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Current Activities of the Online Forum on Synthetic Biology

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Opening of Discussion [#8727]
POSTED ON BEHALF OF NIKOLAY TZVETKOV
---------------------------------------------
Dear participants of the Online Forum on Synthetic Biology,

Welcome to the third round of the forum discussions. My name is Nikolay Tzvetkov and I work in the Bulgarian Ministry of Environment and Water as an expert and I am responsible for regulation of LMOs and other aspects of modern biotechnology. I am the national focal point for the Cartagena Protocol on Biosafety and am a member of the Ad Hoc Technical Expert Group on Synthetic Biology that met in Montreal in 2015. My educational background is in biochemistry and gene and cell engineering. I have worked on cyanobacterial genomics, protein biochemistry technology and neurodegeneration at academic institutions in Japan and Germany.

I have the honour to serve as the moderator of the third round of discussions on “Identifying any living organisms already developed or currently under research and development through techniques of synthetic biology which do not fall under the definition of living modified organisms under the Cartagena Protocol and evaluating the availability of tools to detect and monitor the organisms, components and products of synthetic biology”.

The outcomes of this forum will serve as basis for the discussions of the AHTEG at their face to face meeting, falling under two of the topics listed in their terms of reference as defined by decision CBD/COP/DEC/XIII/17:

o b) [the AHTEG shall…] Identify any living organisms already developed or currently under research and development through techniques of synthetic biology which do not fall under the definition of living modified organisms under the Cartagena Protocol; and
o d) [the AHTEG shall…] evaluate the availability of tools to detect and monitor the organisms, components and products of synthetic biology.

The first subtopic of current discussion aims to identify living organisms that have already been developed or are currently under research and development, including those that may have come up the pipeline since the assessment made by the previous AHTEG in 2015 which do not fall under the definition of living modified organisms as defined under terms of article 3 of the Cartagena Protocol. COP 13 in its Decision CBD/COP/DEC/XIII/17 has already taken note of the conclusions of the first meeting of the AHTEG on Synthetic Biology that:

o living organisms developed through current applications of synthetic biology, or that are currently in the early stages of research and development, are similar to LMOs as defined in the Cartagena Protocol.
o it is not clear, given the current state of knowledge, whether or not some organisms of synthetic biology, which are currently in the early stages of research and development, would fall under the definition of living modified organisms under the Cartagena Protocol
In order to clarify the situation further, you are invited to provide examples, together with the necessary justification, of organisms developed or being developed through techniques of synthetic biology which lie outside of the scope of the definition of living modified organism or whose status is not sufficiently clear. Such examples are very important because they will help to define the scope of the Cartagena Protocol and might identify areas where further action under the Convention is necessary.

The second subtopic of the current discussion aims to “evaluate the availability of tools to detect and monitor the organisms, components and products of synthetic biology”. Efficient methods for detection and monitoring are essential in order to understand if and how such organisms, components and products may affect the conservation and sustainable use of biodiversity in the context of the Convention. It is of particular importance to identify areas where current techniques are not applicable and further work is needed.

In order to facilitate and focus our discussions, I would like to invite you to consider in your interventions the following questions:

• Since the last assessment made by the AHTEG on synthetic biology, have there been any living organisms already developed or currently under research and development through techniques of synthetic biology, which do not fall under the definition of living modified organisms under the Cartagena Protocol?
o If so what are they and what are that characteristics that differentiate them from organisms that fall under the definition of living modified organisms under the Cartagena Protocol

• What tools are available to detect and monitor each of the organisms, components and products of synthetic biology?
o If no tools are available which need to be developed in order to facilitate the detection and monitoring of each of the organisms, components and products of synthetic biology

I fully appreciate from the lively discussions under topics 1 and 2 that a number of participants think that further work by the AHTEG is needed on the definition of Synthetic biology and in some other areas. I have sympathy with many of the arguments raised, but I also believe that third round of discussions should concentrate on the two very important areas at hand. For this reason I would kindly invite you not to discuss any other unrelated topics. I think that the relatively broad definition of Synthetic biology can serve to our advantage, as noted by some participants, as it will allow the current discussions to cover organisms developed through a broader range of modern techniques.

After this round of discussions is over a summary will be prepared by me with the help of the Secretariat on the basis of those interventions that are directly related to items (b) and (d) in the terms of reference of the AHTEG.

Finally, I hope that our discussions will be as lively and productive as those on topics 1 and 2 and I am looking forward your active participation.

Nikolay
posted on 2017-09-04 00:26 UTC by Dina Abdelhakim, SCBD
This is a reply to 8727 RE: Opening of Discussion [#8728]
Dear Nicolay

Congratulations for chairing the third round on synthetic biology. the two former were very exciting. After two days of lunching of the discussion, nobody reacts to the post, I am very worry about. However it is not surprising because the topic is very hard to undertand and to apply in the field. We are familiar with the LMO under Cartagena protocol not those are outside. Personally I hope more from my colleague in order to have notions about such LMO. I would encourage all of us to participate to the debate.

Sincerly

Dr. Jean Bruno MIKISSA
posted on 2017-09-06 12:32 UTC by Mr. Jean Bruno Mikissa, Gabon
This is a reply to 8727 RE: Opening of Discussion [#8729]
POSTED ON BEHALF OF HIROSHI YOSHIKURA
---------------------------------------------------------

I appreciate this occasion that allows us commenting on the topic 3.

To the first question:

1. As the definition of LMO in CBD was drafted in 2000 in the context of conservation of biodiversity (conservation of species is an important element), it was not surprising that genetic exchange overcoming the species barrier was a concern, which is delineated in the clause qualifying bullet points a. and b. of the definition, “that overcome natural physiological reproductive or recombination barriers”. Some products of synthetic biology are, in my view, unsuitable for consideration whether they are LMO or not by using this criterion, such as, organisms created through introduction of a “synthetic sequence” unrelated to sequence of any known organisms, which is frequently practiced: it is unrelated to the issue of “natural reproductive or genetic exchange barriers”. They probably do not fall within the CBD’s definition.

2. I believe, however, that the principles delineated in Annex III, Risk Assessment, of CBD, particularly, bullet points 5 and 6, are fully applicable to synthetic biology, because any product of synthetic biology coming in near future will be derived from existing living organism(s) and because synthesized sequence(s) can be evaluated for the potential risk by comparing with existing genetic sequences; namely comparative safety assessment on a case-by-case basis proposed in the Annex is applicable. As comparator, in addition to the organisms from which the synthetic biology products are derived, the past experience with invasive species, consequence of the eradication trials (e.g., mosquitoes, malaria), experience with the biological control using natural enemies, etc. can be used for the risk assessment.

To the second question:

1. To “detect and monitor”, the organisms should have organism-specific phenotype suitable for easy detection and amplification. Amplification is necessary for increasing the sensitivity of detection. They are, such as, antibiotics resistance, herbicide tolerance, drought tolerance, inundation tolerance, etc. If the organisms have no selection/detection marker, suitable marker genes may have to be added.

2. Added selection marker gene(s), however, may evoke safety concern. Important principle would be use of the OECD’s scale-up principle to gain familiarity step-by-step before environmental release. Monitoring should be conducted only when risk assessment before the release identified what events should be monitored and how they should be monitored.

