Regulatory definition of GMO in relation to organisms developed through genome editing

Seminar, 8 January 2016 FAO offices Budapest, Hungary,

Piet van der Meer • Department of Plant Biotechnology and BioInformatics, Faculty of Sciences,

Ghent University, Belgium • Department of European, Public and International Law, Faculty of Law,

Ghent University, Belgium • Faculty of Science and Bio-Engineering Sciences,

Free University of Brussels (VUB), Belgium

Topics

• Modern Biotechnology and Biosafety – historical highlights

• Biosafety Regulations

• Definitions

• Genome editing

Modern Biotechnology – Historical highlights

1850 - 1900: Gregor Mendel and others: the rules of inheritance 1900 - 1920: new techniques, embryo rescue, hybrids,

radiation/chemical induced mutagenesis. 1920 - 1953 Discovery of ‘genes’, their building blocks, DNA helix.

1950 - 1970: Discovery of restriction enzymes, DNA ligases, etc.

1972: First publications on ‘recombinant DNA’; Jackson, D.; Symons,

R.; Berg, P. 1983: First transgenic plants, University of Gent

Conventional breeding and rDNA

Conventional

breeding x

“elite”

variety

Related

variety

rDNA

any

gene

source

Biosafety – Historical highlights

1974: ‘Berg Letter’ published in Science, anticipated benefits and potential risks, calling for a voluntary moratorium.

1975: Asilomar Conference: technique not inherently risky, but

potential for novel combinations, safety assessed ‘case by case’, end of moratorium.

1976: US-NIH Guidelines for rDNA micro-organisms in laboratories 1978: Other countries follow, e.g. UK (1978), Netherlands (1979) 1986: OECD “Blue Book”, rDNA safety recommendations; key

concepts for risk assessment and risk management

Biosafety – Historical highlights

1986 - 1990: discussions expand: • Technical aspects of the discussion broadened to include

– plants and animals – releases into the environment – other molecular methods to make novel genetic combinations

• Discussions increasingly international:

– OECD, UN (Agenda 21, CBD), EU, CoE, etc

• Discussions increasingly get a regulatory character

Biosafety Regulations

1986: US coordinated framework for regulation of biotechnology

(USDA, EPA, FDA) 1990: EU Directives on Contained Use and Release of GMOs into the

Environment

1996: Canada (CFIA, Health Canada)

2000: Cartagena Protocol on Biosafety - transboundary movement of LMOs to countries that do not yet have biosafety

frameworks

Biosafety Regulation - Cycle

• Developing regulations, e.g. – Objective, e.g. internal market, safety – Scope: e.g. contained use, release, placing on the market of

GMOs – Regulatory mechanism(s): e.g. conditions, authorisations,

notifications

• Implementing, e.g. risk assessment, decision making

• Reviewing, evaluating, and assessing – Review of legal text: e.g. scope and definition clear vis a vis new

developments such as NBTs? – Evaluation: is implementation effective and efficient? – Assessment of impacts on sectors such as R&D and farming

Definitions

Definitions - History

1986: OECD “Blue Book”, rDNA safety recommendations:

Definition of genetic manipulation and recombinant DNA varies in detail between countries, but rDNA organisms are described as constructed by introducing DNA from a “donor” organism into a “recipient” E,g: UK: formation of new combinations of heritable material by the insertion of nucleic acid molecules, produced outside the cell, via vector system so as to allow their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation.

1986: US coordinated framework for regulation of biotechnology

Definitions – Historical highlights

Debate as of the mid 80s: which organisms should be subject to prior risk assessment? Observations put forward:

• Conventional breeding and rDNA can both result in novel genetic combinations that can cause adverse effects

• rDNA techniques themselves do not confer risky characteristics • rDNA can make a broader range of novel combinations

Options discussed: • Include conventionally produced organisms? • Focus on ‘risky’ organisms? • Focus on ‘novel’ organisms ?

Which organisms subject to prior risk assessment?

Considerations:

• Conventional organisms: is there an unmet need to include them?

• Risky organisms: how to assess which are the risky organisms?

• Novel organisms: How to define “novel”? – Genotypic and/or phenotypic? – ‘Does not exist’ or ‘is unlikely to happen’?

