ANNOUNCEMENTS AND REPORTS

Event report: SynBio Workshop (Paris 2012) – Risk assessmentchallenges of Synthetic Biology

Katia Pauwels • Ruth Mampuys • Catherine Golstein • Didier Breyer •

Philippe Herman • Marion Kaspari • Jean-Christophe Pages •

Herbert Pfister • Frank van der Wilk • Birgit Schonig

Received: 27 May 2013� Bundesamt fur Verbraucherschutz und Lebensmittelsicherheit (BVL) 2013

Abstract In Europe and beyond, several advisorybodies have been monitoring the developments inthe field of Synthetic Biology. Reports have been sentto national governments for information on thedevelopments and possible regulatory and riskassessment questions raised by this field. To put theissues in a broader perspective, four national bio-safety advisory bodies (the French High Council forBiotechnology, the German Central Committee onBiological Safety, the Netherlands Commission onGenetic Modification and the Belgian Scientific Insti-tute of Public Health (Biosafety and BiotechnologyUnit)) decided to join forces and organize an inter-national scientific workshop to review some of the

latest scientific insights and look into possible chal-lenges in the risk assessment of Synthetic Biology. TheSynBio Workshop (Paris 2012) – Risk assessment chal-lenges of Synthetic Biology took place on the 12th ofDecember 2012 and gathered scientists from biosafetyadvisory bodies from fifteen European countries,from the European Food Safety Authority as well asrepresentatives of the European Commission, to-gether with research scientists selected for theirexcellence in the field. The workshop was divided intotwo sessions: the first session gave an overview of fourmajor fields in Synthetic Biology. The second sessionwas set up for discussion with a scientific panel andthe audience to identify and address relevant ques-tions for risk assessment raised by recent and futuredevelopments of Synthetic Biology. An overview ofthe workshop and the discussion points put forwardduring the day are discussed in this document.

Keywords Synthetic Biology � Risk assessment �Biosafety � GMOs � New techniques � Emerging risks

The views or positions expressed in this event report do notnecessarily represent the official opinion of any of the advisorybodies that initiated the SynBio Workshop (the French HighCouncil for Biotechnology (HCB); the German CentralCommittee on Biological Safety (ZKBS); the Belgian ScientificInstitute of Public Health (WIV-ISP, Biosafety andBiotechnology Unit (SBB)) and the Netherlands Commission onGenetic Modification (COGEM)). The advisory bodies assume noresponsibility or liability for any errors or inaccuracies thatmay appear in this event report.

K. Pauwels � D. Breyer � P. HermanBiosafety and Biotechnology Unit (SBB), Scientific Instituteof Public Health, J. Wytsmanstraat 14, 1050 Brussels,Belgium

R. Mampuys � F. van der WilkNetherlands Commission on Genetic Modification(COGEM), PO box 578, 3720 AN Bilthoven,The Netherlands

C. Golstein � J.-C. PagesHigh Council for Biotechnology (HCB), 244 BdSaint-Germain, 75007 Paris, France

M. Kaspari � H. Pfister � B. Schonig (&)Office of the Central Committee on Biological Safety(ZKBS), Federal Office of Consumer Protection and FoodSafety, Mauerstr. 39-42, 10117 Berlin, Germanye-mail: birgit.schoenig@bvl.bund.de

J.-C. PagesINSERM U966, 10 Bd Tonnelle, 37000 Tours, France

H. PfisterKlinikum der Universitat zu Koln, Institut fur Virologie,Furst-Puckler-Str. 56, 50935 Cologne, Germany

J. Verbr. Lebensm.DOI 10.1007/s00003-013-0829-9

Journal fur Verbraucherschutz und LebensmittelsicherheitJournal of Consumer Protection and Food Safety

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1 Introduction

Synthetic Biology (SB) is a rapidly evolving field com-bining different disciplines that go beyond biology,including engineering, chemistry, physics, computerscience and bioinformatics. It can be described as therational design and construction of new biologicalparts, devices and systems with predictable and reli-able functional behaviour that do not exist as such innature, and the redesign of existing natural biologicalsystems, for basic research and targeted purposes.Major SB approaches consist of engineering nucleicacid-based biological circuits, defining minimal gen-omes and/or minimal living organisms, constructingprotocells, synthetic genomes and/or synthetic cells.SB also includes a novel approach based on thedevelopment of orthogonal biological systems, inwhich the genetic information is encoded by differentchemical structures (xenobiology).

Most current developments in SB involve geneticmodification. In Europe, products of genetic modifi-cation techniques (genetically modified organisms orGMOs) are specifically regulated under Directive2001/18/EC for deliberate release and Directive2009/41/EC for contained use and are submitted todefined risk assessment procedures. The regulatorydefinitions of GMO and some of the key concepts inthe GMO risk assessment relevant to this report areoutlined in Fig. 1.