Best regards,
Hiroshi Yoshikura, former chair of Codex Task Force on Foods Derived from Modern Biotechnology, Emeritus member of National Institute of Infectious Diseases Japan.
posted on 2017-09-06 13:51 UTC by Dina Abdelhakim, SCBD
This is a reply to 8727 RE: Opening of Discussion [#8730]
Please find my suggestions for the first question attached. It contains a table that shows what products of SynBio are not covered by the CP definition of LMOs.
(edited on 2017-09-06 18:46 UTC by Mr. Gerd Winter)
posted on 2017-09-06 17:26 UTC by Mr. Gerd Winter, University of Bremen
This is a reply to 8728 RE: Opening of Discussion [#8731]
I also agree with Mr. Jean Bruno Mikissa, Gabon, I do not understand the actual meaning of this topic.
Can you please explain this topic in a simplified manner, so that we can follow the discussion.
posted on 2017-09-06 22:03 UTC by Mr. Sampson K. P. Chea, Liberia
This is a reply to 8730 RE: Opening of Discussion [#8732]
The moderator implores us not to discuss the (non/)useful and (in/)operational AHTEG definition of synthetic biology [#8727]. Any similar plea about the definition of an LMO will frustrate resolution of Topic 3, as evidenced by “Kinds and parts of SynBio products not covered by the LMO definition” tabulated by Prof. Gerd Winter [PDF attachment to #8730]. One may infer from the eight lacunae identified by Prof. Winter that something is very wrong with the definition of an LMO. Consider letter (h) of that definition: “‘Living organism’ means any biological entity capable of transferring or replicating genetic material, including sterile organisms, viruses and viroids” (Article 3, Cartagena Protocol).

In a 2016 submission to the Secretariat, The Peruvian Society of Environmental Law examined the relationship of the LMO definition to synthetic biology:

“Thinking like a physicist about the definition of ‘living organism’...is as dispiriting as thinking like an economist about competition and the definition of ‘genetic resources’ in the CBD. ‘Material’, genetic or otherwise, cannot be ‘replicated’. Replicating material is a violation of the First Law of Thermodynamics, more commonly known as the Conservation of Mass and Energy (or more reductively, as the Conservation of Energy). The natural information transmitted in genetic material is replicated in other material, which is drawn from the environment, i.e., nitrogen, carbon, hydrogen and so on... The wry observation of Sir Arthur Stanley Eddington, regarding the Second Law of Thermodynamics, seems all the more apropos for the First:  ‘[I]f your theory is found to be against the second law of thermodynamics I can give you no hope; there is nothing for it but to collapse in deepest humiliation’ (1958, 74).

Would that humiliation ensue! Alas, Eddington commits what George Soros (2008) has identified as the Enlightenment Fallacy: ‘assum[ing] that the purpose of reason is to produce knowledge’ (23) and that ‘the pursuit of truth [would] take precedence over the pursuit of power’ (40).

A neophyte to ABS might think that re-defining the object of access from ‘genetic resources’ to ‘natural information’ in the CBD would be low-hanging fruit that would feed the Cartagena Protocol, the Nagoya Protocol and the Decisions of the COPs. After all, the CBD is a framework convention and arguably draws on science. An operational definition of Synthetic Biology with implicit exclusion and inclusion criteria, is in full sight.” (Peruvian Society of Environmental Law, 2016, 3-4).


Eddington, Arthur Stanley. 1958. The Nature of the Physical World. Ann Arbor, Michigan: The University of Michigan Press.

Peruvian Society for Environmental Law (SPDA). 2016. “Submitted View for the ‘Updated Report and Synthesis of Views in Response to Paragraph 7(b) of Decision XII/24; and Report of the Meeting of the Ad Hoc Technical Expert Group on Synthetic Biology’ (UNEP/CBD/SYNBIO/ AHTEG/2015/1/2 4 and UNEP/CBD/SYNBIO/AHTEG/2015/1/3) http://bch.cbd.int/synbio/peer-review/2014-2016/

Soros, George. 2008. The Crash of 2008 and What it Means: The New Paradigm for Financial Markets. New York: PUBLICAFFAIRS.
posted on 2017-09-07 04:14 UTC by Mr. Joseph Henry Vogel, University of Puerto Rico-Rio Piedras
This is a reply to 8729 RE: Opening of Discussion [#8733]
Dear Nokolay and participants,

Thanks Nikolay for accepting to moderate this third round. As a previous moderator of a similar discussion within the Risk Assessment and Risk Management AHTEG of the Cartagena Protocol, I know this is no easy task.  I strongly support the many useful insights shared by Dr. Vogel in this  (#8732) and in previous rounds.

It is easy to anticipate that opinions  in this round will follow the same patterns of previous discussions: they will be both fragmented and opposed, strongly colored or biased  by the participants background, social and cultural  conditioning  and professional training (example: social scientists or activist vs. molecular biologist),  just to name a few.  By the way, I am not suggesting that one group has a “better” or “worse” opinion than the other. They will simply be different.  Formal reports issued after these discussions need to acknowledge that every single participant will have a certain bias and we are “experts” in different fields.  We humans cannot be totally objective and devoid of emotion. Computers driven by AI can, but we are not inviting them (yet!).  Thus, reports issued following these discussions have to transparently report this fact.

In my opinion and wearing my multiple hats as an AHTEG member and moderator, a participant of several similar CBD and Cartagena Protocol online fora and also as a party delegate from a developing country, I think that party representatives going to COP-MOPs, would benefit  when reading these reports, from knowing that these biases exist and how difficult it is to reach consensus during these discussions. In the past, “sanitized versions” of difficult discussions have been summarized, issued and presented to party members as “the AHTEG concluded” or “a definition for synthetic biology was agreed”  … and so on.

Having said this, I can also anticipate that the opinions of this third round of discussions will span the same spectrum of opinions of the previous AHTEG discussions on risk assessment of synthetic biology (which I had the privilege to moderate last year) and that reaching consensus will be very difficult. It is not surprising that some members are confused about the current task (# 3731).

However, in the spirit of attempting to contribute to the specific questions asked by our moderator, I support the views of Hiroshi Yoshikura’s statement (#8729)  that “ …some products of synthetic biology are, in my view, unsuitable for consideration whether they are LMO or not by using this criterion, such as, organisms created through introduction of a “synthetic sequence” unrelated to sequence of any known organisms, which is frequently practiced: it is unrelated to the issue of “natural reproductive or genetic exchange barriers”. They probably do not fall within the CBD’s definition“.

Regarding the second question of tools to “detect and monitor products of synthetic biology” we will go round in circles again with a flawed definition of synthetic biology, as Dr, Vogel has pointed out on several posts.

To end on a positive point, I look forward to reading different opinions and wonder where this third discussion will take us. It promises to be confusing, difficult but very interesting.

Best regards,

Maria
posted on 2017-09-07 05:39 UTC by Ms. Maria Mercedes Roca, PRRI
This is a reply to 8733 RE: Opening of Discussion [#8734]
Dear colleagues
Many thanks to Mr Tzvetkov for moderating this new topic. I would like to ask a question: do you know whether it is planned, or it has been tried, to create genetically modified viroids and prions? and whether those fit under the Cartagena definition or not (I do not believe so).
posted on 2017-09-07 06:29 UTC by Ms. Barbara LIVOREIL, France
This is a reply to 8730 RE: Opening of Discussion [#8735]
The overview by Prof. Winter provides an excellent clarification of the tasks for this round of the debate. I attach a few comments on his list of potential developments regarding living organisms that might fall outside the scope of the CP definition of LMOs. These mostly concern potential diversity in the interpretation of the definition and the scope of the topic, rather than disagreements about the actual developments themselves. It might be good to clarify these issues before starting a more detailed discussion of individual examples.
posted on 2017-09-07 08:27 UTC by Mr. Rainer Breitling, University of Manchester
This is a reply to 8735 RE: Opening of Discussion [#8736]
Helo every one!!
Dear Nikolay thank you for moderating the third round discussion.
As clearly noted by the moderator there are works of synthetic biology. The most relevant example for this discussion is the work of Venter and his colleagues, the first synthetic cell,  “Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome” (http://science.sciencemag.org/content/early/2008/01/24/science.1151721) Where short oligonucleotides form parts, parts combined to get devices and the devices produce systems.
A water mark is inserted in the non coding regions to identify the organism from the native counterpart.
posted on 2017-09-08 12:33 UTC by Mr. Taye Birhanu, Ethiopia
This is a reply to 8727 RE: Opening of Discussion [#8737]
POSTED ON BEHALF OF NIKOLAY TZVETKOV
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Dear Colleagues,

I am very happy to see that the discussion is picking up after a few days of thinking. The topics of current discussion are rather technical in nature from both biological and legal points of view.
Allow me to make some observations that might be useful to further our discussions and facilitate further understanding of the topic at hand.

