In contrast to the formal regulations im- might require information regarding the expected dietary exposure plementing practices related to the regulation of genetically engi- to any risk groups, such as children or immuno-suppressed patients, neered plants that were issued by the USDA and the EPA, the FDA or particular religious or ethnic groups that might be overly exposed released guidance documents and draft guidances. Guidances to the new food. It is important to remark that the FDA has not merely reflect the current thinking of the FDA and are not legally identified any new food or feed produced by rDNA technology with binding on the agency or the crop developer; however, in practice, unexpected changes in composition or in the presence of allergens the recommendations in guidance documents are followed by de- and other toxicants when compared to the naturally occurring com- velopers. parator [83]. In 1992, the FDA issued a policy statement (Federal Register, After review, the FDA will issue a letter or memorandum indi- May 29, 1992, 57 FR 22984) indicating that it would regulate foods cating the characteristics of the new food and summarizing the and feeds without regard to breeding method and it would not re- possible safety concerns. They do not formally approve the new quire special labeling of genetically engineered foods unless the food or feed to be safe per se. Instead, they indicate it is not materi- composition of the food differed significantly from its conventional ally different from the unmodified version with respect to composi- comparator [78, 81] The basic principle of the FDA policy is that tion or safety as it is “as safe as its non-modified counterparts” [71]. the traits and characteristics of the foods/feeds should be the focus of safety regardless of the technique used to develop them. FDA 5.2. Specific Regulations for Molecular Farming regulates not only the presence of allergens but also any changes in The USDA and FDA have included a few specific references to composition, or levels of nutritional and anti-nutritional substances. Plant-Made Pharmaceuticals (PMP) and Plant-Made Industrials The FFDCA requires premarket approval by the FDA of any food (PMI) in policy statements, regulations, and proposed guidances. In additive (i.e., substances intentionally added to food) that is not its 1992 policy statement, the FDA recognized that new genetic generally recognized as safe (GRAS) or otherwise exempt from modification techniques may develop plants that produce nonfood premarket approval such as pesticides which are regulated by the chemicals, such as polymers and pharmaceuticals. In crops not EPA. Generally, most fruit, vegetables, or grain meet GRAS re- intended for food use, the developer is required to ensure that the quirements as they have been consumed for many years. Thus, nonfood-use varieties do not cross with food-use varieties. This bioengineered foods composed of the same or nearly the same sub- requirement, though, is applied equally to crops developed through stances as their available counterpart (i.e., they are substantially conventional breeding practices and those genetically engineered equivalent) are not considered to be changed or adulterated [77, 78, [81]. In instances where plants or materials derived from plants are 82]. Nonetheless, the FDA in 1992 recommended that developers used to make nonfood chemicals and also will be used for food, the voluntarily consult with FDA about bioengineered foods or PMPs crop developer should consult with FDA in advance for a determi- or PMIs in food crops under development. In 2006 guidance, the nation of whether the nonfood chemical is a food additive that re- FDA indicated that it expected developers of new bioengineered quires premarket approval. plant varieties to participate in the consultation process. Further, the FDA recommended developers consult with the FDA about the As a result of several incidences where APHIS permits were early food safety evaluation of new non-pesticidal proteins pro- violated and non-genetically engineered food crops became “adul- duced by new plant varieties for food use at an early stage and be- terated”, the USDA revised its regulations. In October 2002, fore there is a possibility that the new proteins might inadvertently ProdiGene, Inc. was investigated for failing to remove tasseled enter the food supply. This recommendation was especially relevant volunteer corn plants from a Nebraska field in which the Company to assuring the public of safety in the case of inadvertent entry of grew a genetically engineered crop in the previous year. The field plant made pharmaceuticals into the food supply. Since 1992, all had been planted with soybeans in 2002. The volunteers may have developers of GE foods have routinely consulted the FDA. A list of contained a gene for a vaccine, but the volunteers could have been the FDA consultations is available on the FDA website: negative for the trait because at this stage of their development, the http://www.fda.gov/Food/Biotechnology/Submissions/default.htm; trait was segregating in a 1:1 ratio. Nevertheless, compliance a list of the new protein consultations pursuant to the 2006 guidance documents required that all volunteers be pulled from the field prior is available at the FDA website: http://www.fda.gov/Food/Bio- to flowering and destroyed following standard operating proce- technology/Submissions/ucm222595.htm. dures. Thus, pollen from the tasseling corn “adulterated” the soybeans in the field, making them unsellable. Also, some of the soybeans from this same field had been delivered to an elevator, and The FDA is divided into centers that focus on different regula- were mixed with soybeans in the elevator. This situation created tory targets such as food, feed, or pharmaceuticals. Within these, another 500,000 bushels of adulterated soybeans. ProdiGene was the Center for Food Safety and Nutrition (CFSAN) regulates food fined for this compliance violation and made responsible for de- and color additives and has been the primary regulatory location for stroying the soybeans that had been adulterated (http://www. bio- GE crops [78]. CFSAN and the Center for Veterinary Medicine tech.wisc.edu/Education/prodigene.html). In September 2002, a evaluate new foods and feed composition, respectively, including similar violation of permit conditions was found in a ProdiGene those from GE plants. The agency follows the requirements of the field at a 2001 Iowa test site. In this case, tasseling corn was found FFDCA with respect to human foods and animal feeds derived from in a soybean field as well as a pile of volunteer corn plants that had new plant varieties and provides guidance to industry on scientific been removed from the field previously. Each of these actions vio- and regulatory issues related to these foods, including varieties that lated conditions of the permit. Under USDA supervision, Prodi- are developed using rDNA technology. Biological products and Gene destroyed 155 acres of surrounding corn at the site. drugs for use in humans are regulated by the Center for Biologics 12 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.