Taking into account the current GMO risk assess-ment methodologies, it is likely that sufficientinformation will be available to assess the potentialrisks for human health and the environment associ-ated with SB products developed using well-characterised organisms and genetic material. It isalso expected that in the short term, activities in SBwill focus on research and development or commer-cial production of substances in contained facilities.However, it should be emphasized that SB offers theperspective to develop organisms that could differfundamentally from naturally occurring ones, hencepotentially raising specific issues or challenges asregards the risk assessment principles and method-ologies currently applied to evaluate GMOs.

For these reasons, research and developments inSB have been closely followed by risk assessors ofGMOs. The field of SB is expanding rapidly and couldraise challenges as regards the identification ofappropriate comparators (well-characterized organ-isms with a given risk potential, which are used forrisk assessment of yet-uncharacterized organisms bycomparing their properties), gathering of relevantinformation allowing characterization of the

potential hazards and/or prediction of the behaviourof such engineered organisms in case of intended orunintended release into the environment.

In 2012, four EU biosafety advisory bodies (theHigh Council for Biotechnology (HCB, France), the

Fig. 1 Definitions and key concepts for GMO risk assessment inthe European legislation

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Central Committee on Biological Safety (ZKBS, Ger-many), the Netherlands Commission on GeneticModification (COGEM, The Netherlands) and the Sci-entific Institute of Public Health, Biosafety andBiotechnology Unit (WIV-ISP, SBB, Belgium)) haveshared views on whether the principles and meth-odologies currently enforced for GMO riskassessment could be challenged when applied onorganisms or products developed by means of SB. Itwas concluded that it would be beneficial to gatherexpertise at large by inviting risk assessors andresearch scientists to a one-day scientific workshopon the risk assessment challenges of SB.

This report gives an overview of the main issuesand discussion points put forward during the work-shop. Although other aspects such as self-regulation,biosecurity, public engagement, governance andethics also need further consideration in view of aneffective regulatory oversight of SB, it is important tonote that the workshop was specifically designed totackle risk assessment issues.

2 Scope of the workshop/methodology

Most subfields of SB are rooted in genetic modifica-tion, for which a comprehensive regulatory framehas been developed in the past two decades. Whiledifferent initiatives and programmes have dealt withdiverse biosafety aspects of SB, no coordinated ini-tiative of advisory committees for biosafety has takenplace up to now. These advisory committees have acentral role in GMO risk assessment in Europe anddeal first-hand with risk assessment of productsderived from SB research and development, whichmight prove to be more challenging than the riskassessment of current gene technology products dueto their possible novelty and/or complexity.

Having pursued their SB-related activities sepa-rately before, the four advisory bodies broughttogether risk assessors, researchers and regulatorsfrom European advisory committees on biosafety, theEuropean Food Safety Authority (EFSA) and theEuropean Commission to exchange on how proce-dural elements, general principles and/or criteriapertaining to risk assessment might be challenged bythe fast-paced progress in SB. Thus, this one-dayworkshop created an opportunity for coordinationand dialogue at the European level. Speakers, scien-tific panel and steering committee members aredetailed in Fig. 2.

After an introductory talk, which reminded theprinciples and methodologies currently enforced for

GMO risk assessment and potential challenges posedby SB development, the first part of the programmewas dedicated to the current scientific developmentsin four major fields of SB (metabolic pathway engi-neering, synthetic genomics, protocells andxenobiology). Distinguished speakers were invited togive an overview of their fields, highlight recentdevelopments and share their views on possiblebiosafety issues (Fig. 2).

In the second part, a panel of experts with signif-icant expertise in risk assessment, risk managementand/or research was gathered to discuss with theaudience a set of questions articulated by the work-shop steering committee. This discussion wasmoderated by Prof. Herbert Pfister, the chairman ofthe ZKBS. The aim of the discussion was to addresselementary questions across the different subfields ofSB, i.e. (i) which developments could possibly chal-lenge the comparative approach and/or the case-by-case approach, (ii) which data would be particularlycritical or challenging for performing a proper riskassessment, (iii) how uncertainty should be dealt within contained use and in deliberate release, and (iv)whether and how SB should be dealt with in thecurrent GMO regulatory framework.

The closing session was designed to highlight dif-ferent considerations and consensus reached in thediscussion.

3 Scientific developments in Synthetic Biology

This section provides an overview of the oral com-munications given during the workshop on thescientific developments and possible biosafety issuesin four different subfields of SB: metabolic pathwayengineering, protocells, synthetic genomics andxenobiology.

3.1 Metabolic pathway engineering

Prof. Jean-Loup Faulon (University of Evry/Institute ofSystems & Synthetic Biology, Genopole, France)introduced the field of metabolic pathway engineer-ing from a historical perspective of metabolicengineering achieved through strain selection ordirect pathway modification. As these techniqueshave been used for many years, he did not considermetabolic pathway engineering as a typical subfieldof SB. Besides, while metabolic engineering aims atthe bioproduction of chemicals, SB has the broadergoal of engineering biological components and sys-tems that do not exist in nature.