On Subtopic 1 “Living organisms already developed or currently under research and development through techniques of synthetic biology which do not fall under the definition of living modified organisms (LMO) under the Cartagena Protocol (CP)”

Let me start with stating the use of terms in CP as defined in Article 3:

****
"Living modified organism" means any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology;
"Living organism" means any biological entity capable of transferring or replicating genetic material, including sterile organisms, viruses and viroids;
"Modern biotechnology" means the application of:
a. In vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA) and direct injection of nucleic acid into cells or organelles, or
b. Fusion of cells beyond the taxonomic family,
that overcome natural physiological reproductive or recombination barriers and that are not techniques used in traditional breeding and selection;
****

With this understanding, the AHTEG, at its last meeting, deliberated on whether living organisms derived from synthetic biology fall under the scope of the CP by considering both the similarities and the differences between LMOs and the living organisms developed through current and near-future applications of synthetic biology.

The AHTEG agreed that living organisms developed through current and near future applications of synthetic biology are similar to LMOs as defined in the Cartagena Protocol and noted, however, that it is not clear at the current stage whether or not some organisms of synthetic biology, which are currently in the early stages of research and development, would fall under the definition of LMOs under the Cartagena Protocol.

The aim of this discussion is therefore to deliberate on whether or not there have been any recent developments that have led to the development of organisms falling outside the definition of an LMO in the CP. In your interventions you may either provide concrete examples of such organisms or any criteria that could facilitate identifying them.

As I see it, some of the interventions that have been made so far propose examples where an organism developed through synbio may not be an LMO as defined in the CP. In the following paragraphs, I would like to outline some of the examples that were raised for the benefit of further facilitating the discussion on this topic. However, let me please be very clear that, in providing these examples, I am in no shape or form suggesting that the organisms should or should not be considered LMOs as defined in the Protocol.

-> 1. The entity in question is a living organism that possesses a novel combination of genetic material, but is NOT obtained through the use of modern biotechnology

The discussion on gene editing falls under this category and is probably the trickiest of all examples provided. Here, let me please try to deconstruct the process of creating an organism (i.e. gene editing), which is the part that is relevant to our discussion, from the discussion on whether or not biosafety measures (e.g. risk assessment, labelling, etc) should be imposed under such organisms.

Recently developed techniques for gene editing, such as CRISPR, Meganucleases, Zinc-finger nucleases (ZFN), Transcription activator like effector nucleases (TALEN) or oligonucleotide directed mutagenesis, all use nucleic acid techniques to trigger modifications in genomes.

Gene editing normally requires the introduction of new genetic material (expression construct) into the cell. The organism thus generated will be an LMO. But that expression construct can be removed from the cell after the modification is accomplished. In some of those cases when only single point mutations, deletions or small insertions are introduced and if the recombinant construct is no longer present it may not be possible to distinguish whether a modified organism was produced by use of classic mutagenesis or by modern biotechnology.

In addition some of the modifying agents used for gene editing, such as site-specific nucleases or oligonucleotides, can be introduced into the cell directly for example with the help of liposomes or electroporation. Then the resulting modified organism might fall outside of the scope of the definition of an LMO as it was generated with the help of a sophisticated mutagenic reagent.

When we discuss gene editing vis-à-vis the definition of modern biotechnology (“In vitro nucleic acid techniques … that overcome natural physiological reproductive or recombination barriers …”), I trust that we may all agree that the different methodologies all involve a “nucleic acid” technique in one step or another of the process. However, I can foresee some controversy on whether or not the techniques “overcome natural physiological reproductive or recombination barriers”. As such, it might be hard or practically impossible, with the current definition of an LMO under the CP, to determine whether or not an organism is an LMO from a legal point of view.

-> 2. The entity in question is a living organism that possesses genetic material from the same species or sexually compatible species

It is possible to introduce changes in that already occur in the same species or in closely related sexually compatible species, through a process usually known as cisgenesis. If those are the only modifications introduced the resulting organism may be considered equivalent to an organism that has been produced by traditional breeding or selection. In such cases the use of modern biotechnology would not be considered as a means to “overcome natural physiological reproductive or recombination barriers”.

-> 3. The entity in question is a living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology, but that genetic material has no similarity with any naturally occurring genetic material

This is the scenario outlined by Dr Yoshikura [#8729] and Dr Roca [#8733]. I am personally well aware of an example of that kind. The laboratory of Dr Michael Hecht designed de novo proteins on the basis of simple amino acid pattern that share no similarity to any natural proteins and whose only significant physicochemical property is to form β-amyloid (DOI: 10.1073/pnas.96.20.11211). It was shown by Dr Ulrich Hartl, Dr Martin Vabulas and their colleagues that when produced inside the cell those proteins can have profound effects on cellular processes (DOI: 10.1016/j.cell.2010.11.050).

In such cases it would be considered that any organism carrying such novel genetic material would be an LMO, because they fulfil all conditions in the definition of the Cartagena Protocol ,including the fact that natural physiological reproductive or recombination barriers would have to be overcome as they include limits to which random genetic material can be introduced into the genome.

-> 4. The entity in question is a living organism that has been synthesised entirely in vitro

If the whole genome has been synthesised entirely in vitro (possible for viruses and some bacteria, expected to be possible for higher eukaryotes soon) we have in vitro use of nucleic acid techniques. So if it possesses novel combination of genetic material it would be an LMO, if it is entirely identical to naturally occurring genome it may not be considered an LMO. I am happy to see that some of the issues discussed above have already been considered by Dr Winter [38730] and Dr Breitling [#8735]

-> 5. The entity in question is a living organism that possesses a novel combination of genetic material, but only in cells and tissues that do not participate in sexual and non-sexual reproduction under natural conditions

It is possible to generate an organism where only some of the cells or tissues are modified, e.g. by grafting. Those modified tissues or cells might not be able to participate in sexual (as germ cells) or asexual reproduction under the natural conditions for the species in question nor to exchange genetic material with other cells that can participate in such reproduction. The second point is not trivial because it was shown in the laboratory of Dr Ralf Bock grafting can result in exchange of genetic material (DOI: 10.1038/nature13291) and chloroplast genomes can be transferred horizontally as well (DOI: 10.1073/pnas.1114076109). The status of such organism as an LMO is not very clear, because it won’t be able to transfer the modified genetic material, but it still might be able to replicate it even when it is sterile. Therefore, only in situations where the cells carrying the novel combination of genetic material are not capable of transferring or replicating would the organism not be considered an LMO.

-> 6. The entity in question in not living organism

For examples under this category to be excluded from the LMO definition, the entity in question must not be capable of transferring or replicating genetic material. One such example may be prions, which does not contain genetic material as such and are not specifically included in the definition of living organism. To answer the question of Dr Livoreil [#8734] modified prions have been studied in great detail, e.g. in the labs of late Dr Susan Lindquist (see for example DOI: 10.1038/nature03679) and Dr Jonathan Weissman (DOI: 10.1038/nature06108).

On Subtopic 2 “Tools are available to detect and monitor each of the organisms, components and products of synthetic biology”

Here I would outline two specific problems that I see:

-> 1. As mentioned above in cases of gene editing, cisgenesis and fully synthetic genomes the changes introduced can be very minor compared to the size of the genome, perhaps even single point mutations. It may be very difficult to detect such modifications even when whole genome sequencing is used. On a more conceptual level it might not be possible to prove on the basis of sequence alone whether those modifications where introduced by classical mutagenesis, traditional breeding or selection or with the help of modern biotechnology. What can be done to detect such organisms/LMOs is a matter of discussion.

