New Plant Breeding Techniques:
Legal Classification & Regulatory Implications
Drew L. Kershen
Earl Sneed Centennial Prof. Emeritus
Crop Bioengineering Consortium
Iowa State University
November 5, 2013
Site-Directed Nuclease Techniques
• Meganucleases (MNs)
– Restriction enzyme source: many species
• Zinc-finger Nucleases (ZFNs)
– Protein source: many species (including humans)
– Synthetic biology – customized ZFNs
• Transcription Activator-Like Effector Nucleases (TALENs)
– Effector source: Xanthomonas genus
• Clustered regularly interspaced short palindromic repeats
(CRISPRs)
– Cas9 sources: Streptococcus thermophilus & Staphylococcus
aureus
• Construct components
– Promoter (CaMV 35S); Terminator (NOS); nuclear localization
signal (SV40 – simian virus 40)
United States Law
USDA-APHIS
• Plant Protection Act (2000, as amended), 7 U.S.C. §§ 77017772
• Regulated article. Any organism which has been altered or
produced through genetic engineering, if the donor organism,
recipient organism, or vector or vector agent belongs to any
genera or taxa designated in § 340.2 and meets the definition
of plant pest, or is an unclassified organism and/or an
organism whose classification is unknown, or any product
which contains such an organism, or any other organism or
product altered or produced through genetic engineering
which the Administrator, determines is a plant pest or has
reason to believe is a plant pest. Excluded are recipient
microorganisms which are not plant pests and which have
resulted from the addition of genetic material from a donor
organism where the material is well characterized and contains
only non-coding regulatory regions. 7 C.F.R. 340.1
United States Law
USDA-APHIS
• Plant pest. Any living stage (including active and dormant
forms) of insects, mites, nematodes, slugs, snails, protozoa, or
other invertebrate animals, bacteria, fungi, other parasitic
plants or reproductive parts thereof; viruses; or any organisms
similar to or allied with any of the foregoing; or any infectious
agents or substances, which can directly or indirectly injure or
cause disease or damage in or to any plants or parts thereof,
or any processed, manufactured, or other products of plants. 7
C.F.R. 340.1
• Organism. Any active, infective, or dormant stage or life form
of an entity characterized as living, including vertebrate and
invertebrate animals, plants, bacteria, fungi, mycoplasmas,
mycoplasma-like organisms, as well as entities such as viroids,
viruses, or any entity characterized as living, related to the
foregoing. 7 C.F.R. 340.1
United States Law
USDA-APHIS
•
•
•
•
§ 340.2 Groups of organisms which are or contain plant pests and exemptions.
(a) Groups of organisms which are or contain plant pests. The organisms that are or contain
plant pests are included in the taxa or group of organisms contained in the following list.
Within any taxonomic series included on the list, the lowest unit of classification actually
listed is the taxon or group which may contain organisms which are regulated. Organisms
belonging to all lower taxa contained within the group listed are included as organisms that
may be or may contain plant pests, and are regulated if they meet the definition of plant pest
in § 340.1 4
4 Any organism belonging to any taxa contained within any listed genera or taxa is only
considered to be a plant pest if the organism “can directly or indirectly injure, or cause
disease, or damage in any plants or parts thereof, or any processed, manufactured, or other
products of plants.” Thus a particular unlisted species within a listed genus would be
deemed a plant pest for purposes of § 340.2, if the scientific literature refers to the organism
as a cause of direct or indirect injury, disease, or damage to any plants, plant parts or
products of plants. (If there is any question concerning the plant pest status of an organism
belonging to any listed genera or taxa, the person proposing to introduce the organism in
question should consult with APHIS to determine if the organism is subject to regulation.)
NOTE: Any genetically engineered organism composed of DNA or RNA sequences,
organelles, plasmids, parts, copies, and/or analogs, of or from any of the groups of
organisms listed below shall be deemed a regulated article if it also meets the definition of
plant pest in § 340.1.
United States Law
USDA-APHIS
• § 340.3 Notification for the introduction of
certain regulated articles
– (b) Regulated articles eligible for introduction
under the notification procedure. Regulated
articles which meet all of the following six
requirements and the performance standards …
• (6) The plant has not been modified to contain the
following genetic material from animal or human
pathogens:
– (i) Any nucleic acid sequence derived from an animal or
human virus, or
– (ii) Coding sequences whose products are known or likely
causal agents of disease in animals or humans.