In response to these violations and to strengthen the confidence field trials and ultimately to the regulatory approval path taken by in the regulatory system, APHIS strengthened field-testing re- the developer. quirements for genetically engineered crop plants that express Specific selected transgenic plants expressing the enzymes in genes for out-put traits such as vaccines, pharmaceutical proteins, seed were bred into elite germplasm lines to provide material for or industrial products. New safeguards were added that included hybrid seed for production fields. Germplasm development is being comprehensive confinement procedures, performance standards and carried out by Infinite Enzymes, LLC, in Arkansas, USA. Several required monitoring/auditing practices for ensuring that out- permits have been issued for field-based crossing nurseries for this crossing or commingling with other seeds and commodities cannot material. happen. In September 2002, the FDA and APHIS issued a draft guidance for industry on “Drugs, Biologics, and Medical Devices Containment requirements: GE seed movement should be per- Derived from Bioengineered Plants for Use in Humans and Ani- formed according to 7 CFR340.8: “All seeds shall be transported in mals” (FDA-2002-D-0135-0001). While this draft guidance has a sealed plastic bag of at least 5 mil thickness, inside a sealed metal never been implemented, it provides insight into USDA and FDA container, which shall be placed inside a second sealed metal con- regulatory considerations related to PMP and PMIs. For example, tainer. Shock absorbing cushioning material shall be placed be- the draft guidance recommends the implementation of measures tween the inner and outer metal containers. Each metal container that will prevent inadvertent mixing of a bioengineered pharmaceu- shall be independently capable of protecting the seeds and prevent- tical plant with plant material intended for food or feed, facilitate ing spillage or escape. Each set of metal containers shall then be rapid and easy identification of the pharmaceutical plant including enclosed in a sturdy outer shipping container constructed of corru- strategies that readily distinguish between the pharmaceutical plant gated fiberboard, corrugated cardboard, wood, or other material of and its food or feed counterpart. equivalent strength.” The developer may propose an alternate protocol to APHIS and have that approved for use instead of the above Aside from this draft guidance and the revised APHIS field- protocol in 340.8. The goal is to prevent spillage of GE seed in the testing requirements, no specific regulations have been imple- event of an accident during transport. mented that are focused on PMPs or PMIs. The primary difference for PMPs and PMIs over input traits for GE crops entails the level GE corn grown outside should be isolated from seed production of containment required when a crop expressing one of these output fields by 1320 feet (~400 m) and from grain production fields by traits is in the field. For example, fields are subject to multiple in- 660 feet (~200 m). The entire nursery or production acreage should spections, the field must be surrounded by a 50-foot fallow zone, be surrounded by at least 4 rows of non-transgenic border corn as a isolation distance from similar crops is strictly enforced with wind- pollen trap. An additional 50-foot fallow zone surrounds the border pollinated crops at a larger distance than self-pollinated crops, rows to allow space to monitor for volunteers during the growing equipment must be dedicated to the field trial for the entire field season. Because production fields are open pollinated, additional season then cleaned using USDA approved Standard Operating isolation can be achieved by delayed planting so that pollen shed Procedures. Numerous reports are required before planting, during occurs at later times than other corn in the surrounding area. All production and after harvest, including a volunteer monitoring re- personnel should be trained in the handling of GE materials using port for the following year. No crop can be planted on the plot the USDA-approved protocols. All equipment is cleaned using standard following year so that volunteers can