-> 2. When the LMO contains non-natural nucleotides that can be maintained during replication then standard techniques for detection and identification might have to be modified.

I hope that this rather long intervention will help to clarify the topics being discussed and further the already developing interesting discussion.

Best Regards,
Nikolay
posted on 2017-09-08 17:34 UTC by Dina Abdelhakim, SCBD
This is a reply to 8727 RE: Opening of Discussion [#8738]
Dear all,
Thanks to Nicholas for chairing this session and taking up the brave task to guide us through another round of interesting discussions. You have asked us to be to the point and we will try to do this. But we want to state two points first:

• We would like to avoid starting a legal discussion on what is considered to be a LMO or not, this does not seem a useful way forward.
• We consider synthetic sequences not to be different from ‘natural’ sequences only based on the fact that they are synthetic. The main aspect for this discussion is the function these sequences encode.


1.      Since the last assessment made by the AHTEG on synthetic biology, have there been any living organisms already developed or currently under research and development through techniques of synthetic biology, which do not fall under the definition of living modified organisms under the Cartagena Protocol?

Answer:
No, according to our interpretation of an LMO, we do not know of any existing living organism or living organism under development  through techniques of synthetic biology that does not fall under the definition of an LMO.

There is however scientific interest in developing bottom-up, artificial cells. Recently a consortium of Dutch Universities acquired substantial research funds for research into the development of an artificial and self-replicating cell (see e.g.: https://www.tudelft.nl/en/2017/tu-delft/dutch-researchers-join-forces-to-build-synthetic-cell/) . But even for this future development the definition of LMO still seems to be applicable.

2.      What tools are available to detect and monitor each of the organisms, components and products of synthetic biology that is not considered a LMO?

Answer:
In case there would be examples of such organisms, it should of course be ascertained whether or not these organisms would lead to potential adverse effects on biodiversity (including human health and the environment). Specific monitoring tools will very much dependent on the type of organism in question. As the specifics of this organism are as yet unknown, no statements can be made about monitoring tools at this stage. If these specifics are known there are, however, no fundamental reasons that preclude the development of dedicated monitoring tools.

With kind regards, also on behalf of Mrs. Boet Glandorf,

Jaco Westra
RIVM
The Netherlands
posted on 2017-09-09 07:23 UTC by Mr. Jaco Westra, Netherlands
This is a reply to 8738 RE: Opening of Discussion [#8739]
Dear all

I would like support the point of view from Mr Jaco. He did a good analysis about our discussion and from his intervention we can go ahead.

Best regards

Jean Bruno MIKISSA
posted on 2017-09-09 14:37 UTC by Mr. Jean Bruno Mikissa, Gabon
This is a reply to 8738 RE: Opening of Discussion [#8740]
The posting of Dr. Jaco Westra begins with two bullet points:
"• We would like to avoid starting a legal discussion on what is considered to be a LMO or not, this does not seem a useful way forward.
• We consider synthetic sequences not to be different from ‘natural’ sequences only based on the fact that they are synthetic. The main aspect for this discussion is the function these sequences encode" [#8738].

The first point refers to postings the made by Professors Winter, Breitling, and myself [respectively, #8730 and #8735 and #8732] and the moderator’s response [#8737]. The point coheres with previous pleas in earlier threads to accept the status quo of the brokered definitions of the CBD, NP and CP.

The second point seems to undercut the first. To consider “synthetic sequences not to be different from ‘natural’ sequences” calls into question the definition of  “genetic material” as  “any material of plant, animal, microbial or other origin containing functional units of heredity” (Article 2 of the CBD), which is the linchpin in both the definition of an LMO and synthetic biology.
posted on 2017-09-11 03:40 UTC by Mr. Joseph Henry Vogel, University of Puerto Rico-Rio Piedras
This is a reply to 8727 RE: Opening of Discussion [#8741]
Dear all—

Let me add my thanks to Nikolay for moderating this rather technical session.  I am Bob Friedman from the J. Craig Venter Institute (JCVI) and joined by John Glass, one of the senior scientists in JCVI’s synthetic biology group.

We would like to add our agreement with the concise conclusion by Jaco Westra and Boet Glandorf [#8738]:  We also do not know of any living organisms already developed or currently under research and development through techniques of synthetic biology that do not fall under the definition of living modified organisms (LMO) under the Cartagena Protocol.

Because JCVI’s research has been directly mentioned by Taye Birhanu [#8736] and referred to by Gerd Winter [#8730] (minimal cell), we would like to clarify the difference between synthesizing a bacterial genome, using a synthesized genome to create a “synthetic cell”, and a “minimal cell” (also based on a synthesized genome). 

In [#8736], Taye Birhanu mentions a bacterial genome synthesized by JCVI scientists in 2008. This synthetic genome by itself is not an LMO because it is not alive.  It is just a very large molecule of DNA that cannot replicate. 

In 2010, JCVI synthesized the genome of a different bacterium, Mycoplasma mycoides (a very close, but not identical copy to that found in nature); installed the synthetic genome into a cell of another closely related species so that the resulting cells have the synthetic genome and not the genome of the recipient cell; and observed the resulting cell take on all the characteristics defined by the synthesized genome.  For simplicity, we called this bacterial cell controlled by a chemically synthesized genome a “synthetic cell”.  http://science.sciencemag.org/content/329/5987/52  This bacterium, which lives and reproduces in our labs, is an LMO.

We subsequently redesigned the synthesized genome over the next 5 years, and synthesized a new genome comprised of about half of the genes present in the original M. mycoides genome. That set of genes included only those that were essential to the survival and reproduction of the cell.  In 2016, we published a scientific paper describing the highly modified version of Mycoplasma mycoides with a genome about half the size of the original.  http://science.sciencemag.org/content/351/6280/aad6253 For simplicity, we called this a “minimal cell”, because if we removed any of its remaining genes, the bacterium would no longer be able to live and reproduce.  This bacterium constructed through the techniques of synthetic biology is an LMO as defined by the Cartagena Protocol.

Use of “xenobiology”, a subdiscipline of synthetic biology, is sometimes mentioned as a way that a living organism might not fall under the Cartagena Protocol’s definition of LMO.  We know of no approach under consideration by any research group that is not based on nucleic acids, that is, that would not be an LMO.  The only current example of a reproducing organism with an expanded genetic alphabet has been developed by the Romesberg group at The Scripps Research Institute, which they call a “semisynthetic organism”.  It also is an LMO as defined by the Cartagena Protocol.
http://www.pnas.org/content/114/6/1317.full?sid=8fd587ac-be94-40c0-b27f-f3320986ad6c .

Finally, Westra and Glandorf mention scientific interest in developing bottom-up, artificial cells, often referred to as “protocells”.  We also know of no example of a self-replicating protocell today.  In addition, most of the research groups working in this area are exploring protocells based on nucleic acids, which if successful, would also be considered an LMO under the Cartagena Protocol.

Best regards to all,
Bob Friedman and John Glass
posted on 2017-09-11 06:11 UTC by Mr. Robert Friedman, J. Craig Venter Institute
This is a reply to 8741 RE: Opening of Discussion [#8742]
Dear all

I fully agree with the analysis done by Bob Friedman and John Glass [# 8741], it is very usefull for our discussion. Their intervention gives more clarity about Living organism outside LMO defined in Cartagena Protocol. I think that, from this intervention, we can build something reasonnable for the following of the discussion.

With best regards

Jean Bruno MIKISSA
posted on 2017-09-11 08:30 UTC by Mr. Jean Bruno Mikissa, Gabon
This is a reply to 8735 RE: Opening of Discussion [#8743]
I have been told that my earlier comments on Prof. Winter's document may not have been readable for everyone. I here upload a newly formatted version of the comments, although I realize that the discussion has moved on. Just to clarify: my interpretation at the moment agrees with the assessment by Westra & Glandorf [#8738] and Friedman & Glass [#8741] that  all living organisms already developed or currently under research and development through techniques of synthetic biology fall under the definition of living modified organisms (LMO) under the Cartagena Protocol.
posted on 2017-09-11 08:57 UTC by Mr. Rainer Breitling, University of Manchester
This is a reply to 8727 RE: Opening of Discussion [#8744]
I agree with previous posters, thank you to Jaco Westra and Profs Friedman & Glass for the clarification which seems sensible to me.

