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New Biotechnology Volume 31, Number 6 December 2014 RESEARCH PAPER Research Paper Synthetic biology and its regulation in the European Union Hans-Jörg Buhk1 Federal Ministry of Food, and Agriculture, Division Research and Innovation, Wilhelmstraße 54, D-10117 Berlin, Germany The term synthetic biology is used increasingly, but without a clear definition. Most of the recent research carried out in this field is genetic engineering, as defined by current GMO-legislation in the EU. Synthetic biology has developed its own language. In vitro synthesis of DNA also carries the label synthetic biology. It is important to analyze whether present and future activities of synthetic biology are within the scope of existing EU-legislation. Introduction The term synthetic biology is used increasingly, but without a clear definition. Most of the recent research carried out in this field is genetic engineering, as defined by current GMO-legislation in the EU. This legislation regulates activities by which organisms are genetically modified and by which the resulting genetically modified organisms (GMOs) are used in any other way, including marketing the GMOs or their products. Synthetic biology has developed its own language. For example, the recipient organism is called a chassis and the introduced modifying DNA is called a bio brick. In vitro synthesis of DNA also carries the label synthetic biology. New breeding methods applying different molecular methods have been developed since the introduction of the EU legislation on GMOs. This raised the question whether they are within or outside the scope of the GMO-legislation. Similarly, it is important to analyze whether present and future activities of synthetic biology are within the scope of existing EU-legislation. Genetically modified organism In the EU the Council Recommendation concerning the registration of work involving recombinant deoxyribonucleic acid (DNA) (82/472/EEC) was established in 1982. National guidelines and the Council 1 Permanent address: Federal Office of Consumer Protection and Food Safety, Department of Genetic Engineering, Mauerstraße 39-41, D-10117 Berlin, Germany. Corresponding author: Buhk, H.-J. ([email protected], [email protected]) 528 www.elsevier.com/locate/nbt Recommendation provided the basis for the subsequent development in 1990 of two Directives: one was Directive 90/219/EEC on the contained use of genetically modified micro-organisms (GMMs); the other was Directive 90/220/EEC on the deliberate release of genetically modified organisms (GMOs). For the interpretation of the current EU legislation on GMOs it is necessary to bear in mind its history. According to the Directives, a genetically modified (micro-)organism (GMM or GMO) means a (micro-) organism, with the exception of human being, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination (Article 2, Directive 2009/41/ EU and Directive. 2001/18). Interpreting these definitions frequently provokes the unanswered question whether the technique by which the GMM or GMO is produced or the presence of a modified nucleic acid is crucial for the resulting organism to be called a GMM or GMO. Within the terms of this definition genetic modification refers to the use of the techniques listed in Annex I A Part 1 of Directive 2001/18/EC and Annex I Part A of Directive 2009/41/EU; the techniques listed in Annex I A Part 2 of Directive 2001/18/ EC and Annex I Part B of Directive 2009/41/EU are not considered to result in genetic modification. GMMs derived by cell fusion are fully exempted. GMMs derived by self-cloning are only exempted under certain conditions from current Directive 2009/41/EU on the contained use of GMMs but not from Directive 2001/18/EC on the deliberate release of GMOs. Since the ancestors of both Directives were drafted in parallel by the same committee and since these differences have been 1871-6784/$ - see front matter ß 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nbt.2014.02.007 New Biotechnology Volume 31, Number 6 December 2014 retained unchanged by the amendments of the ancestors of both Directives, it is a clear indication that self-cloning and cell fusion were deliberately excluded from the scope of Directive 2009/41/EU but not from the scope of Directive 2001/18/EC. For the purpose of the Directives (micro-)organism means any biological entity capable of replication or of transferring genetic material, including cellular and non-cellular micro-organisms such as viruses, viroids and animal and plant cells in culture. Synthetic biology The term synthetic biology is not clearly defined. Some groups have concluded that synthetic biology presents a self-defining community of researchers from a variety of disciplines who are articulating themselves around the term synthetic biology and related terms such as synthetic genomics [1]. They