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Metabolic engineering is a discipline aiming atengineering cell factories for the bioproduction ofchemical and pharmaceutical products (Stephano-poulos 2012). According to Prof. Faulon, this field ismoving forward from the improvement of the pro-duction of native metabolites to the production ofnearly any desired (bio-)chemical, pharma- andneutraceutical component. The field of SB is providinguseful tools to metabolic engineering for buildingnon-natural pathways through synthetic DNA con-structs that are difficult to produce using traditionalgenetic engineering techniques. Although the actualsize of the ‘‘chemical space’’ (i.e. the number ofchemicals that can be accessed by metabolic engi-neering) is not known, it is believed that SB cangreatly enhance the number of components that canbe produced in engineered microorganisms. Fur-thermore, modular constructions of genetic devicessuch as advanced molecular switches can be usedfor the combinatorial optimization of metabolicpathways.

Continuing, Prof. Faulon distinguished three typesof engineering:

– Natural heterologous: using an insert or pathwayfrom one donor organism in a host organism.

– Non-natural: using a pathway with different partsfrom different donor organisms.

– New chemistry: evolving enzymes allowing newreactions with slightly different products andinserting genes coding for these enzymes into ahost organism (Curran and Alper 2012).

In his presentation, attention was also given to theincreasing role of computational tools, as in

retrosynthesis (Carbonell et al. 2011; Planson et al.2012), and experimental approaches in the designprocess of heterologous chemicals.

Prof. Faulon ended his presentation by concludingthat metabolic engineering can greatly benefit fromdevelopments made in SB, and in particular for thesynthesis and control of non-natural biochemicalpathways. SB can too benefit from the methods ofmetabolic engineering in the areas of pathwayanalysis, optimization and design (Zhang et al. 2012).He stressed as crucial the future availability of moregenetic codes for different compounds, more modelor chassis strains, improvements in synthesis effi-ciency and the streamlining of design andproduction processes.

3.2 Protocell models as a step towards syntheticcellularity

Prof. Stephen Mann (University of Bristol, UnitedKingdom) reviewed recent approaches involving theuse of protocell models as a step towards the designand construction of synthetic cellularity. Most of thepresented work dealt with the generation of com-partmentalized chemical reactions. At this momentthe so-called protocells could be viewed as sophisti-cated nano-bioreactors. These approaches also aim atproviding elements in the future to achieve thetransition from nonliving to living matter. Heexplained how a continuum might be seen fromchemical origins onwards to protocells (basic auton-omy), minimal life and finally Biology (life as weknow). Whereas efforts towards the construction ofminimal cells have been driven by simplification of

Fig. 2 Speakers, scientific panel and steering committee members of the SynBio Workshop (Paris 2012)

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biological cells, the study and use of protocell modelsfocus on the design and (re)constitution of cellularfunctions and synthetic cellularity by compartmen-talization (Dzieciol and Mann 2012; Mann 2012).

Achieving compartmentalization is a key require-ment for engineering protocells. It is sought bydifferent self-organisation means, as shown by thethree following examples:

– Lipid-based vesicles, where various componentsare trapped inside organic bilayer vesicles (e.g.phospholipids or fatty acids) to circumvent theproblem of impermeability (Stano et al. 2011),

– Inorganic nanoparticle based membrane vesicles(silica nanoparticles that contain pores of 20 nm),

– Membrane-free peptide/nucleotide droplet for-mation by phase separation that enables thediffusion of molecules.

Several examples of gene-free systems were alsodiscussed as part of protocell research, including:

– The reconstitution of cellular functions such ascytoskeletal formation in vesicles of phospholipidsand membrane proteins,

– The enzyme catalysis in bioinorganic compart-ments or membrane-free droplets enabling forinstance the production of a compound inside theprotocell-like system upon the addition of asubstrate in the outside medium,

– The enzyme-mediated nucleic acid synthesis inbilayer vesicle membrane allowing for RNA rep-lication, transcription and PCR (Polymerase ChainReaction).

In addition, recent studies with cell-free geneexpression in synthetic vesicles were presented. Thesesystems have the advantage of using purifiedrecombinant compounds: the mere addition of DNAor RNA molecules to the system is sufficient to gen-erate the desired corresponding products.

At the end of his talk, Prof. Mann concluded thatprotocells are most interesting bioreactor models forconducting basic research. He also emphasized thatthe current protocell-like systems have no evolu-tionary capacities and that developments in this fieldare still far away from constructing artificial life withautopoiesis properties (Stano and Luisi 2010; Noi-reaux et al. 2011).

3.3 Promise and peril of synthetic genomics

According to Dr. Steffen Mueller (Stony Brook Uni-versity, NY, USA), the continuing improvements inDNA synthesis technology hold serious potential to

transform the biological sciences. De novo gene andgenome synthesis liberates the investigator from therestrictions of the pre-existing template and allowsfor the rational design of any conceivable newnucleotide sequence. In his presentation, he empha-sized that the status of synthetic genomics is not theresult of a single transformative technology butrather a result of incremental improvements of manytechniques, methods and tools that have been used inmolecular biology and genetic engineering for over30 years.