It brings me back to my question of the previous strand, learning from nano and other technologies, which is not, as Jaco suggests, to spend further time on legal and further definitional discussions, but to consider what are we worried about and what are we not worried about in relation to LMOs in their various forms, particularly in application and use.

Thank you for the organisers, I am finding this a helpful forum and wish there were similar international groups to debate the issues around the issues in relation to robotics, AI and quantum tech, the social issues around which I am also exploring with stakeholders.
posted on 2017-09-11 09:37 UTC by Ms. Hilary Sutcliffe, MATTER
This is a reply to 8741 RE: Opening of Discussion [#8745]
I would like to thank Nicolay for moderating this discussion forum and would like to add to the comments posted so far and to add to those by colleagues at JCVI.

Another major international project (US, UK, China, Singapore, Australia) is the synthetic yeast Sc2.0 project (see http://syntheticyeast.org/). The aim is to redesign all 16 chromosmes from the common Bakers yeast or S. Cerevisiae and incorporate them into a yeast cell such that the cell is controlled and regulated by the synthetic chromosomes.

The redesigns are very similar to the natural yeast strain but are computaionally designed and assembled using DNA synthesis / molecular biology technqiues. This year the project made significant progress which is covered in a special issuie of Science magazine (see http://science.sciencemag.org/content/355/6329) where 5 new synthetic  chromosomes were reported.

It should be noted that part of the design process was to provide an ability to scramble the chromosomes in the lab strains to explore evolution and adaptation within this unicellular and simple eukaryote. This experimental  approach will inevitably lead to a much greater understanding of chromosome architecture, dynamics, stability and regulation.

Like other cells with synthetic chrosomes the Sc2.0 strains would be considered as LMO's under the Cartagena Protocol.
posted on 2017-09-11 10:49 UTC by Mr. Paul Freemont, Imperial College London
This is a reply to 8741 RE: Opening of Discussion [#8746]
I am Jeremy Sweet of JT Environmental consultants with long term experience of risk assessment and studies of the agro-eco impacts of new technologies. My thanks to Nickolay and all contributors to this discussion.  I would like to add my agreement with Bob Freeman and John Glass, and commend them on their informative response. I also agree the conclusions by Jaco Westra and Boet Glandorf [#8738]:  I am not aware of any living organisms already developed or currently under research and development through techniques of synthetic biology that do not fall under the definition of living modified organisms (LMO) under the Cartagena Protocol.
The only grey area that I see is drawing the boundary between constructed genetic elements and  genetic elements forming a viable organism, as some non-viable genetic elements may have inheritance or viability characters in some associations. For example if free non-viable DNA links with viral or bacterial DNA.
posted on 2017-09-11 13:07 UTC by Mr. Jeremy Sweet, JT Environmrntal Consultants Ltd
This is a reply to 8727 RE: Opening of Discussion [#8747]
Dear all

My name is Christian R. Boehm and I am Chair of the European Association of Synthetic Biology Students and Postdocs (EUSynBioS), a Scientist at the Max Planck Institute and a Research Affiliate at the Cambridge Centre for the Study of Existential Risk. Many thanks for all the contributions made and to Nikolay for moderating this discussion.

Are there any living organisms already developed or currently under research and development through techniques of synthetic biology which do not fall under the definition of LMOs under the Cartagena Protocol? If we apply the definition of LMOs as living organisms that possess a novel combination of genetic material obtained through the use of modern biotechnology, most probably not.

However, several techniques have been developed which allow manipulation of living organisms at the level of RNA or protein only. At the level of RNA, these include injection or spraying of functional RNA molecules for vaccination, immunotherapy, or agricultural pathogen control. At the level of protein, these include a technique known as supplementation-based incorporation of non-canonical amino acids (SPI), for example. By starving E. coli for a specific canonical amino acid, the organism can be forced to incorporate a - structurally similar - synthetic amino acid into its proteins instead. As an instance of xenobiology, SPI is frequently referred to as a synthetic biology technique.

While organisms manipulated in this way may currently not be considered LMOs, it is worthwhile staying aware of synthetic biology-related efforts in this area due to their potential impact on biological diversity.
posted on 2017-09-11 14:10 UTC by Mr. Christian R. Boehm, European Association of Synthetic Biology Students and Postdocs
This is a reply to 8727 RE: Opening of Discussion [#8748]
POSTED O BEHALF OF ANGELA LOZAN
-------------------------------------------
Dear All,

I would like to thank Nicolay for guiding the discussion and bringing to us some useful examples.

I am of opinion that the cases of gene editing, cisgenesis and fully synthetic genomes perhaps might be considered to be off of the LMOs definition under the CP or very minor compared with LMOs. Some of them involve technics of classical mutagenesis or traditional breeding along with modern biotechnology. It is also suggested by some of researchers that the detection and identification technics as well as risk assessment on biodiversity and human health for the mentioned above types of Synbio might be different from those used for LMOs and should be modified. It request that additional guidng documents should be developed that will cover the mentioned cases.

With best regards,

Angela Lozan
MD
posted on 2017-09-11 14:27 UTC by Dina Abdelhakim, SCBD
This is a reply to 8741 RE: Opening of Discussion [#8749]
Thanks Mr. Nikolay for moderating this third session and alos thanks contibutors for their statements about the two key questions.  I am Hideyuki Shirae, working in Japan Biological Informatics Consortium.   I agreed with Mr. Jaco Westra's Opinion [#8738], I have not been known that any existing living organisms or living organisms developed in the ongoing research projects of synthetic biology, which do not fall under the definiton of living modified organisisms under the Cartagena Protocols.   I also agreed with the analyses done by Dr. Bob Friedman and Mr. John Glass [#8741].  In case of xenobiology, some research groups reported some organisms which have unnatural nucleic acid in their genome constructs, but such unnatural nucleic acids do not exist in natural world and alos the specific artificial replication enzymes, which never existed in natural world, are needed to grow for such organisms with unnatural nucleic acids.  Therefore, such organisms do not live and not be replicable in nature, and do not affect on any biodiversity.   Regarding genome editing and cisgenesis technologies, the basal techniques derived from the classical genetic engineering method which is classfied in "modern biotechnology" defined in  Cartagena Protocol, and such technologies are applied by some existing risk assessment methods, too.  The problem on whether organisms by genome editing can be detecting and monitoring or not is a ethical issue, but not an issue on methologies.   Finally, I supporse that the argument on proteins involving in non-canonical amino acids is out of scope in this session because those are not replicable in natural, referred to [#8747].
posted on 2017-09-12 02:07 UTC by Mr. HIDEYUKI SHIRAE, Japan
This is a reply to 8749 RE: Opening of Discussion [#8750]
Dear All,

Thanks to Nikolay for moderating this round. Always great if a moderator has a technical background that is directly relevant to the topic at hand.

To introduce myself: name is Piet van der Meer, and I have been involved in the negotiations for the CBD and the CPB from the late 80s to 2000, while serving as the Netherlands competent authority for biosafety/GMOs. As of 2000, I provide support to governments and public sector organisations about biosafety and I teach about biosafety at two universities.

Many countries are currently reviewing their regulatory frameworks for biosafety, and one of the elements of those reviews is similar to the current on-line discussion, i.e.: are the regulatory definitions adequate vis a vis new technological developments such as genome editing and synthetic biology? The specific questions to be answered are a) whether there are organisms developed through these new technologies that are not covered but should be covered, and b) whether there are organisms developed through these new technologies that are covered but should not be covered.