have reviewed several descriptions and classifications of synthetic biology. The question whether synthetic biology is something new or a mere extension of genetic engineering and therefore covered by the EU-legislation regulating GMOs needs to be considered [2,3]. If the existing legislation is applicable, for how long will it be sufficient? New techniques of genetic modification have evolved since the introduction of the legislation in 1990. The EU Commission set up a specialized Working Group in December 2008 to consider new biotechnological techniques being applied in plant breeding or the modification of other organisms [4]. The Working Group has examined a range of new techniques to assess whether they should be considered to lead to GMOs or GMMs as defined under Directive 2001/18/EC or Directive 2009/41/EU, respectively. They are implemented in the EU-member states by national legislation. The following techniques were identified as the starting point for the consideration: 1. Zinc Finger Nuclease Technology (ZFN) (comprising ZFN-1, ZFN-2 and ZFN-3 as defined in the report) 2. Oligonucleotide Directed Mutagenesis 3. Cisgenesis (comprising Cisgenesis and Intragenesis) 4. RNA-dependent DNA methylation via RNAi/siRNA 5. Grafting 6. Reverse Breeding 7. Agro-infiltration 8. Synthetic Biology The EU Commission will consider both the results of the Working Group and the analysis of their final report from December 2011 (European Commission, unpublished data) by the Competent Authorities of the member states. In the final report the majority of the Working Group came to clear recommendations concerning which of the organisms resulting from the new techniques considered are within the scope of the definition of a GMO or GMM, respectively. The final report does not indicate the need for amending the Directives in order to cover new techniques considered to result in genetic modifications that can be identified as such. A modification of a single base pair within the genome of a given organism can be detected, but such detection does not indicate whether this modification occurred just by chance or whether it has been introduced intentionally. The Task Force on Detecting and Identifying Crops Produced with the New RESEARCH PAPER Plant-Breeding Techniques expressed its opinion that a genetic modification must comprise at least 20 nucleotide pairs (NPs) in order to allow identification of the resulting organism based on the modification [5]. Statistically, a specific sequence of 20 NPs within a nucleotide sequence with a random distribution of the NPs occurs once in 420 NPs (1.1 1012 NPs). Hence, any specific sequence of less than 20 NPs is to be expected to arise by chance in large genomes such as that of maize (its haploid genome comprises 2.5 109 NPs) with a certain degree of probability. A deliberate alteration of less than 20 NPs cannot be distinguished with sufficient certainty from an incidental occurrence of this sequence, so although specific sequences of less than 20 NPs can be detected, they are not suitable for determining their origin. They cannot be differentiated from genetic modifications arising from conventional mutagenesis or natural mutation (incidental occurrence) [6]. A mutation that is induced by mutagenesis techniques does not constitute a genetic modification according to Annex 1 B (1) of Directive 2001/18/EC and Annex II Part A No. 1 of Directive 2009/41/EU. The generation of synthetic biological processes that start with a natural product that is then modified chemically to generate a biological process that does not occur naturally is also considered as synthetic biology. Depending on the nature of the process and the chemicals involved, certain provisions of the worker protection legislation may apply. The European Framework Directive on Safety and Health at Work (Directive 89/391/EEC) ensures minimum safety and health requirements. In addition there are several sector-specific directives related to worker protection at the EU level. The most relevant for the sector including synthetic biology is Directive 2000/54/EC on the Protection of Workers from Risks Related to Exposure of Biological Agents at Work. The term biological agent refers mainly to micro-organisms (bacteria, fungi and viruses), but also includes GMMs, cell cultures and human endoparasites. The list of biological agents provides indications of allergenic potential and toxic effects. Measures proposed include containment categories for laboratory work and industrial processes. The Directive also lays down requirements for notification of selected activities to national authorities. The requirements are minimum requirements and have been implemented into national legislation. The following were members of the New Techniques Working Group: Austria: Alois Haslinger, Dietmar Vybiral; Belgium: Didier Breyer, Philippe Herman, Katia Pauwels; Bulgaria: Genoveva Nacheva; Czech Republic: Milan Bartos, Jaroslava Ovesna; Denmark: Jan Pedersen; Estonia: Hannes Kollist; Finland: Kirsi Törmäkangas, Matti Sarvas; France: Olivier Le Gall, Jean-Christophe Pages; Germany: Detlef Bartsch, Hans-Jörg Buhk, Wilfried Wackernagel; Republic of Ireland: Tom McLoughlin, Bernadette Murray, Donal Grant; Italy: Elena Sturchio, Latvia: Isaak Rashal; Lithuania: Odeta Pivoriene; The Netherlands: Boet Glandorf, Hanneke Bresser; Norway: Eirik Biering, Casper Linnestad, Tove Loken; Portugal: Teresa Borges, Clara Fernandes, João Lavinha; Romania: Călina Petruţa Cornea; Slovakia: Zdenka Balatova, Piet van der Meer; Slovenia: Borut Bohanec, Marko Dolinar; Spain: D. Rafael Pérez Mellado; Sweden: Katarina Eskils, Marie Nyman; United Kingdom: Louise Ball, Michael Paton. www.elsevier.com/locate/nbt 529 RESEARCH PAPER Members of the Task Force on Detecting and Identifying Crops Produced with new Plant-Breeding Techniques were as follows: Belgium: Sylvia Broeders, Katia Pauwels, Marc De Loose; Czech Republic: Jaroslava Ovesna; Germany: Hans-Jörg Buhk; The Netherlands: Theo W. Prins; Poland: Slawomir Sowa; Slovenia: Mojca Milavec; United Kingdom: Christine Henry. Synthetic genomics With regard to synthetic genomics (and more generally synthetic biology) the New Techniques Working Group focused on specific and already available applications involving the recombination of DNA fragments. The construction of minimal genomes and their use as a basic chassis for introduction of DNA modules could be used for production purposes. Complete synthetic bacterial genomes can be introduced into living cells [7]. Apart from such transplantation into a host (or ‘chassis’) derived from existing bacteria, no example of a synthetic genome being able to sustain life and to reproduce has been reported in the literature. The New Techniques Working Group concluded that the creation of synthetic genomes and their transplantation into recipient hosts do meet the definition of genetic engineering techniques. They further considered as follows. Synthetic genomes can be introduced into receiving environments such as cell extracts or protocells. Since these environments as such are not capable of continuous replication (in the meaning of the Directives) or of transferring genetic material, they do not meet the definition of an organism or microorganism. In this case the technique falls outside the scope of the Directives. Point 2 of Annex I Part A of Directive 2009/41/EU (and of Annex I A Part 1 of Directive 2001/18/EC) refers to techniques involving the direct introduction of heritable material prepared outside the organism. This can be interpreted as including introduction of genetic elements and full genomes into a recipient structure. If the resulting entity is capable of replication or of transferring genetic material in the meaning of the Directives, it meets the definition of an organism or micro-organism. In this case, the technique falls under the scope of the Directives in the meaning of Point 2 of the abovementioned annexes. When recombinant nucleic acid molecules are used, Point 1 of the above-mentioned annexes may also apply. Synthetic DNA Increasing the expression of genes and stabilization of gene products through optimized nucleotide sequences synthesized in vitro has become common praxis. Not only the use of in vitro synthesized DNA (synthetic DNA) in genetic engineering and synthetic genomics, but also the production of synthetic DNA is summed up under the term synthetic biology, at least by the enterprises involved in the production of synthetic DNA and associated in the International Gene Synthesis Consortium, the International Council for the Life Sciences and the International Association for Synthetic Biology [8–10]. Gene sequencing technology is getting faster, cheaper and more available. The supporting developments in bioinformatics are increasing knowledge of genomics and are available over the web and other open sources. In addition it is possible to mail 530 www.elsevier.com/locate/nbt New Biotechnology Volume 31, Number 6 December 2014 order specific genes from gene synthesis companies. Such outsourcing makes synthetic DNA easily available. To address the problem of outsourced gene synthesis, the International Gene Synthesis Consortium (IGSC) and the International Association for Synthetic Biology (IASB) have separately developed Codes of Conduct for their members to seek to ensure that they do not inadvertently fulfill gene synthesis order for those who would misuse the science. These Codes have much in common and essentially require members to check orders against databases of dangerous gene sequences and suspect clients [11,12]. A further Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA has been issued by the US Department of Health and Human Services [13]. This is in line with what the gene synthesis industry has been calling ‘. . . upon both the United States and Europe to require all makers of synthetic genes to screen according to a list of restricted sequences . . .’ and ‘. . . governments should be able to provide the most up-to-date and accurate list of restricted sequences’ [14]. Gene synthesis companies in the EU follow their Codes of Conduct. Besides that, their customers and any person importing synthetic genes into the EU have to comply with above-mentioned Directives 2009/41/EC and 2001/18/EC when they insert those synthetic genes into living organisms where they can replicate and be passed on. In 2000 the EU introduced legislation to control the export of certain synthetic genes to non-EU countries as dual use goods. In August 2009 the EU re-issued this regulation as Council Regulation (EC) No 428/2009 [15]. This was amended by Council Regulation (EU) No 388/2012 and contains the latest version of the EU Dual-Use List of controlled items. Council Regulation (EU) 428/2009 is usually updated on an annual basis to decontrol certain items or to introduce controls on new items, thus following agreement in international control regimes. As amended, it sets out the scope, authorization, control measures, custom procedures and other measures concerning the control of Dual-Use goods across the EU. Under the EU export control regime, controlled items may not leave the EU customs territory without an export authorization issued by the national competent authority. This regulation comprises several annexes. Annex I is known as the EU Dual-Use List. In control category ‘1’ of Annex I ‘materials, chemicals, micro-organisms and toxins’ are listed. Its sub-category ‘C’ contains a list of ‘materials’ and regime origin ‘3’ refers to the ‘Australia Group’ as the international control regime for those materials [16]. Section 1C353 of Annex I covers genetic elements and genetically modified organisms, as follows: Genetically modified organisms or genetic elements that contain nucleic acid sequences associated with pathogenicity of organisms specified in 1C351.a., 1C351.b., 1C351.c., 1C351.e., 1C352 or 1C354; Genetically modified organisms or genetic elements that contain nucleic acid sequences coding for any of the ‘toxins’ specified in 1C351.d. or ‘sub-units of toxins’ thereof. Technical notes to section 1C353 provide further explanation: Genetic elements include inter alia, chromosomes, genomes, plasmids, transposons and vectors whether genetically modified or unmodified. New Biotechnology Volume 31, Number 6 December 2014 Nucleic acid sequences associated with the pathogenicity of any of the micro-organisms specified in 1C351.a., 1C351.b., 1C351.c., 1C351.e., 1C352 or 1C354 mean any sequence specific to the specified micro-organism that: in itself or through its transcribed or translated products represents a significant hazard to human, animal or plant health; or is known to enhance the ability of a specified micro-organism, or any other organism into which it may be inserted or otherwise integrated, to cause serious harm to humans, animals or plant health. RESEARCH PAPER In addition to dual-use goods controlled by the EU Dual-Use Regulation, there may be a small number of dual-use items that are controlled by national legislation of EU member states. Conclusion Directives 2001/18/EC and 2009/41/EU on one hand and Regulation (EU) 428/2009 on the other hand present a framework of regulation of synthetic biology in the EU that ensures sufficient biosafety and biosecurity at present and for the near future. References [1] Oldham P, Hall S, Burton G. Synthetic biology: mapping the scientific landscape. PLoS ONE 2012;7(4):1–15. e334368. [2] Directive 2009/41/EU on the Contained Use of Genetically Modified Microorganisms, http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L: 2009: 125:0075:0097:EN:PDF [accessed 12.12.13]. [3] Directive 2001/18/EC on the Deliberate Release of Genetically Modified Organisms into the Environment, http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2001:106:0001:0038:EN:PDF [accessed 12.12.13]. [4] http://ec.europa.eu/food/food/biotechnology/docs/wk_gp_new_technics_ 2627012011_terms_of_reference.pdf [accessed 12.12.13]. [5] Lusser M, Parisi C, Plan D, Rodriguez-Cerezo E. 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[12] http://iclscharter.wpengine.netdna-cdn.com/files/2012/11/The-IASB-Code-ofConduct-for-Best-Practices-in-Gene-Synthesis.pdf [accessed 12.12.2013]. [13] http://iclscharter.wpengine.netdna-cdn.com/files/2012/09/US-Guidelance-toSynthetic-Double-stranded-DNA-Providers.pdf [accessed 12.12.2013]. [14] Minshull J, Wagner R. Preventing the misuse of gene synthesis. Nat Biotechnol 2009;27:800–1. [15] http://trade.ec.europa.eu/doclib/docs/2009/june/tradoc_143390.pdf [accessed 12.12.2013]. [16] http://www.australiagroup.net/en/biological_agents.html [accessed 12.12.2013]. www.elsevier.com/locate/nbt 531