In his presentation, Dr. Mueller first gave an over-view of different synthesis methods and discussed thechallenges and limitations of the current state of theart of synthetic genomics. The oligonucleotide syn-thesis costs have dropped and the price gap betweenoligonucleotide-reconstructed genes and clonedgenes is still narrowing. Unfortunately, it is not pos-sible to synthesize long pieces of DNA without errors(Carr et al. 2004). The correct assembly of oligonu-cleotides is a limiting factor that will become moreand more critical as SB projects become more com-plex. Dr. Mueller stated that at the moment there areno promising techniques on the horizon regardingthe improvement of the accuracy of oligonucleotidesynthesis. This is also the case for the developmentsof methods without the need for syntheticoligonucleotides.

Two milestones of synthetic genomics were dis-cussed from their scientific and societal/politicalperspectives: the synthesis of poliovirus by StonyBrook University (Cello et al. 2002) and the synthesisof a full bacterial genome (Mycoplasma mycoides) bythe J. Craig Venter Institute (Gibson et al. 2010).Besides the achievements in the synthesis of wholegenomes, the role of synthetic genomics in large-scale mutagenesis was further explored based on theexample of Synthetic Attenuated Virus Engineering(SAVE) and its role in vaccine development (Colemanet al. 2008; Mueller et al. 2010).

SAVE is based on codon deoptimization, leading toattenuated viruses that can be used as vaccine can-didates. Several codons are known to encode thesame amino acid. However, in a defined organism,some codons and tRNAs occur more often than oth-ers (they are more ‘optimal’). The statistical frequencyat which a specific codon pair is used can be pre-dicted. Viruses tend to follow the coding biases oftheir host genomes. Consequently, by using a com-puter algorithm, the genetic code of the virus can beredesigned into a deoptimized state, leading to 100 %identity on protein level, but being significantly dif-ferent on nucleotide level. The resulting viruses are

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far less efficient regarding viral translation and rep-lication. In turn, this inefficiency increases thepossibilities of the cell to detect and respond to theviral invader. Similarly, released viral loads are lowand thus more susceptible to adaptive immunity.Theoretically, natural selection processes may lead tosome parts of the virus evolving back into their old‘optimized’ state over time, but because the attenu-ation is based on many hundreds of nucleotidechanges, reversion to its wild type virulent form ishighly unlikely (‘‘death by a thousand cuts’’).

Dr. Mueller concluded his presentation byaddressing other challenges and concerns in the fieldof synthetic genomics. A frequently expressed con-cern is the potential of dual use and the creation ofhuman pathogens (Carlson 2003). Dr. Mueller ques-tioned our collective capacity to regulate thesynthesis of oligonucleotides because it will becomeincreasingly difficult to oversee as the componentsare easily available and accessible to everyone. Itwould thus make more sense to focus on preparingto face a threat rather than to focus on prevention. Alist of viruses really constituting a bioterrorist threatshould be defined and vaccines against them shouldbe developed. The chemical synthesis of polioviruswas a wake-up call that viruses can never be con-sidered extinct. Consequently, ending specificvaccination programmes for existing viruses mightalso make viruses more interesting as bioterroristagents. Rather than the possibility of a completelynew pathogen that is unlike anything we have seenthus far, according to Dr. Mueller, it is more probablethat something will emerge that we are alreadyfamiliar with, and thus can be prepared for.

From a broader perspective, Dr. Mueller pointedout that there is no unambiguous definition of SBand that several technologies that now fall under itsscope have been used for many years. The term‘‘Synthetic Biology’’ should perhaps best be regardedas an accumulation of tools rather than as a newdiscipline in itself. It is a logical continuum emergingout of the more traditional realms of recombinantDNA technology.

3.4 Xenobiology

Prof. Ned Budisa (Berlin Institute of Technology/TU,Berlin, Germany) first introduced the audience todifferent concepts of life, from Gottfried WilhelmLeibniz and Erwin Schrodinger to Tibor Ganti andJohn von Neumann. He presented the central dogmaof molecular biology, which postulates that geneticinformation flows from DNA transcribed into RNA,

which in turn is translated into proteins, and relies onthe genetic code universally used on Earth. Thisgenetic code builds on the four nucleotides guanine,adenine, thymine and cytosine. This explains how allcells share a common set of chemistry, macromole-cules, information processing and organization ofmetabolic pathways.

He then explained that artificial life could be cre-ated either by the ‘‘bottom-up’’ approach (creation oflife from non-living matter) or the ‘‘top-down’’approach (reduction of pre-existing life forms withthe possible subsequent introduction of novel traits).The bottom-up approach was discussed as early as1911, when Jacques Loeb formulated the goal that thecreation of artificial living beings would once suc-ceed, and if not, that the reasons for this impossibilityshould be found out. The top-down approach hasbeen followed since the 1970s, when classical genetechnology was introduced.

According to Prof. Budisa, SB and xenobiologydiffer in that SB assembles novel living systems bycombining interchangeable parts from natural bio-logical systems (creating GMOs) whereas xenobiologyuses non-natural (xeno-)molecules for the productionof novel biological characteristics and systems, cre-ating Chemically Modified Organisms (CMOs).Amongst other techniques, the CMOs using newnucleotides can be created by applying a direct evo-lutionary pressure to cells of choice (Marliere et al.2011).