United States Law
USDA-APHIS
• Regulated Article Letters of Inquiry
– http://www.aphis.usda.gov/biotechnology/am_i_reg.shtml
– Transformation method
– Construct (each element – promoter, gene, terminator and
source from which derived)
– Recipient Organism
– Donor Organism
– Trait (phenotype)
• Nineteen letters of inquiry posted through 13 March
2013
– One letter on ZFN-1; one letter on MN-1
• Not regulated articles – Knockout effect
– No Letters of Inquiry on TALENs or CRISPRs
USDA-APHIS
Analysis – The Plant Pest Trigger
• Transformation method – Agrobacterium
tumefaciens (yes); biolistics (no); electroporation
(no)
• TALENs – Binding domain from Xanthomonas
– Presumptively a “regulated article” 7 C.F.R. 340.2
• CRISPR – Binding domain from Streptococcus
thermophilus or pyogenes – not listed in 340.2
• Yogurt and cheese
– CRISPR – Nuclear Localization signal from Staphylococcus
aureus (human pathogen) – not listed in 340.2 but
340.3(b)(6)(ii)
– Constructs – several components derived from viruses or
bacteria
USDA-APHIS
Analysis – The Classification
• No Regulated Article Letters of Inquiry
– CRISPR appears better than TALENS for regulatory avoidance
– CRISPR (Streptococcus t.) compared to CRISPR (Staphylococcus a.)
– Need Biolistic transformation & NLS, promoters & terminators
from non pathogen sources to avoid the underlying plant from
being regulated
• Null segregants from the construct, not regulated article
• TALENs – avoidance approaches
– Binding domain by synthetic biology (customized chimeric
binding domain) – not an organism? – an analog of a listed 340.2
pest?
– Null-segregant plants – back crossing with confirmation that no
Xanthomonas DNA/RNA
• Several Letters of Inquiry on null segregant plants
– Underlying plant regulated (keep in laboratory)
– Offspring plants, not regulated article
United States Law
Environmental Protection Agency
• Federal Insecticide Fungicide Rodenticide Act (as amended through
2012), 7 U.S.C. §§ 121-136y
• PESTICIDE.—The term ‘‘pesticide’’ means (1) any substance or
mixture of substances intended for preventing, destroying,
repelling, or mitigating any pest, (2) any substance or mixture of
substances intended for use as a plant regulator, defoliant, or
desiccant, and (3) any nitrogen stabilizer, … 7 U.S.C § 136(u)
• PLANT REGULATOR.—The term ‘‘plant regulator’’ means any
substance or mixture of substances intended, through physiological
action, for accelerating or retarding the rate of growth or rate of
maturation, or for otherwise altering the behavior of plants or the
produce thereof, … 7 U.S.C. § 136(v)
• Analysis
– The intended purpose of the deletion, recombination or insertion, not
the SDN method per se
– EPA has pondered expanded FIFRA regulation of GE plants, e.g., RNAi
and as “new chemicals” using Toxic Substances Control Act (TSCA).
United States Law
Food & Drug Agency (FDA)
• Food, Drug & Cosmetic Act, 21 U.S.C. §§
341-350l-1 (as amended to 2012)
– “Voluntary” Consultation
– Substantial Equivalence (149 foods to date)
• Data analysis of the food -- TALENs
– Generally Recognized as Safe (GRAS)
• CRISPR Cas9 and binding domain from a bacterium
used to produce yogurts and cheese – assuredly
GRAS
European Union Law
2001/18/EC – Deliberate Release
• Article 2 -- Definitions
– For the purposes of this Directive:
– (1) "organism" means any biological entity capable of replication or of
transferring genetic material;
– (2) "genetically modified organism (GMO)" 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;
– Within the terms of this definition:
– (a) genetic modification occurs at least through the use of the
techniques listed in Annex I A, part 1;
– (b) the techniques listed in Annex I A, part 2, are not considered to result
in genetic modification;
• Article 3 – Exemptions
– This Directive shall not apply to organisms obtained through the
techniques of genetic modification listed in Annex 1B.
European Union Law
2001/18/EC – Deliberate Release
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ANNEX I A -- TECHNIQUES REFERRED TO IN ARTICLE 2(2)
PART 1: Techniques of genetic modification referred to in Article 2(2)(a) 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.
PART 2: Techniques referred to in Article 2(2)(b) which are not considered to result in
genetic modification, on condition that they do not involve the use of recombinant
nucleic acid molecules or genetically modified organisms made by
techniques/methods other than those excluded by Annex I B:
– (1) in vitro fertilisation,
– (2) natural processes such as: conjugation, transduction, transformation,
– (3) polyploidy induction.
European Union Law
2001/18/EC – Deliberate Release
• ANNEX IB--TECHNIQUES REFERRED TO IN
ARTICLE 3
– 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.
The Cartagena Protocol
• Super-imposed on European law
• Binding in 167 countries
• Regulates “Modern biotechnology”
– “In vitro nucleic acid techniques, including
recombinant deoxyribonucleic acid (DNA) and
direct injection of nucleic acid into cells or
organelles”
• What makes the CP so nefarious is that
nothing says the nucleic acid must remain in
the cell
– Null segregants are still covered
2001/18/EC
Analysis
• No mechanism in the EU directly comparable to USDAAPHIS Regulated Article Letters of Inquiry
– Working groups (advisory)
– Discussion of ZFNs, but not TALENs or CRISPR
• Annex IA Part One – “inter alia”
– TALENs and CRISPR probably covered
• New introduced genetic material
• Technique used for insertion of genetic material prepared
outside the organism
• Null segregant plants – very unclear under EU law
– Covered by Cartagena Protocol if will be released to the environment
• Like mutagenesis? Especially SDN-1 techniques
– Precautionary Principle
• Whereas Clause (8)
European Union Law
Reg. (EC) No. 1829/2003 (22 Sept 2003)
• Whereas Clause 16) -- This Regulation should
cover food and feed produced ‘from’ a GMO but
not food and feed ‘with’ a GMO. The determining
criterion is whether or not material derived from
the genetically modified source material is present
in the food or in the feed. Processing aids which are
only used during the food or feed production
process are not covered by the definition of food or
feed and, therefore, are not included in the scope
of this Regulation.