be destroyed. protocols after each use with GE material. While in storage, all regulated articles covered under each per- 5.3. Case Studies for Experimental Field Releases of PMP and mit should be kept in a locked storage facility with limited access to PMI Producing Plants only authorized personnel. Storage containers must be identified as A. Recombinant Cellulases in Transgenic Maize Seed: Cellu- containing a genetically engineered regulated article. At least one lases (endo and exo) are the enzyme activities required in the great- sign stating "Authorized Personnel Only" must be posted in each est abundance for the conversion of lignocellulosic materials into area where the regulated article is stored. Upon completion of re- simple sugars so that the sugars can be used for biobased products search, all regulated articles (except those retained for future stud- and biofuels. In addition to the huge volumes, because biofuels are ies) should be rendered non-viable by an appropriate method (e.g., a commodity product, the cost of these enzymes must be the lowest heat or steam sterilization, bleach treatment, etc.). There is to be no ever produced for an industrial process. Therefore, producing the further distribution of this regulated article under this permit with- cellulases in a plant system was a logical step for over-production out prior approval from State (intrastate movement) and Federal for this cost-sensitive industry. The advantages of the plant seed- regulatory officials (interstate movement). APHIS/BRS and/or based system are large scale production without building infrastruc- APHIS/PPQ personnel may conduct inspections of facilities and/or ture [84] and the ability to achieve high levels of expression [69]. records at any time (taken directly from permit 12-109-101rm). The genes for an endo-cellulase (E1 from Acidothermus cellu- Reporting requirements: Several reports pertaining to the field lolyticus) and an exo-cellulase (cellobiohydrolase I, CBH I, from trials are required during the lifetime of the permit. The first is a th Trichoderma reesei) have been transformed into corn [85]. They planting report that is due the 15 day of the month following the are expressed from the maize globulin-1 promoter [86] and targeted planting of the regulated article and must include data describing either to the vacuole (E1) or the cell wall (CBH I). The vacuolar the plants, the field conditions and the GPS coordinates. A second targeting sequence is from barley aleurain [87], the cell wall target- report, the Field Test Report, is due within six months after the ing sequence is the barley alpha amylase signal sequence (BAASS) expiration date of the permit. Field Test Reports provide the final [88], and the pinII terminator is from the potato proteinase inhibitor status and observations at each location. APHIS considers these II gene [89]. The 35S promoter and terminator are from Cauliflower data reports as critical to assessment of plant pest risk and devel- Mosaic Virus, and the selectable marker is moPAT, a maize- opment of regulatory policies based on the best scientific evidence. optimized phosphinothricin N-acetyltransferase gene from Strepto- Finally, a volunteer monitoring report must be filed. The report myces viridochromogenes [90] offering resistance to the herbicide should include the permit number, the state, the county, location Bialaphos. Each of these elements is assembled into an Agrobacte- name(s), location unique ID(s), dates when the field site and pe- rium tumefaciens T-DNA unit between the left and right borders rimeter fallow zone were inspected for volunteers, number of vol- (Fig. 1). Transformants that are resistant to the herbicide will have unteers observed, and any actions taken to remove or destroy volun- the cellulase transcription unit because transfer begins from the teers. The final monitoring report is due no later than three months right border sequence and ensures that the gene of interest goes in from the end of the volunteer monitoring period (taken directly first. Each of these elements and their source are relevant to the from permit 12-109-101rm). During the field test, permittees are Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 13