Finally, he presented options to engineer andexpand the genetic code in order to be able toengineer proteins consisting of non-canonical aminoacids or even to adapt entire proteomes. This caneither be achieved by engineering components byreprogramming the flexibility and tolerance of cel-lular systems or by orthogonalization (introducingnon-interacting aminocyl tRNA-synthetase:tRNApairs or metabolic pathways without cross-reactivitywith the native metabolism). According to Prof Bud-isa, xenobiology offers the opportunity to generate a‘‘genetic firewall’’ as a biosafety tool as organismswith heritable material based on non-canonicalnucleic acids would not be able to exchange geneticmaterial through horizontal gene transfer or sexualreproduction (Acevedo-Rocha and Budisa 2011;Schmidt 2010; Marliere 2009). Nevertheless, theseso-called xeno-organisms could interact and com-pete for resources within the environment. He hypo-thesized that any escape of a xeno-organism fromdirect human control would automatically leadto the death of that organism, as it would betotally dependent on external supply of essential

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biochemical building blocks. He finished withemphasizing that the scientific development ofxenobiology is in an early phase and has beenrestricted to contained use so far.

4 Discussion

Following the oral presentations, the audience wasinvited to exchange views with the panel memberson whether and how procedural elements, generalprinciples and/or criteria in the current GMO riskassessment methodology could be challenged whenapplied on organisms/products developed by meansof SB.

4.1 Data relevant for risk assessment

The identification and gathering of data relevant fora comprehensive risk assessment raised severalquestions. For SB applications, some participantsunderlined that information gathered according tothe current risk assessment approach for GMOs wassufficient. On the other hand, some discussion pointsalso illustrated potential challenges for the riskassessment of organisms generated by SB. As SBencompasses different approaches and techniques,the level of data requirement for the assessment ofresulting organisms was found to differ accordingly.

In cases where synthetic biological parts areassembled to enable ‘‘metabolic pathway engineer-ing’’, the approach could be considered as anadvanced extension of classical recombinant DNAtechniques. However, the SB approach offers thepotential to build whole systems using an unlimitednumber of traits derived from different donororganisms. Even if the sources of all parts of a syn-thetic organism are known and every new geneticcircuit understood, it could be difficult to assess theinteractions between all of these parts or circuits andto predict whether the organism would have anyunexpected emergent properties. For example, oneof the participants pointed out that many metabo-lites have a signalling function in distinct metabolicpathways, underscoring the need to ensure thatpleiotropic effects are properly assessed in terms oftheir outcome and potential risk to human healthand the environment. Therefore, the higher order ofcombination and complexity could make risk assess-ment more difficult.

Within this regard it was considered whether thequalitative approach of performing risk assessmentshould gradually be complemented by a quantitative

approach. The risk assessment of GMOs is currentlymainly based on a qualitative approach, involving aweight-of-evidence approach and using qualitativeestimates to formulate the level of risk (high, mod-erate, low or negligible). A more quantitativeapproach could be of particular relevance fororganisms with a higher order of combination ofparts and an increased number of new interactions tobe assessed. Risk assessment could benefit fromcomputational aids in order to improve the quanti-tative approach. However, it would also necessitategathering relevant data to build appropriate baselineinformation, i.e. information related to naturalcomparators. This would be necessary to fulfil one ofthe current principles of the GMO risk assessmentmethodology: the comparative approach (Fig. 1). Thiscomparative approach could be particularly chal-lenged in cases where molecules not known to bepresent in nature are produced. For those caseswhere an appropriate comparator will be lacking, acomprehensive safety assessment will be necessarytaking into account the scope of the use of theorganisms (contained use versus deliberate release).

‘‘Omics’’ technologies have been proposed as onepossibility to generate data useful for risk assessmentof GMOs or organisms derived from SB. ‘‘Omics’’technologies refer to high-throughput technologiesenabling the parallel analysis or profiling of variouskinds of macromolecules such as DNA molecules ingenomics, transcripts in transcriptomics, proteins inproteomics and metabolites in metabolomics. Tech-nical aspects in collecting ‘‘omics’’ data sets arecontinuously improving and profiling techniquesnow serve several distinct purposes. ‘‘Omics’’ canprovide complementary tools to study potentialintended or unintended differences between GMOsand their comparators (e.g. in nutrient, anti-nutrient,endogenous toxicant or allergen levels) or to char-acterize the GMO’s responses to environmentalfactors. However, the current value of ‘‘omics’’ data inrisk assessment is limited since a considerable partstays uncharacterized and genomes, transcriptomes,proteomes and metabolomes are far from beingthoroughly understood. Collecting data will be valu-able provided that tools are at hand to interpret andunderstand them in a proper way.

Contrary to the expectations that the develop-ments in pathway engineering will increase thecomplexity of biosafety permit applications due tothe number of interactions to be assessed, it wasargued that the ‘‘quantity of changes’’ regardingmetabolic pathway engineering should not be over-estimated in terms of introducing additional hazards.