• Article 2: Definitions -- 10. ‘produced from GMOs’
means derived, in whole or in part, from GMOs,
but not containing or consisting of GMOs;
Reg. (EC) No. 1829/2003
Analysis
• EFSA opinion of October 2012
– p. 2 “With respect to the genes introduced, the SDN-3 technique
does not differ from transgenesis or from the other genetic
modification techniques currently used with respect to any
hazards associated with introduced genes. The SDN-3 technique
can be used to introduce transgenes, intragenes, or cisgenes.”
• Null segregant plants – produced “with” (not covered) or
“from” (covered by regulations)?
– Wine (GM yeast) is “with”; Wine (GM grapes) is “from”; Wine
from GM rootstalk but non-GM scion is unclear and undecided
– My opinion on null segregant plants: likely produced “from”
GMO plants (covered)
• Reg. (EC) No. 1830/2003 – labeling and traceability
– Applies once 1829/2003 determines something to be a GMO
food or feed
Exceptions
• Vitamins extracted from GM soy are
covered
• Vitamins extracted from GM
microorganisms are exempt
• The point is that Europeans do make
exceptions when convenient for them
Legal Issues arising from
differences between EU & US laws/regulations
• Regulatory costs to get multiple approvals
• Asynchronous approval
• Legal Liability for impact on international
trade with Europe
– Voluntary Contracts
– Coexistence
• World Trade Organization Treaties
– Sanitary and Phytosanitary Treaty (SPS)
– Technical Barriers to Trade Treaty (TBT)
Policy Issue
New Plant Breeding Techniques
• NAS (1987): “Several conclusions can be drawn from this
review of the relationship between traditional genetic
manipulation techniques and the R-DNA techniques developed
during the last 15 years, and of the experience gained from the
application of each:
– There is no evidence that unique hazards exist either in the use
of R-DNA techniques or in the movement of genes between
unrelated organisms.
– The risks associated with the introduction of R-DNA engineered
organisms are the same in kind as those associated with the
introduction of unmodified organisms and organisms modified by
other methods.
– Assessment of the risks of introducing R-DNA engineered
organisms into the environment should be based on the nature
of the organism and the environment into which it is introduced,
not on the method by which it was produced.”
Policy Issue
New Plant Breeding Techniques
• ACRE (2013): Executive Summary
– “Our understanding of genomes does not support a
process-based approach to regulation. The continuing
adoption of this approach has let to, and will increasingly
lead to, problems. This includes problems of consistency,
i.e. regulating organisms produced by some techniques and
not others irrespective or their capacity to cause
environmental harm.”
– “Our conclusion, that the EU’s regulatory approach is not fit
for purpose for organisms generated by new technologies,
also applies to transgenic organisms produced by
‘traditional’ GM technology. … the potential for
inconsistency is inherent because they may be
phenotypically identical to organisms that are not
regulated.”
References
• Lusser, M. & Davies, H.V., (2013) Comparative
regulatory approaches for groups of new plant
breeding techniques, New Biotechnol. (in press)
• Minikel, E. (2013) TALENs and ZFNs,
www.cureffi.org
• Pennisi, E. (2013) The CRISPR Craze, Science
341:833-836
• Podevin, N. et. al, (2013) Site-directed nucleases: a
paradigm shift in predictable, knowledge-based
plant breeding, Trends in Biotechnology, 31:375383.
References
• European Commission, Directorate-General
Environment, Working Group on the Establishment of a
List of Techniques Falling under the Scope of Directive
2001/18/EC (unpublished, available as a leaked
document, August 2013)
• Lusser, M. et al., (2011) New Plant Breeding
Techniques: State-of-the-art and prospects for
commercial development (JRC Scientific and Technical
Reports).
• EFSA Panel on GMOs: Scientific opinion addressing the
safety assessment of plants developed using ZFNs-3
and other SDN with similar functions, EFSA Journal
2012, 10(10:2943 (31 pp.)
References
• National Academy of Science (USA),
Introduction of Recombinant DNAEngineered Organisms into the
Environment: Key Issues (1987)
• Advisory Committee on Releases to the
Environment (ACRE), Report 2: Why a
modern understanding of genomes
demonstrates the need for a new
regulatory system for GMOs. (Sept. 2013)
Wayne Parrott, Ph.D., Professor, Plant Breeding and
Genomics, Crop & Soil Sciences, University of Georgia is
coauthor of this PowerPoint.
Thank you.
dkershen@ou.edu