Fig. (1). Plant transcription units driving expression of an exocellulase (CBH I) or an endocellulase (E1). The exocellulase is targeted to the cell wall with the barley alpha amylase signal sequence (BAASS) and transcription is driven by the maize globulin-1 promoter. The endocellulase is targeted to the vacuole using the barley aleurain signal sequence. Transcription is also from the maize globulin-1 promoter. The pinII terminator is from the potato proteinase inhibitor gene. Selection was achieved with phosphinothricin acetyl transferase conferring resistance to bialaphos. 35S promoter and terminator are from Cauliflower Mosaic Virus. required to report any unusual occurrences, any unintended effects, The field trial conditions for a plant-made pharmaceutical or any accidental releases of the regulated articles. (PMP) are more stringent than the standard permit conditions cited B. Recombinant Hepatitis B Surface Antigen in Transgenic above for the cellulases. The following information on containment Maize Seed: The production of vaccines in edible plant materials and procedures is taken directly from the issued permit for the may allow the oral ingestion of a vaccine rather than administration grow-out of the regulated pharmaceutical-producing corn lines. The by injection. To achieve this, the vaccine should be produced in an distance from the edge of the field test site to reproductively com- edible plant part such as corn seed. The advantages of such a sys- patible corn, Zea mays, will be by one of the following two meth- tem include ease of digestibility, a palatable delivery system, no ods: allergenic agents, as well as seed providing a stable and bioencap- • No corn grown within 1.0 mile of the field or sulated environment for the antigen [91]. In addition, preparations • Corn tassels will be bagged, or removed, and no corn grown for oral delivery can be inert, pelleted products that do not require a within 0.5 mile of the field test, and the transgenic corn will be cold chain, a major advantage for delivery to individuals in devel- planted no less than 28 days before or 28 days after any corn oping countries. growing in a zone extending from 0.5 mile to 1 mile from the The Hepatitis B surface antigen (HBsAg) has been expressed in field test, and if tassels are bagged, plants will be checked and characterized from recombinant maize seed [91]. The construct weekly for intact bags. of interest that has been employed in mice feeding studies is ex- In this test site, two endangered species have been identified pressed from the maize globulin-1 promoter and targeted to the and measures to avoid harm to the California red-legged frog and apoplast using the maize-optimized barley alpha amylase signal tiger salamander must be taken. The permittee is required to moni- sequence (BAASS) [88] similar to the cellulase construct above tor the field test site according to a "USFWS (US Fish and Wildlife (Fig. 2). The construct contains an additional transcription unit for Service) Measures to Avoid Harm to the California Red-Legged the selectable marker encoded by the moPAT gene isolated from Frog and Tiger Salamander" and the "APHIS CRLF (CA red- Streptomyces viridochromogenes which produces the enzyme legged frog) Information Sheet." phosphinothricin N-acetyltransferase (see cellulase case study and Fig. 1). This gene is constitutively expressed throughout the plant The procedures, processes, and safeguards used to prevent es- and is the same gene used for many transgenic plants including cape, dissemination, and persistence of the regulated article as de- commercialized corn plants used in crop improvement. The surface scribed in the permit, in APHIS-approved Standard Operating Pro- antigen from the hepatitis B virus (HepBsAg) is a structural protein cedures (SOPs) and, in the supplemental permit conditions must be and has no enzymatic or toxic activity. This protein is currently strictly followed. The permittee must maintain records sufficient to used to make the commercial vaccine for hepatitis B (e.g. Recom- verify compliance with these procedures, including information bivax® HB by Merck) and millions of people have been injected regarding who performed the activity. Persons performing such with this protein. activities shall have received training as described in a training program submitted to and approved by APHIS. These records are First generation seed from plants expressing this construct were subject to examination by APHIS. APHIS/BRS and/or APHIS/PPQ bred into elite germplasm lines to improve agronomic growth char- (Plant Pest Quarantine) personnel may conduct inspections of the acteristics and to produce higher-expressing HBsAg seed. High- test site, facilities, and/or records at any time. APHIS may invite the expressing individuals from the breeding program were self- FDA or State Regulatory Officials to participate in these inspec- pollinated to achieve homozygosity for HBsAg and complementary tions. Inspections will likely correspond to the beginning of the inbreds were crossed to produce hybrid seed for hybrid grain pro- field test, mid-season or during flowering, and/or following harvest, duction (Hayden et al., in press, Plant Biotechnology Journal). and during the post-harvest monitoring period. Inspections will Using defatted germ (embryos) from these homozygous trans- include examination of records to verify compliance with regula- genic lines as a source of HBsAg feed material, mice that were tions and SOPs. previously injected with a commercial Hep B vaccine (Recombi- To ensure that transgenic plants are not inadvertently commin- vax®) ingested the corn-derived material and produced high anti- gled with plants to be used for food or feed, a perimeter fallow zone body titers of IgG and IgA from the orally administered boost [91]. of at least 50 ft. (unless a variance is granted by APHIS) must be The success of this study warrants continued growth of the corn maintained around the transgenic test site in which no crops are lines with the view to produce large quantities for product devel- grown that would be used for food or feed. The permitted border opment. rows of non-transgenic plants that are the same as, or sexually- compatible with, the regulated article are considered part of the 14 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.