• Scope and definitions of existing regulations

>> Different approaches in different systems, most focus on novelty

United States Department of Agriculture

Biotechnology Regulatory Services

Animal and Plant Health Inspection Service

Department of Agriculture (USDA-APHIS-BRS)

• PPA: Protecting against damage from plant pests and noxious weeds

Environmental Protection Agency (EPA) • FIFRA: Regulating the safe use of pesticides • FFDCA: Setting allowable levels of pesticides in food • TSCA: Regulating toxic substances

Food and Drug Administration (FDA) • FFDCA: Regulating safety of food, drugs, and cosmetics

US Biotechnology Regulation under the Coordinated Framework

Definitions – USA

USDA (1987, based on FPPA and PQA, which were later incorporated into the Plant Protection Act, which includes also noxious weeds): Regulatory trigger:

– the use of genetic engineering in combination with – the use of plant pest as recipient, donor or vector, in the

transformation process.

Definitions – USA

FDA: Authority based on Federal Food, Drug and Cosmetic Act

(FFDCA) Guidance in the Federal Register of 1992

“The 1992 policy applies to foods developed from new plant varieties, including varieties that are developed using recombinant deoxyribonucleic acid (rDNA) technology (which is often referred to as "genetic engineering" or "biotechnology"). This guidance document refers to foods derived from plant varieties that are developed using rDNA technology as "bioengineered foods."

Definitions – USA

EPA: authority based on the Federal Insecticide, Fungicide and

Rodenticide Act (FIFRA), FFDCA, and Toxic Substances Control Act (TSCA)

Plant-Incorporated Protectant (PIP, 2001) - a pesticidal substance that is intended to be produced and used in a living plant, or in the produce thereof, and the genetic material necessary for the production of such a pesticidal substance. It also contains any inert ingredient contained in the plant, or produce thereof." PIPs developed through conventional breeding will remain exempt from all FIFRA and FFDCA requirements, with the exception of adverse effects reporting requirements for manufacturers.

Definitions - EU

EU (1990): GMO means an organism in which the genetic material has been altered in a way that does not occur naturally;

• genetic modification occurs at least through the use of:

– rDNA techniques; – direct introduction of heritable material; – fusion techniques.

Definitions – Canada

CFIA (1996, based on the Seeds Act and Feed Act) and Health Canada (Food and Drug Act) Plants with novel traits fall under the regulatory trigger of the Seeds regulations if:

– They have a novel trait that was not present prior to December

1996 in germplasm of the same species in Canada, and – that plant poses an environmental risk.

Definitions – CPB

Cartagena Protocol on Biosafety: (2000) • "Living modified organism" means any living organism that

possesses a novel combination of genetic material obtained through the use of modern biotechnology;

• Modern biotechnology" means the application of: - In vitro nucleic acid techniques, including recombinant

deoxyribonucleic acid (DNA) and direct injection of nucleic acid into cells or organelles, or

- 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;

Definitions - EU

Question whether and to what extent organisms developed through NBTs such as genome editing fall under the definition of GMO. Discussion: “Altered in a way…that does not occur naturally…?” Refers to: • Process/technique ? • The product/resulting organisms ? • Both?

[ See also Callebaut 2015]

Definitions - EU

Legal clues: The law itself, explanatory notes, Jurisprudence, doctrine The final word is with the ECJ. Interpretation by the ECJ: “the wording, general scheme and spirit” • Literal interpretation, text of the definition and the annexes

When the wording in itself is not conclusive: • Systemic interpretation (general scheme), internal and external

consistency (e.g. CPB) • Dynamic interpretation, e.g. teleological or functional, see ERA,

pushing an interpretation ‘to its limits’ [See also Callebaut 2015]

Definitions - EU

Conclusion

For an organisms to fall under the EU definition of GMO, the use of certain techniques and a certain level of novelty of the resulting combinations of genetic material are required. E.g. small point mutations obtained through genome editing would not result in a GMO.

Definitions - EU

Views of others and other views:

• European Commission: – formulating views by taking into account reports of a WGNBT

(2012), EFSA, JRC and Legal Services – Confirmed in reply to MEPs, both process and product relevant

• D and SE authorities have formally concluded that certain

organisms developed through ODM and CRISPR are not GMOs • D, UK, IRL and have confirmed in a letter to the EC that the EU

GMO definition relies on both the technique and the resulting organism.

