Download New techniques and the GMO-legislation

New techniques and the GMO-legislation
Mistra Biotech
Workshop
2014-05-23
The EU has a common legislation that regulates the
use of GMO e.g. Directive 2001/18/EC on the
deliberate release into the environment of
genetically modified organisms.
The Directive regulates:
 all organisms, except humans
 both experimental and commercial activities
The Directive is process-based, so it is the technique
used that decides if a certain product should be
regulated or not.
The definition and the list of techniques of genetic
modification are the same as in Directive
90/220/EEC from 1990 (repealed by 2001/18/EC).
Several new techniques has been developed since
then and the development continues.
It is unclear if certain technique results in a
GMO/GMM that should be regulated or not.
What defines a GMO?
Article 2 (2) in the Directive 2001/18/EC
Definition
"genetically modified organism" means an
organism, with the exception of human beings,
in which the genetic material has been
altered in a way that does not occur naturally
by mating and/or natural recombination.
Excludes everything except fertilization!
Annex 1A, part 1
Techniques of genetic modification referred to
in Article 2(2) are inter alia:
1) recombinant nucleic acid techniques involving
the formation of new combinations of genetic
material by the insertion of nucleic acid
molecules produced by whatever means outside
an organism, into any virus, bacterial plasmid or
other vector system and their incorporation into
a host organism in which they do not naturally
occur but in which they are capable of continued
propagation.
2) techniques involving the direct introduction into
an organism of heritable material prepared outside
the organism including micro-injection, macroinjection and micro-encapsulation.
3) cell fusion (including protoplast fusion) or
hybridisation techniques where live cells with new
combinations of heritable genetic material are
formed through the fusion of two or more cells by
means of methods that do not occur naturally.
Annex 1A, part 2
Techniques referred to in Article 2(2)(b) which are
not considered to result in genetic modification….
1)
in vitro fertilisation,
2)
natural processes such as: conjugation,
transduction, transformation
3)
polyploidy induction
Annex 1B
Techniques/methods of genetic modification yielding
organisms to be excluded from the Directive, on the
condition that they do not involve the use of
recombinant nucleic acid molecules or genetically
modified organisms other than those produced by one
or more of the techniques/methods listed below are:
1) mutagenesis,
2) cell fusion (including protoplast fusion) of plant
cells of organisms which can exchange genetic
material through traditional breeding methods.
In the GMO:s approved so far a DNA-sequence
has been integrated into the genome om the
recipient organism -> recombinant DNA.
Several of the new techniques results in an
organism that do not have any foreign DNA
integrated -> no recombinant DNA in the final
organism.
Working Group on New Techniques
Established at the request of the competent
authorities in October 2007.
Two experts from each Member State nominated.
Evaluate a list of eight new techniques.
Discussed in the light of:
• the definition of a GMO/GMM;
• the techniques listed in the Annexes
• the most recent available scientific data
A report was submitted to the commission in
January 2012.
Examples of New Techniques
Oligonucleotide-Directed Mutagenesis (ODM)
Cibus LLC: http://cibus.com/
 Site-specific mutation
 No foreign DNA in the resulting organism.
Organisms developed through mutagenesis are
excluded from the Directive, on the condition that
the technique do not involve the use of recombinant
nucleic acid molecules.
Key issues discussed
 Are the synthetic oligonucleotide a recombinant
nucleic acid molecule?
 Are synthetic oligonucleotides heritable
material?
Different oligonucleotides can be used e.g.
Capped ssDNA (Cibus)
CY3-dye
Peptide Nucleic Acid
Idc (reverse base)
N-(2-aminoethyl)-glycine units linked by peptide bonds.
Locked Nucleic Acid
bridge between 2‘-O and 4‘-C
PNA – not a nucleic acid in a biological sense
Conclusions from the Working Group
All experts
ODM results in changes that can be obtained with
other forms of mutagenesis.
ODM is expected to generate fewer unintentional
changes than traditional mutagenesis.
The majority of the experts
Oligonucleotides in this technique cannot be
considered as recombinant nucleic acids or heritable
material and that they are not capable of continued
propagation.
