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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])
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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
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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.
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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
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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.
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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.
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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.
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