The current online discussion focuses on the question a) and I think that a systematic approach of ‘dissecting’ the LMO definition that for example the moderator and Winter followed can be very helpful, be it that Winter’s table goes beyond the question of this debate as Breitling rightly pointed out.

Having conducted a similar exercise, I come to the same conclusion as Westra, Glandorf, Friedman, Yoshikura, Breitling, Boehm and others: I do not know of any living organism currently under development through techniques of synthetic biology that does not -or would not -fall under the CPB definition of an LMO.

However, as was also expressed in earlier online discussions and discussions of the AHTEG, it will be advisable to keep monitoring scientific developments, and I therefore commend Westra, Glandorf, Boehm and Sweet for their forward looking contributions. 

Lastly, some general observations:
1) We should avoid confusing Synthetic Biology with the application of new breeding techniques (see for example EPSO: http://www.epsoweb.org/webfm_send/2329). For example, genome editing can
a) result in deletions or base-pair changes that are not distinguishable from what can be achieved with conventional breeding and which would not result in LMOs,
b) use templates to introduce genes from unrelated species, which would result in LMOs but which are not necessarily Synthetic Biology, and
c) develop organisms with novel or de novo genetic combinations that would constitute LMOs and which also fall under the description of synthetic biology.
2) A request to all to try to use wording in these online discussions that is also comprehensible for non-native English speakers, who are at a disadvantage since these discussions are conducted in English only.

Looking forward to the remainder of this debate

Piet
posted on 2017-09-12 05:49 UTC by Mr. Piet van der Meer, Ghent University, Free University of Brussels, Belgium, PRRI
This is a reply to 8737 RE: Opening of Discussion [#8751]
Dear Nikolay and and other colleagues,

Hello. My name is Jim Thomas. I work with the ETC Group – an international civil society organization that tracks emerging technologies including Synthetic Biology and I am fortunate enough to serve on the AHTEG on Synthetic Biology. I come to this set of questions as a non-scientist from a policy perspective but with a long familiarity with the field of synthetic biology and associated topics.. First, many thanks to Nikolay for opening up this discussion, which as i understand it is an exercise to determine how far the concept of ‘LMO' under the biosafety protocol legally captures the use of new techniques and approaches that are clustering under the term ‘synthetic biology’ when applied to living organisms

. I have 3 observations.

1)  I would first  in passing point out that not all uses of synthetic biology techniques give rise to living “organisms” but may nonetheless result in significant impacts for biodiversity conservation, sustainable use and equitable sharing of benefits form genetic resources. For example  there appears to be increasing interest in cell-free systems for protein synthesis  (and synthesis of other molecules using nucleic acids and other biomolecules as the production ‘machinery’). See for example this interesting article: https://www.economist.com/news/science-and-technology/21721560-new-type-biological-engineering-should-speed-up-innovation-cell-free-biotech. While these cell-free systems are not ‘living’ in the sense of reproducing, they use genetic resources and ‘bioparts’ and may potentially be scaled up to produce ingredients, drug molecules and other compounds that could replace naturally sourced natural products thereby impacting sustainable use and equitable sharing of biodiversity. In so far as these systems will rely on  genetic codes taken originally form natural sources there are also significant questions of  access and ‘equitable sharing of benefits’. I am not arguing here that synthetic cell-free production systems are ‘organisms’ but that it would be unwise to overlook them and their potential impact.

2) My  main request to this group, in light of the question asked of us, is that it would be helpful to have an agreed understanding of which are the ‘techniques of synthetic biology’ that are under discussion. As reflected in the definition of synthetic biology under the CBD and elsewhere, this field is not simply defined by ‘techniques’ - it is also defined by an approach (that is to say a deliberate ‘ engineering' approach to altering genetic systems) as well as by notions of "design, redesign, manufacture and/or modification of genetic materials, living organisms and biological systems”. Nonetheless i think it would be helpful in the discussions under the convention to have developed an illustrative list of techniques used in the practice of synthetic biology so we can have a common understanding of what we are discussing. For the purposes of initiating the creation of such an illustrative list i would offer the following as a starting point encourage others to add, edit and alter so that the AHTEG can have a crowd-sourced 'list of techniques’ to refer to. There is a useful list of techniques in Technical Series 82 that this draws from.  Other good sources acting as a review of available techniques include: Raj Cheri and George Church : Beyond editing to writing large genomes -  http://www.nature.com/nrg/journal/vaop/ncurrent/full/nrg.2017.59.html

Adrian Mackenzie - What is design in synthetic biology? From techniques to reflexive meta-materials - http://www.lancaster.ac.uk/staff/mackenza/papers/Mackenzie_synbio_design-biosoc-may09-web

An illustrative list of “ Techniques of Synthetic Biology”:

a) Gene Editing techniques 
Sequence-specific nucleases (SSNs)
Site-specific recombinases - e.g. tyrosine and serine- recombinases
Cre recombinase  
 Recombinase-mediated cassette exchange (RMCE)
 Meganucleases/ homing endonucleases
 Zinc finger nuclease (ZFN) / Synthetic Transcription Factors
 Directed Mutagenesis via oligonucleotides 
 CRISPR-Cas system /Cpf1
 TALENs
 Oligonucleotide directed mutagenesis (ODM)
Triple Helix / Peptide nucleic acid (PNA) gene-editing 
 Rapid Trait Development System (RTDS) (Cibus)
multiplex automated genome engineering (MAGE) using λ-red recombination
conjugative assembly genome engineering (CAGE),
Transfection of single-stranded oligonucleotides
Argonaute proteins (Ago) e.g. from Thermus thermophilus (TtAgo) and  Natronobacterium gregoryi, NgAgo,
 Group II introns / retrohoming
Gene transfection by femtosecond laser and optical tweezers


b) Gene Silencing and gene regulation:
RNA-dependent DNA methylation (RdDM)
gene Silencing via RNAi pathway
RNAi sprays/pesticides
optogenetics
Epigenetic modification (eg chromatin modification)/ epigenetic editors


c) Nucleotide Synthesis:
Gibson Assembly/ combinatorial assembly of synthetic gene cassettesSynthetic chromosomes 
Bacterial Artificial Chromosomes

d) Genetic (re)design tools:
codon optimization
Synthetic Metabolic Pathway Engineering
Biobrick assembly or other Standard Modular synthetic DNA ‘parts’
Directed Evolution
DNA-based Genetic Circuits 
Genome Shuffling
minimal genome assembly
refactoring genomes
modular protein assembly
Genetic design software - e.g. GenoCAD, Synbioss, 
Machine learning for genomic design.

e) xenobiology
Expanded Genetic Alphabets 
artificial nucleotides
reduced amino acids- e.g. XNA, PNA
Mirror biology
Protocell Construction

I would be glad to hear comments, additions, editing of this list..



3) Thirdly to answer the charge to identify techniques of synthetic biology that give rise to living organisms but may escape technical  consideration as LMO’s I suggest we look carefully at developments in epigenetic engineering - for example histone modification and engineering of chromatin intended for the purpose of altering gene expression An interesting review of epigenome engineering from 2016 is   Minhee Park, Albert J. Keung, Ahmad S. Khalil, The epigenome: the next substrate for engineering, Genome Biology, 17:183, 2016.    . To quote from the abstract to this paper:

"We are entering an era of epigenome engineering. The precision manipulation of chromatin and epigenetic modifications provides new ways to interrogate their influence on genome and cell function and to harness these changes for applications.”  