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The natural chemical space is so large and still inparts undiscovered that it is probable that so-called‘‘new’’ metabolites are already produced in nature.Another point of view was that while the scale andspeed at which new and complex organisms will begenerated might considerably increase, our knowl-edge of new systems may not increase just as fast.From the risk evaluators’ point of view, this couldchallenge the case-by-case approach in the futuresince they might be confronted with an increasingnumber of dossiers, each with an increased com-plexity. This could necessitate replacing the case-by-case approach with a more generalized assessment ofgroups of different products or organisms developedin SB. Within the context of contained use applica-tions, a possible way forward to partly reduce theburden for risk evaluators would be to distinguishbetween applications that necessitate a comprehen-sive risk assessment and those applications that cangenerally be regarded as safe. More particularly, thequestion was whether a regulatory mechanism couldbe applied, which allows specific microorganisms tobe exempted from some parts of Directive 2009/41/EC, provided they can be shown to be safe and tofulfil a given list of criteria (cfr Part 1 (b) of Article 3within the Directive). Alleviating unnecessary regu-latory burden of selected technology applicationscould foster innovations.

Currently developed protocells or protocell-likesystems should be considered as chemical mattersrather than living organisms. Accordingly, as for mostsystems currently assessed for potential chemical risks,data requirement should essentially be focused on theway they are assembled. Most participants agreed thatthese systems are currently not covered by the GMOregulation due to their inability to replicate. Futuredevelopments in the field will have to consider whe-ther these systems are sterile or latent, and willdemand an assessment of their capacity of replicationand transfer of their own genetic material. Since cur-rent protocell developments will take place inchemical rather than in biological laboratories, therewere also concerns as towhether developments can beproperly monitored and regulated. Some participantsmade the parallel with nanotechnology, which isregulated at the level of applications rather than onthe basis of the technique. Taking into account thatprotocells are currently essentially a model for basicresearch, the question of whether these systems arecapable of evolution was judged premature. It wasalso noted that this field should not be overregulateddue to their inability to propagate and the limited (ifany) risks for human health and the environment.

Notwithstanding the fact that protocells andprotocell-like systems are not likely to confer specifichazards in the short term, some participants opinedthe necessity for GMO safety advisory committees toevaluate them on a case-by-case basis. In their view,criteria for assessment could be based on the poten-tial to confer risk rather than focusing on theproperties of living organisms. For example, referringto prion-like proteins that transmit and propagatemisfolded states of proteins (tau aggregates as anexample), it was noticed that aspects of transmissi-bility and propagation of ’’information’’ couldnecessitate an assessment of protocell-like systemseven when no genetic material is present.

With regard to xenobiology, most of the exchangesduring the discussion concurred with the observationthat applications in this field are still far away. Twomain perspectiveswere brought up: someparticipantsopined that xenobiology will use small modificationsto develop products with new beneficial properties,whereas others claim that xenobiology can also have amuch larger impact in the future as new artificial bio-systems are created, thus adding a new level of com-plexity tonature. For the future, aproper assessment ofpotential interactions between organisms generatedby means of xenobiology and natural organisms wasidentified to be crucial, even though xenobiologymaygive rise to organisms that are not regulated under thecurrent GMO regulation. The need to characterize SBorganisms in terms of their interaction (e.g. competi-tiveness) with natural organisms was a recurrent issuefor most of the approaches discussed.

Anapplication thatwouldneed consideration in theshort term is the use ofminimal genomes that serve as‘chassis genomes’ to be expanded by genes not presentin the parental genome. Such chassis organisms cre-ated for industrial purposes are usually generatedfrom non-pathogenic organisms or organisms with anegligibly low pathogenicity. Moreover, it was notedthat most of these organisms are expected to beauxotrophs and thus unlikely to propagate outsidedefined laboratory conditions. Another field ofresearch consists in genome minimization aiming atexploring the smallest number of genes necessary for acell to survive.Most of the participants opined that thisapproach is unlikely to generate organisms that aremore pathogenic than their respective parentalorganisms but the potential deletion of genes involvedin pathogenicity or virulence will remain a specificpoint of attention in the risk analysis. Within thisregard, some examples were brought up where thepathogenicity of the resulting organisms wasincreased upon the deletion of single genes, thereby

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illustrating that the deletion of single genes couldsometimes lead to otherwise silent but deleteriousproperties. It should be noted that the increased effi-ciency of an organism’s functions in consequence ofgenomic deletions is not specific to SB or geneticmodification, nor does it by definition encompass anincreased risk. Oneof theparticipants also emphasizedthatminimal organisms should not only be assessed interms of their potential pathogenicity but should alsobe evaluated for other potentially changed properties(e.g. their possible environmental impact, as done forGMO risk assessment).

4.2 Is the GMO regulatory framework applicableto all fields of SB?

As a significant part of SB research is based on geneticmodification techniques, it was generally assumedthat SB should be regulated under the GMO regula-tory framework. One participant suggested that theongoing discussion on SB regulation could help toestablish a ‘‘better’’ regulatory frame for GMOs, as thedecision on how to regulate a new technology hadsignificant consequences on the development of thistechnology. One possible way to reach this goal couldbe to exempt certain SB organisms from the Directive2009/41/EC as already mentioned above. Another viewwas that products of SB should be regulated based onthe resulting product and not on the process by whichthey have been developed.