ODM is captured by Annex IB -> excluded
Herbicide tolerant rapeseed developed using ODM
 Commercially available in the US where the
authorites consider it to be a non-GM.
 Recently approved in Canada (March 2014).
 Field trials in the UK where the authorites
consider it to be a non-GM.
 Cibus asked the competent authority in
Sweden if they need to apply for field trials
according to Directive 2001/18/EC.
The answer was no.
Zincfingernuklease 1 (ZFN1)
Protein complex consists of:
 DNA-binding domain: Zincfingers
(designed to bind to a specific site in the genome)
 DNA-cleaving domain: Nuklease
Zincfingers
Nuklease
Zinkfingernuklease1
Nukleotides lost/added -> mutation
Repair
Double-strand break
DNA
Non-replicating
DNAPlasmid
GMO?
mRNA
GMO?
NOT GMO!
Transient presence of ZFN-encoding
recombinant plasmid
No recombinant DNA
Plant cell
X
A site-specific
mutation is created
The plasmids/mRNA/
proteins are degraded
ZFN-encoding
non-replicating
plasmid
Plant cells in culture are GMM
(Directive 2009/41/EC)
No recombinant DNA
X
Conclusions from the Working Group
All experts
Results in changes that can be obtained with other
forms of mutagenesis.
Is expected to generate fewer unintentional
changes than traditional mutagenesis.
If ZFN-proteins are introduced directly the
technique is fully captured by annex 1b -> excluded
If plasmids/mRNA is used it should be excluded.
Two different views:
Already excluded/inclusion in Annex 1B needed.
Classical GMO
Classical mutagenesis
DNA-sequence
integrated into the
genome.
Recombinant.
Random mutations over
the entire genome.
(radiation or chemicals)
Not recombinant.
Regulated
Not regulated
New technique
Site-specific mutagenesis
eg. ODM, ZFN,TALEN,
CRISPR. Recombinant DNA
can be used early in
breeding process.
Regulated?
Cisgenesis
DNA from the same species or a species´ that
the recipient organism can hybridize with through
conventional breeding techniques.
The Working Groups conclusions
All experts
 Captured by Directive 2001/18/EC -> regulated
 May in some cases meet the criteria of
self-cloning in Directive 2009/41/EC on
contained use of GMM -> excluded.
Animal and plant cells in culture = GMM
Reverse breeding
Gene technology
Contained use
Heterozygote elite plant
modified with gene that
inhibit recombination (RNAi)
Traditional breeding
Microspores -> haploid
plants.
Selection for non-GM.
Contained use
Chromosom doubling
-> homozygote.
Sexual crossing.
Heterozygote without any
foreign DNA.
X
Deliberate release
Conclusions from the Working Group
The resulting plants and their offspring may be
considered as not within the scope of the
Directives based on:
 The resulting organisms have never contained any
inserted foreign DNA.
 The genetic composition of the offspring is the
same as the original plant material.
 The resulting organisms can be obtained by
traditional breeding techniques.
Other activities at the EU-level
The European Food Safety Authoritys GMO-panel
 Determine whether there is a need for new guidance or
whether the existing guidance on risk assessment should
to be updated or further elaborated.
 What are the risks in terms of impact on humans, animals
and the environment that the techniques could pose,
irrespective of whether or not they fall under the GMO
legislation?
After the evaluation of two of the techniques,
the Commission announced that the panel do not have to
continue their work with the other techniques.
DG Joint Research Center (2011)
New plant breeding techniques
State-of-the-art and prospects for commercial development
Some conclusions
Companies and research institutes based in the EU
play a prominent role in research and development.
Commercialization likely within 2-3 years (i.e. now!).
The main constraints for the adoption of the
techniques are the regulatory uncertainty and
the potentially high costs for risk assessment
and registration (if the crops derived by these
techniques are classified as GMOs).
The result of several of the techniques cannot be
distinguished from those produced by conventional
techniques or by natural variation.
A detection method is a regulatory requirement.
GMO or not GMO?
Excluded or not excluded?
These are the questions.
Thanks for listening!
Marie Nyman
www.genteknik.se