Fig. (2). Plant transcription units driving expression of Hepatitis B Virus “S” antigen. The S antigen is targeted to the cell wall with the barley alpha amylase signal sequence (BAASS) and transcription is driven by the maize globulin-1 promoter. The pinII terminator is from the potato proteinase inhibitor gene. Se- lection was achieved with phosphinothricin acetyl transferase conferring resistance to bialaphos. 35S promoter and terminator are from Cauliflower Mosaic Virus. field test. The perimeter fallow zone shall start outside the border cles to become mixed with the proposed food or feed crop during rows. The perimeter fallow zone shall be managed in a way that harvesting. allows detection and destruction of volunteer plants that are the Reports and notices are required to be submitted at various same as, or sexually compatible with, the transgenic plants. intervals during the field test. At least 7 calendar days before plant- To ensure that the regulated article is not inadvertently removed ing, a Pre-Planting notice that includes information for each field from the site, planting and harvesting equipment must be dedicated test site should be submitted. A planting or environmental release for use in the permitted test site(s) from the time of planting report is the second report due to BRS. Planting reports must be through the end of harvesting. After harvest, permittee will not be submitted to BRS by the 15th of the month following the month in required to obtain APHIS authorization to use this equipment on which the environmental release was started As above, ample data APHIS-permitted sites (same sites or different sites) planted with to describe the planting site and plants involved. same transgenic crop, with the target protein(s) authorized under A third report is the Pre-Harvest/Termination Notice. At least this permit, in subsequent growing seasons under an extension of 21 calendar days prior to the anticipated harvest or termination, the this permit or a different permit. Authorization is required from permittee must submit a Notice indicating the planned date of har- APHIS before this planting and harvesting equipment can be used vest or termination and the contact information for each field test on sites planted to crops not included under this permit. The permit- site. For multiple harvests, the notice must be submitted prior to the tee must notify APHIS/BRS and the State Regulatory Official at initial harvest. least 21 calendar days in advance of cleaning this equipment for this purpose so that APHIS may schedule an inspection to ensure The two final reports are the Field Test Report and the Volun- that the equipment has been cleaned appropriately. teer Monitoring Report. Within six months after the expiration date of the permit, the permittee is required to submit a Field Test Re- To minimize the risk of seed movement and commingling, port. Field Test Reports provide the final status and observations at equipment used for planting and harvesting, as well as other field each location. equipment (e.g. tractors and tillage attachments, such as disks, plows, harrows, and subsoilers) used at any time from the time of APHIS encourages the inclusion of other types of data if the planting through the post-harvest monitoring period must be applicant anticipates submission of a petition for determination of cleaned in accordance with procedures submitted to and approved non-regulated status for their regulated article. APHIS considers by APHIS before they are moved off of the test site. Equipment these data reports as critical to our assessment of plant pest risk and used to transport seeds or harvested material must be cleaned prior development of regulatory policies based on the best scientific evi- to loading and after transportation to the authorized site in accor- dence. Failure by an applicant to provide data reports in a timely dance with procedures submitted to and approved by APHIS. Seed manner for a field trial may result in the withholding of permission cleaning and drying must be performed in accordance with the pro- by APHIS for future field trials. cedures submitted to and approved by APHIS to confine the plant The Volunteer Monitoring Report must include the statistics on material and minimize the risk of seed loss, spillage, or commin- the trial as for the Field Test Report. In addition it should include gling. dates when the field site and perimeter fallow zone were inspected Dedicated facilities (locked or secured buildings, bins, or areas, for volunteers, number of volunteers observed, and any actions posted as restricted to authorized personnel only) must be used for taken to remove or destroy volunteers. The final monitoring report storage of equipment and regulated articles for the duration of the is due no later than three months from the end of the volunteer field test. Before returning these facilities to general use, they must monitoring period. be cleaned in accordance with procedures submitted to and ap- In cases where a regulated article is stored on a production site, proved by APHIS. The permittee must notify APHIS/BRS and the a report must be filed with APHIS each year the regulated article State Regulatory Official at least 21 calendar days in advance to remains in storage at the production site. The report, which must allow for