Yet, it was remarked that creating a special statusfor SB would mean to overestimate that field which isat the moment firmly rooted in GM technology,whereas others emphasized that attention should bepaid not to underestimate SB and its fast-pacedscientific developments which could exceed GMtechnology, making necessary amendments to theexisting regulatory framework.

In the past, the SB community proposed a systemof self-regulation, meaning that scientists themselvesshould develop and adapt appropriate guidelines forrisk assessment and risk management of theirresearch. This approach was doubted to succeed, asconcerns were raised that systems of self-regulationcan only work until they are too time- or money-consuming and stand in the way of commercialinterest.

During the discussion, no concrete examples wereidentified where current research may not be cov-ered by GMO legislation, except for protocells, whosepresent developments are likely to fall within a reg-ulatory framework covering chemicals rather thanwithin the current GMO regulatory framework.

Yet, challenges to the regulatory framework maywell lie ahead in the future. Xenobiology, with themodification of basic chemistry underlying geneticinformation, is likely to generate a specific challengeand a regulatory status on its own, unless the currentGMO regulatory framework is amended to includethis new type of modification.

5 Conclusions and perspectives

From the early 2000s on, when many scientistsassigned their field of research with the contempo-rary significance of ‘SB’, the definition of the field hasbeen subject to debate. Today there is still no inter-nationally agreed consensus about the definition.The speakers of the workshop ‘‘Risk assessmentchallenges of Synthetic Biology’’ identified thedevelopments in SB as a tool to introduce innovativeelements and broaden the perspectives of potentialapplications in their specific domain of research. Thelack of an internationally agreed consensus defini-tion of SB should form in no case an obstacle todiscuss potential risk assessment challenges. We areof the opinion that we should be careful assigning‘new’ hazards to approaches of SB although thismultidisciplinary field may give rise to an additionallevel of emerging and unintended hazards in thefuture that need further exploration.

Current developments in SB mainly involve theuse of well-characterized microorganisms andgenetic material and focus on research and devel-opment or on commercial production of substancesin contained facilities. Sufficient knowledge andappropriate comparators are available and the cur-rent GMO risk assessment methodology provides agood framework to assess potential risks. Based onthe experiences of our national advisory bodies andthe results of the workshop, it is hardly conceivablethat microorganisms or entities will be generated inthe next few years that are far different from existingorganisms. Therefore, the manipulation of syntheticorganisms in the laboratory or their accidentalrelease in the environment are unlikely to representadditional risks in the near future. This conclusion isin line with earlier reports (Pauwels et al. 2012; CO-GEM 2013; DFG, acatech and Leopoldina 2009; ZKBS2012).

In the long term, developments in SB could gen-erate organisms that will differ more fundamentallyfrom naturally occurring ones. Several potentialchallenges to procedural elements, general princi-ples and/or criteria in the current GMO risk

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assessment methodology can be distinguished, basedon the outcome of the discussion during theworkshop.

One such general principle potentially challengedby developments in single subfields is the compara-tive approach of risk assessment. It could be moredifficult to identify an appropriate ‘natural’ compara-tor in cases where unknown artificial sequences orcomplex combinations are used (pathway engineer-ing), xeno-molecules or orthogonal systems areemployed (xenobiology) or different cellular func-tions and cellularities are reconstituted (protocells).New organisms could be generated that are funda-mentally different from those found in nature. Themore an organism departs from a host or donororganism, the more difficult it will prove to assess thecharacteristics of the organism based on the charac-teristics of the different single parts of the host/donororganism. For example, it could become increasinglydifficult for applications in the field of metabolicpathway engineering to assess the interactionsbetween all novel parts/circuits. In those cases, thenovelty and complexity of the resulting organismwill demand a more comprehensive assessment. Thismight include the gathering of relevant informationrelated e.g. to its potential pathogenicity, possibletoxic or allergenic effects or its capacities for survival,multiplication and dispersal in potential receivingenvironments. Furthermore, the increase of scale andspeed in SB applications, for example with high-throughput technologies, may impede the case-by-case approach of risk assessment from a practicalperspective. This might present a possible pitfall forthe regulatory framework, as it might challenge riskassessors, both from the point of view of havingenough workforces to deal on a case-by-case basiswith a greater number of applications and of therising complexity of the genomic changes.

On the other hand, we do not expect the com-parative approach to be challenged in other subfieldsof SB such as genome minimization, insertion of a(limited number) of well-characterized genetic cir-cuits using isolated and characterized ‘standardbiological parts’ or reconstitution of knownmicroorganisms.

Regarding data required for performing a thor-ough risk assessment, and similar to the assessmentof GMOs, the relevance of establishing ‘‘omics’’ pro-files to SB developments could be considered. Wethink that the development of standardized andvalidated methods is a prerequisite and that theinterpretation of data necessitates a good insight inthe baseline of natural variations. Ideally, in order to

beneficially use profiling techniques in risk assess-ment, it will be crucial to identify the appropriatequestions and to tackle the potential gaps of data, inother words to distinguish what is ‘‘nice to know’’from what is ‘‘needed to know’’.