The same tools used for DNA editing (e.g. zinc Fingers, TALENS, CRISPR) are now also being targeted to deliberately modify chromatin and other epigenetic factors. the same paper points out that these tools open new directions as synthetic biology techniques:

"synthetic control over chromatin provides new capabilities in the field of synthetic biology, the engineering of functional biological systems from genetically encoded “parts”. New possibilities include engineering higher-order transcriptional control in cells and programming cellular memory states through the manipulation of epigenetic marks. The development of engineered readers, writers, and erasers that can effectively process the reversible modifications made to chromatin will expand the synthetic biology toolkit available for establishing synthetic linkages in cellular networks, enabling a better understanding of the function of these networks and control of complex cellular behaviors”

Another relevant paper is Keung, A.J., Joung, J.K., Khalil, A.S., and Collins, J.J. (2015) Chromatin regulation at the frontier of synthetic biology. Nat. Rev. Genet., 16 (3), 159–171.

I think its still very appropriate to consider an epigenetically engineered organism to be an LMO since epigenetic material is increasingly being recognized as ‘genetic material’ and as Nikolay correctly pointed out the definition of an LMO under the protocol  "means any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology;“ (i.e. not just modified nucleic acids)  and for now techniques such as ZFN/CRISPR/TALENS are firmly ‘modern biotechnology’ since they truck in nucleic acids (RNA/DNA)  but it may be that techniques arise to alter chromatin for example that do not use nucleic acids to achieve this.  A list of chromatin modifiers representing a epigenetic engineering 'toolbox' is listed at table 1 of this paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472160/

Another area to pay close attention to may be the optical methods of manipulating genomes (and maybe epigenomes?). On the one hand researchers have used femtosecond lasers combined with optical tweezers to transfect a genome with novel synthetic DNA. (see for example this description: http://www.osa.org/en-us/about_osa/newsroom/news_releases/2013/new_high-tech_laser_method_allows_dna_to_be_insert/). Other researchers use lasers in combination with gold nano shells an siRNA to carry out gene silencing
 Again I think this is modern biotechnology because here synthetic DNA and RNA (nucleic acids) are being employed but if this approach was to be used to alter an organisms genome in some way without also involving new DNA /RNA sequences then it may fall outside of that definition. Additionally optogenetic researchers are exploring means to turn genes on and off and control gene regulation remotely using light -this potentially may also give rise to deliberately altering genetic functioning in a novel way in organisms without introducing nucleic acids to do so.


The second question posed by Nikolay concerns detection of organisms, components and products of synthetic biology. While I presume that identifying organisms should be reasonably straightforward (e.g. by PCR where the altered sequence is known and can be identified), the more interesting question is whether the products of synthetic biology can be detected.

For example is it possible to distinguish if a vanillin or resveratrol molecule is derived from a  synthetic biology production method or from a vanilla pod or knotweed plant (or indeed from synthetic chemistry). My sense from conversations with others , but I am not an analytical chemist, is that such tests may be possible based on detection of co-arising proteins from fermentation. For example a bioengineered ‘heme’ protein produced by Impossible Foods was recently put n the market, derived from an engineered yeast Pichia pistoris. Documents provided to the US Food And Drug Administration by Impossible Foods indicate that the heme ingredient they are putting into the marketplace is not simply a single protein but in fact contains 46 additional proteins arising from the biological production of this product in a bioengineered yeast. This group of 46 compounds in effect form a ‘fingerprint’ that theoretically could allow this bioengineered heme to be distinguished from an identical heme derived from the root nodules of soybeans. Thats to say theoretically this product of synthetic biology could be identified and tested for. i presume the same would be true for other ‘syn Bio’ derived ingredients on the market (e.g. those listed at http://database.synbiowatch.org. )

I hope these observations are helpful to move this discussion along and once again thank Nikolay for his moderation.
 
With best wishes
Jim Thomas, ETC Group
posted on 2017-09-12 06:38 UTC by Mr. Jim Thomas, ETC Group
This is a reply to 8751 RE: Opening of Discussion [#8752]
I would like to come back to the list provided by Jim Thomas. Most of the methods and resulting organisms / products mentioned fall within the definition of synthetic biology as chosen by the ATHEG, as well as the definition of LMOs as given by the CBD.

On the other hand, I saw opinions that I would not agree with because the criteria those opinions are based on do not seem well defined. For example, it does not matter if additional DNA is inserted into an organism or not: If we take a look at multiplex deletions in the genome of polyploid plants that concern multiple, redundant genes and gene family networks as typically established with the help of CRISPR-Cas, they do not necessarily require the insertion of additional DNA. In many cases such a pattern of deletions are unavoidable if nucleases like CRISPR are applied in plants which are known to inherit many redundant DNA sequences. Contrary to the arguments of some stakeholders, such deletions are not likely to occur under random processes. In any case, the resulting organisms have to be considered as LMOs, derived by means of synthetic biology.

In addition, some other methods might require further discussion. For example, bringing about changes in the epigenome by means of synthetic biology (such as synthetic RNA or modified CRSPR-Cas) can result in organisms that are significantly changed in their biological qualities. At the same time, these organisms might not show “a novel combination of genetic material” if 'genetic material' is strictly reduced to the level of DNA. So maybe we need to discuss the definition of genetic material to be sure that these epigenomic changes do not escape our attention.

In regard to the availability of tools to detect and monitor the organisms, components and products of synthetic biology, it is evident that comprehensive information is needed to enable their identification and traceability. Many of the newly developed organisms are not as easy to detect as previous genetically engineered organisms. Therefore, we will need more robust systems for registration, identification and monitoring than currently are available.
posted on 2017-09-14 13:57 UTC by Mr. Christoph Then, Testbiotech
This is a reply to 8727 RE: Opening of Discussion [#8753]
POSTED ON BEHALF OF NIKOLAY TZVETKOV
------------------------------------------------------
Dear Colleagues,

Thank you very much for your contributions to the Forum so far. I am happy to see that many of them go into great technical detail to explain the positions of the authors and cite relevant literature. This is really important because the questions posed are technical in nature. In particular, since my last intervention, a few posts were shared explaining some of the most recent developments in the field and why they result in organisms that must be regarded as LMOs [#8737, #8741, #8743, #8749] as well as a very comprehensive list of the recent developments in the field of molecular biotechnology [#8751].

As we are entering the last few days of our discussion, I would like to encourage everyone to continue with the excellent work. I hope more contributions will be coming.

I would like to make an observation: the definition of LMO under Cartagena Protocol is based on the process that creates the organism, rather than on the properties of the final organism. At present it seems to me, based on your contributions and my own experience, that very few, if any, modifications do not involve use of in vitro nucleic acid techniques at one step or another. If that is indeed the case, the final organisms will then be LMOs. Additionally, “natural physiological reproductive or recombination barriers” are not absolute. In principle horizontal gene transfer can occur between any two organisms; genes with any sequence can evolve if necessary and if unlimited time is available. Modern biotechnology overcomes barriers not so much in the sense of making impossible things possible, but rather by allowing us to do things that would take an unrealistic amount of time if we used other methods.

This shifts the question more towards detection, especially when very similar results can be obtained by using classic techniques and with modern biotechnology. I think this is why those two subtopics are discussed together. For that reason I would encourage you to comment also on “Tools are available to detect and monitor each of the organisms, components and products of synthetic biology”.

As moderator of the Forum on this topic, I do not plan on providing any detailed comment based on my personal views, but allow me a little digression and a brief note on vanillin as I am keen on exotic flavourings, as an example. Distinguishing between chemically synthesized vanillin and vanillin that is a product of synthetic biology could, in principle, be done if minor contaminants can be detected that could be indicative of one or the other. If the product is too pure, then, in principle, an isotope signature can be used as the two different products may have slightly different isotope ratios.

The reason for this side comment is related to Subtopic 2. If we have to detect and distinguish LMOs developed through whole genome synthesis, cisgenesis or gene editing from those from classical breeding, paradoxically the tell-tale sign might be that the first group is too pure. It may contain too few other changes in the genome other that those specifically introduced. Classical breeding may introduce many other extra changes, because of mutations and recombination, which would be too hard to purge within the time-span and budget of a breeding program.

Once again thank you for your contributions so far and I am looking for further ones.