APHIS to schedule an inspection to ensure that the facili- include the permit number, storage location, crop, event, and quan- ties have been cleaned appropriately. APHIS authorization should tity of each regulated article, must be filed prior to the anniversary be received before facilities are returned to general use. of the expiration date of the permit under which the regulated arti- The field test site including the perimeter fallow zone must be cle was produced. monitored for the presence of volunteer corn plants for one year C. Glucocerebrosidase - the first plant-made pharmaceutical to after termination of the field test on a monthly basis. Production of receive FDA approval for systemic administration. As a third ex- food and feed crops at the field test site and the perimeter fallow ample, it is worth mentioning the lysosomal enzyme replacement zone is restricted during the growing season that follows harvest or therapeutic, glucocerebrosidase, produced in plants. This enzyme termination of the field test. Permission must be obtained from was first produced in transgenic tobacco in the early 1990’s in the APHIS/BRS prior to planting any food or feed crop at the field test Cramer group at CropTech Corp. partnering with Virginia Tech site and perimeter fallow zone during the post-harvest monitoring University. Glucocerebrosidase is deficient or lacking in patients period. Requests for such permission are not encouraged and will with the lysosomal storage disorder referred to as Gaucher’s syn- not be granted in cases where there is a reasonable potential for drome, an orphan disease with fewer than 200,000 patients world- plant material derived from, or originating from, the regulated arti- wide. Enzyme replacement therapy is the only feasible treatment, Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 15 and current treatments based on bioproduction in mammalian cells with cell culture systems being more familiar to the current Pharma have been described as the most expensive drug in the world. The industry (i.e. mammalian, and bacterial culture systems). production of this enzyme in plants is protected by USP# 5,929,304 In the US, the recent regulatory approval of Enogen corn from to VA Tech and CropTech with inventors: David Radin, Carole Syngenta has paved the way for industrial protein/enzyme commer- Cramer, and Deb Weissenborn. The production of this enzyme in cialization from plants grown in the field. Enogen corn contains a plants was licensed by Protalix in Israel in 2005, where the com- heat tolerant amylase in the starchy endosperm, allowing for re- pany produces the enzyme in carrot cell suspension culture. Initial duced cost of saccharification during processing and subsequent clinical trials were also initiated in 2005. In 2009, Pfizer Pharma- ethanol production. Within the context of the US regulatory frame- ceuticals licensed rights for worldwide commercialization of the work, industrial proteins/enzymes that are already commonly used product with Protalix retaining rights in Israel. On May 1, 2012, the in industry, such as amylase and cellulase, are considered to be low FDA approved this drug for use in patients in the US, making it the hazard and are not regulated as “industrials” but under traditional first plant-made pharmaceutical drug approved for human systemic permit conditions, so do not appear in the lists of Pharmaceuticals, administration. Because it is produced in suspension cell culture, it Industrials and Value-Added Proteins, as tallied in (Table 4). Mul- will not be subject to field regulation. tiple field trials of these products - amylase and cellulase in particular, have been conducted without incident. 6. DISCUSSION In the EU project ‘Pharma Planta’ (www.pharma-planta.net), In 2006 a poultry vaccine against Newcastle disease, developed the primary aim was to develop a PMP, gain a better understanding by Dow Agrosciences, became the first PMP to gain regulatory of downstream processing and cGMP compliance issues and take a approval by the US Agricultural department. Although this product formulated product all the way through the regulatory process to was never marketed, it was a significant milestone for PMF in phase I clinical trials. Both a food (maize) and a non-food (tobacco) terms of challenging and road testing the current regulatory sys- host system were investigated at the early stages of the program. tems. In 2012, an enzyme for treating the rare hereditary Gaucher The choice to focus on a non-food crop was partly led by the belief disease (Elelyso, developed by Protalix) became the first PMP for that regulators would favour non-food crops, as any potential risks human use to gain regulatory approval by the US FDA. Both of to the food/feed chain would be minimal. The program also chose a these products were developed using plant cell suspensions grown contained growth production system of whole tobacco plants grown in bioreactors (tobacco and carrot cells respectively). The choice of in glass houses, which therefore also avoided the issues of ‘a delib- production system, i.e. the use of cell culture in contained bioreac- erate release into the environment’ that would have been associated tors, was no doubt taken to ease the regulatory approval process; with a small-scale experimental field trial (Part B of Directive