5.1 Dealing with uncertainties

During the workshop, uncertainty was repeatedlyput forward as a potential issue for future applica-tions of SB. It is possible that the interaction betweennovel parts and circuits within organisms or theinteraction of novel organisms with their environ-ment will not be completely understood. Uncertaintyis inherent to the concept of risk, hence risk assess-ment often deals with uncertainties that may arisefrom limitations or lack of data like limited exposuredata, inadequacy of study design or model systems ordifferent interpretations of existing data. Thisuncertainty can be addressed by gathering moreinformation or by implementing appropriate riskmanagement strategies. Existing risk managementstrategies, such as the division into risk groups,biological and physical containment and aprecautionary attitude towards introduction into theenvironment, are applicable to most applications ofSB. While one strategy to deal with uncertainty couldbe to adopt high levels of containment for organismsfor which the risk assessment proves to be complexand associated with high levels of uncertainties, weare of the opinion that this should be done in arealistic and proportionate manner in order not tohamper research and only if there is sufficient reasonto assume that the organism might have a higher riskpotential.

Addressing uncertainty concerns is even morechallenging when SB applications are proposed to bereleased in the environment. As for any other GMOs,organisms developed in SB should first be character-ized in contained use to gather relevant scientificinformation while minimizing potential risks forhuman health and the environment. This contain-ment can be gradually decreased if the evaluation ofdata shows that potential risks for human health andthe environment are acceptable. Relevant data forthe environmental risk assessment of these applica-tions should include information on the physiologyof synthetic organisms, their survival, their compet-ing and/or evolutionary potential in receivingenvironments and their ability to exchange geneticmaterial with other organisms. The collection ofthese environmental data will be crucial but chal-lenging if organisms are very different from natural

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ones and have only been studied in contained facil-ities. It is also possible that applicants will proposenovel biosafety tools to mitigate potential risks. Forexample, orthogonality/xenobiology is presented bysome researchers as the key to biosafety issuesbecause it aims at preventing any exchange ofgenetic information with the natural world (‘‘geneticfirewall’’). Since there are many uncertainties asregards the capability of the corresponding organ-isms to adapt, interact and evolve, we are of theopinion that these applications should be carefullylooked at before xenobiology could be regarded as atechnique enabling biological self-containment.

Finally, as in GMO risk assessment, remaining risk-related uncertainties following the risk assessment ofSB applications should be addressed through appro-priate monitoring through well-defined surveillanceplans.

Regarding regulatory frameworks, we concludethat current activities involving the development anduse of synthetic organisms make use of techniquesthat fall within the scope of Directives 2009/41/EC and2001/18/EC. Some legislations of contained use ofGMOs also cover non-GMO pathogens (e.g. Belgianregional decrees). In that case, the reconstitution ofpathogenic microorganisms not differing geneticallyfrom their pathogenic archetype also falls within theprovisions of the GMO regulatory framework. How-ever, it should be noted that the use of protocellularsystems unable to replicate or the modification of thebasic chemistry underlying the genetic informationmachinery and processes could raise potential issuesas regards the regulatory status of the resultingproducts or organisms. Along with the furtherdevelopment of these approaches, questions will beraised as to whether the understanding and/or defi-nition of ‘‘organism’’, ‘‘GMO’’ or ‘‘genetic material’’needs to be expanded or reconsidered to includethese activities under the scope of the GMO regula-tory framework. Alternatively, while protocells mayfall under regulatory frameworks covering chemi-cals, the chemically modified products ofxenobiology may fall under a new, specific regula-tory framework.

5.2 Perspectives

This workshop, bringing together participants fromfifteen European countries, representatives fromEFSA and the European Commission, has given anappropriate setting to allow fruitful exchangesbetween scientists involved in research and devel-opment, experts involved in risk assessment/

evaluation, and regulators involved in risk manage-ment. We are convinced that communicationbetween scientists and risk assessors is crucial totimely identify emerging challenges for risk assess-ment and to estimate which information is relevantfor the risk assessment. This is of particular relevancegiven the multidisciplinary and international char-acter of SB, and the possibility that advances in high-throughput technologies of genetic modificationswill soon enable developments in this field that mayoutpace the increase of knowledge on the riskpotential of the organisms created.

As SB research becomes more complex or moredistant from what we know as ‘natural’, mutuallearning processes between these groups will becrucial to avoid overregulation (based on risk per-ception), which might result from a lack ofunderstanding or from assigning new technologieswith new hazards. Overregulation could lead to theapplication of unnecessary precaution and couldsignify a burden to the development of applicationsthat may be beneficial for society. Therefore, whilerecognizing that a precautionary approach is impor-tant in cases of high complexity and uncertainty, weare of the opinion that application of containment,confinement, mitigation measures and monitoringshould be realistic, proportionate to risk and adoptedon a case-by-case basis to allow sufficient flexibility forresearch and development initiatives.

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