Best Regards,
Nikolay
posted on 2017-09-14 17:34 UTC by Dina Abdelhakim, SCBD
This is a reply to 8752 RE: Opening of Discussion [#8754]
Dr. Then suggests that “maybe we need to discuss the definition of genetic material” [#8752]. Any such discussion would begin with Article 2 of the CBD: "‘Genetic material’ means any material of plant, animal, microbial or other origin containing functional units of heredity”. N.B.: Rule 2 from formal logic is violated: “A definition must not be circular” (Copi and Cohen, 2010: 98). The rule also appears in guidelines for legal writing. For example, from the government of the non-Party: “Do not include part or all of the term being defined in the text of your definition” (Federal Register, 2016). Nevertheless, many of us will excuse the circularity in the CBD definition of “genetic material” because the word “material” seems unambiguous. Dr. Then’s suggestion indicates that it is not. The suggestion is also evidence why the rules of logic should be respected in crafting definitions (see [#8393] in Thread “Genetic Resources as Natural Information” of Topic 1).

Many issues relevant to the online discussion concern natural information, which is “non-material”. To interpret  “genetic material” to include its opposite---the “non-material”--- would do violence to language.

Copi, Irving and Carl Cohen.  (2010). Introduction to Logic. New York: Routledge.

Federal Register. (2016). “Drafting Legal Documents, Definitions”
https://www.archives.gov/federal-register/write/legal-docs/definitions.html
posted on 2017-09-15 06:02 UTC by Mr. Joseph Henry Vogel, University of Puerto Rico-Rio Piedras
This is a reply to 8753 RE: Opening of Discussion [#8755]
Dear forum participants,
my name is Swantje Strassheim and I work in risk assessment on synthetic biology at the German Federal Office of Consumer Protection and Food Safety. At first, I would like to thank our moderator Nikolay Tzvetkov for his precisely formulated questions and for his very helpful explanations and the summaries he provided so far.
As many others in this forum have stated, for example posts [#8738], [#8741] and [#8750], I do not know of any living organism that does not fall under the LMO definition according to the Cartagena Protocol.
I would also like to thank Piet von der Meer, who explains in his post ([#8750]), why we shouldn´t mix synthetic biology and new breeding techniques. I can fully agree to his argumentation as, for now, synthetic biology organisms are part of modern biotechnology, but modern biotechnology includes a lot more than synthetic biology. It has been and will be very difficult to find a definition for synthetic biology that includes all concepts, but allows to distinguish synthetic biology from modern biotechnology/genetic engineering for regulatory purposes. I therefore appreciate the approach we have taken with this topic to rely on the existing regulations and to keep monitoring whether any adaptations or modifications to those regulations are necessary.
posted on 2017-09-15 12:11 UTC by Ms. Swantje Strassheim, Germany
This is a reply to 8755 RE: Opening of Discussion [#8756]
Dear all,

Thank you for the moderator and thanks for the interesting discussion. I would like to answer the two proposed questions:

Since the last assessment made by the AHTEG on synthetic biology, have there been any living organisms already developed or currently under research and development through techniques of synthetic biology which do not fall under the definition of living modified organisms under the Cartagena Protocol?

No, there are no organism until the moment that do not fall under the definition of LMO. The principles of risk assessment outlined in Annex III of the Cartagena Protocol includes a scientifically sound, transparent and comparative approach and recognizes that the absence of scientific knowledge or consensus does not indicate a certain level of risk. The Annex III principles remain applicable to the risk assessment of LMOs developed through synthetic biology.


What tools are available to detect and monitor each of the organisms, components and products of synthetic biology?

The current LMOs on the market can be detected by different methodologies. For those organisms developed using biotec techniques that do not result in a novel combination of genetic material the definition of LMO is not applicable and they are similar to organisms obtained by conventional breeding. The monitoring measures are part of risk analysis and should be defined case by case according to the result of risk assessment.   
There is no need to develop any tool at this moment but it is important to keep this kind of discussion in the online forum to monitor biotechnological development and enable the identification of examples that do not fall under LMO definition.

Best regards,

Luciana P. Ambrozevicius
Ministry of Agriculture / Brasil
posted on 2017-09-15 21:29 UTC by Ms. Luciana Ambrozevicius, Brazil
This is a reply to 8727 RE: Opening of Discussion [#8757]
Dear Colleagues,

I had recalled the past discussion by the Risk assessment and risk management group (UNEP/CBD/BS/RARM/AHTEG/2016/1/5). Although this report focused on the risk aspects, those points in the report are related to the gaps in the term of living modified organisms. Also it mentioned the challenge that we faced in detection especially for genomic editing.

I appreciate that colleagues had pointed out xenobiology, which may not be covered in the biosafety protocol. The gene drives derived organisms possessed a kind of genetic material whose inheritance does not follow general segregation. In addition, the completely new synthetic DNA sequence is not the conventional recombinant DNA that defined in the protocol.

Sincerely

Wei
posted on 2017-09-17 15:29 UTC by Mr. Wei Wei, China
This is a reply to 8727 RE: Opening of Discussion [#8759]
Dear forum participants,

Our moderator has invited us to address items (b) and (d) of the terms of reference for the AHTEG on Synthetic Biology in decision CBD/COP/DEC/XIII/17, namely:

(b) [the AHTEG shall…] Identify any living organisms already developed or currently under research and development through techniques of synthetic biology which do not fall under the definition of living modified organisms under the Cartagena Protocol;

and

(d) [the AHTEG shall…] evaluate the availability of tools to detect and monitor the organisms, components and products of synthetic biology

Like other participants have stated in this discussion (#8739, 8741, 8743, 8744, 8746, 8749, 8750), we agree with the comments of Westra (#8738) and Friedman and Glass (#8741) that living organisms already developed or currently under research and development through techniques of “synthetic biology” are LMOs as defined by the Cartagena Protocol. We also note that this topic was previously addressed in the 2015 online discussion (Topic 2) and our views remain unchanged: the tools and techniques that may be used in synthetic biology applications are within the scope of “modern biotechnology” as defined by the Cartagena Protocol, with the resulting living products within the definition of “LMO”. Further, the definition of “genetic material” in the Convention does not exclude synthetically derived nucleic acid sequences (#7133).

We have also commented previously (in agreement with #8750 and 8755), that genome editing is not synthetic biology. In our information submission (http://bch.cbd.int/database/record.shtml?documentid=112053) we note that this oft-cited example of synthetic biology in this forum is better and more correctly described as an enabling tool. The “illustrative list” in #8751 provides many examples of enabling tools, however these cannot be used to define synthetic biology. Like “older” recombinant DNA technologies, genome editing may be used in various applications, which may or may not be labelled (or re-labelled) as “synthetic biology”. For example, in plants, the outcomes of certain genome editing applications are similar to transgenics (LMOs), while others are similar to plants developed with conventional breeding tools (not LMOs). Neither presents a fundamental change in risk.

This discussion on in/out of the LMO definition scope is ultimately concerned with adequate regulation of risk. We should remember the objectives of the Convention on Biological Diversity, and the scope of applicability of the Cartagena Protocol (Article 4):
"This Protocol shall apply to the transboundary movement, transit, handling and use of all living modified organisms that may have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health."
This clearly states that LMO status considered in isolation does not determine risk, and not all LMOs will be within the scope of the Cartagena Protocol – there needs to be an assessment of risk that they “may have adverse effects on the conservation and sustainable use of biological diversity”.

On the question of availability of tools to detect and monitor organisms developed through synthetic biology, we agree with #8738 and 8756 that these are specific to each LMO and cannot be discussed in a generic way. Further, detection and monitoring may not be required – where they are, established and developing technologies can be applied. We do not believe there is a need to develop new tools or guidance for this purpose in the absence of specific examples of organisms where there is demonstrated need.
posted on 2017-09-17 23:30 UTC by Ms. Felicity Keiper, Bayer CropScience