Table 4. US Release Permits for Pharmaceuticals, Industrials and Value-Added Proteins from 2004-2012. Most of the permits are “CBI”, confidential business information deleted, which does not allow the reader to identify the trait within the plant

Crop Trait Number of Trials

Corn Vaccine 4

Corn Industrial trait 4

Corn Pharmaceutical trait 5

Rice Pharmaceutical trait 22

Potato ? 1

Safflower Pharmaceutical trait 7

Tobacco Antibody 6

Tobacco w/TMV Pharmaceutical trait 6

Tobacco Pharmaceutical trait 6

Tobacco Industrial trait 4

Crambe ? 1

Camelina Industrial trait 2

Camelina Bioenergy trait 1

Barley ? 5

Pea ? 1

Aspen Phytoremediation 5

Brassica Sp. Phytoremediation 1

Tobacco Phytoremediation 1

Hybrid Poplar Phytoremediation 7 16 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.

2001/18/EC). The final product, an anti-HIV monoclonal antibody CONFLICT OF INTEREST (MAb) formulated as a solution in saline, gained approval by the The authors confirm that this article content has no conflicts of UK regulatory authority to enter human phase I clinical trials in interest. 2011.. This was the first MAb to be produced in plants and to be tested in humans (although other PMP/PMIs e.g. enzymes and other ACKNOWLEDGEMENTS proteins, for human use, have reached late stage clinical trials [32]. The authors thank Ilse-Marie Jungkurth for carefully proof However, to fully exploit the potential of PMF there is still a reading of the manuscript and COST Action Molecular Farming strong need for the regulatory authorities to develop a policy (FA0804). framework to address the issue of 'large scale', i.e. field-grown agricultural production of PMPs. 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Received: November 30, 2012 Accepted: January 31, 2013