• Some NGOs adhere to a process based interpretation arguing that “these technologies are considered likely to bring negative impacts on human and/or environmental health”.

Definitions - EU

EU (2001): GMO means an organism in which the genetic material has been altered in a way that does not occur naturally; • genetic modification occurs at least through the use of:

– rDNA 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;

– fusion 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.

What does genome editing do?

René Custers

‘New’ modification technologies

1. Oligo-directed mutagenesis

2. ZFN and other SDNs

3. Cisgenesis/intragenesis

4. RdDM

5. Grafting

6. Reverse breeding

7. Agro-infiltration

Genome editing

A T

G C T

G A T

G C A

T G G A C T A

A T C

mutagenic oligonucleotide

Chemically differing from DNA

plant protoplast

Oligo-directed mutagenesis - ODM

Adapted from Keygene

Oligonucleotide lifetime in the cell

After mutagenesis

the oligo is degraded

and disappears from the cell

Source: Keygene

From single protoplast to plant

Source: Keygene

ODM compared to classical mutagenesis

Natural random

mutation

Induced random

mutagenesis

Directed

mutagenesis

UV light,

chemicals

Chemical treatment

or radiation

Oligo

treatment

1 100 5,000

4000 BC 20th century 21st century

Sou

rce:

Key

gen

e

Conclusions ODM

• Only in crops with established plant regeneration from protoplasts

• Current efficiency ≈ 2%

• Mutagenic oligonucleotide disappears within 48 hours

• Extremely specific

• Single point mutation introduced in a precise and directed manner, compared to dozens to thousands of random mutations in natural and classical mutagenesis

Site-Directed Nuclease technology (SDN)

For instance:

• Zink Finger Nucleases (ZFN)

• TAL Effector Nuclease (TALEn)

• CRISPR/Cas9

• (Meganuclease)

Zink Finger Nucleases

CRISPR/Cas9

A Double-strand break

Double-strand break repair

INDELs

Homologous gene with edit

Full (trans)gene addition

Double-strand break repair

INDELs

‘SDN-1’ ‘SDN-2’ ‘SDN-3’

SDN-1 generated INDELs

Generally between -20 to +20 bp

Off-target effects using CRISPR/Cas9

Craddick et al, Nucleic Acids Research, 2013

Off-target effects:

• May introduce one or a few additional INDELs

• Locus dependent and guide RNA sequence specific

• Smart guide RNA design can limit off-target effects

Cas9 nickase to enhance specificity

Ran et al, Cell, 2013

Can result in larger deletions (up to ≈50 bp)

Or multiple small deletions in one locus

SDN-2

SDN-2 not 100% efficient

Competition

SDN-2 generated edits

Any type of edit you want (already existing in nature or not):

• Point mutation ACGTCCGGCATTC -> ACGTCCTGCATTC

• Frameshift mutation ACGTCCGGCATTC -> ACGTCCTGGCATTC

• Stop codon insertion ACGTCCGGCATTC -> ACGTCCTAAGGCATTC

• Small deletion ACGTCCGGCATTC -> ACGT---GCATTC

• Small insertion ACGTCCGGCATTC -> ACGTCCTATCGGCATTC

• Etc, etc..

Reversal back to wild type using SDN-2

Donor DNA: wild-type gene

Cas9 DS break

mutation

SDN-2 can also achieve allele replacement

Donor DNA containing replacement allele

SDN-3: insertion of full genes

Double-strand break

Full gene added, either:

• Transgene

• Intragene

• Cisgene

To conclude: Genetic alterations achieved by genome editing (ODM and SDN-1 and -2)

1. Generation of already existing combinations of genetic material

o Allele replacement

o Introduction of already existing mutations

o Reversion of existing mutations back to wild-type

2. Introduction of small alterations

o Point mutations

o Frameshift mutations

o Introduction of stop codons

o Small deletions (1 to ≈50 bp)

o Larger deletions (>100 bp) (if two Cas9s are used to generate two DS breaks)

o Multiple small deletions in one and the same locus (nickase technology)

o Small insertions (up to 20 bp)

o Small INDELS in different loci (resulting from off-target effects)