Food derived using new technologies: lessons learned from GM food safety assessment Janet Gorst on behalf of Lisa Kelly Risk Assessment – Biological Sciences

Outline Overview of GM food safety assessment Emergence of new technologies/new breeding techniques for modifying genomes The current problem and the FSANZ review Genome editing as an example of issues and questions

Standard 1.5.2 – Food produced using gene technology Establishes a pre-market assessment and approval system for GM foods & imposes mandatory food labelling requirements

Key definitions food produced using gene technology means a food which has been derived or developed from an organism which has been modified by gene technology gene technology means recombinant DNA techniques that alter the heritable genetic material of living cells or organisms

No presumption of safety, formal risk assessment applied Current food regulation is based on process Conventional foods have presumption of safety based on history of safe use Conventional breeding Traditional cross breeding & selection Mutation breeding (plants) Cell culture techniques Gene Technology Recombinant DNA techniques Transgenesis No presumption of safety, formal risk assessment applied No pre-market approval Pre-market approval

GM food: main health & safety concerns Expression or production of new substances as a consequence of the genetic modification new proteins new non-protein substances (e.g. interfering RNAs, novel herbicide metabolites) Unintended changes arising from the insertion of new genetic material in the genome (insertional mutagenesis) Significant (intended) changes to the nutrient composition of foods

GM food safety assessment FSANZ approach consistent with Codex Principles and Guidelines Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants (CAC/GL 45-2003)

Safety assessment approach

Key elements

Safety assessment outcomes FSANZ has assessed more than 70 GM foods Extensive experience and familiarity with foods derived from GM crops A large evidence-base exists No differences have been identified that raise any legitimate food safety concerns Initial (primarily theoretical) concerns about safety particularly in relation to unintended changes are so far unfounded

Outline Overview of GM food safety assessment Emergence of new technologies/new breeding techniques for modifying genomes The current problem and the FSANZ review Genome editing as an example of issues and questions

Emergence of new breeding techniques When the standard for GM foods was first developed, definitions were designed to essentially capture a single technique transgenesis, where (foreign/new) DNA from another species is inserted The new techniques are a game changer - many do not conform to the transgenesis or “GM” stereotype

Examples Genome editing Site-directed nucleases (ZFN, TALENS, CRISPR/Cas9) Oligo-directed mutagenesis Cisgenesis/Intragenesis GM Rootstock Grafting Agro-infiltration (including non-germline and germline tissue) Transient transgenics Accelerated breeding following early flowering Reverse Breeding Hybrid Seed Production Technology (SPT)

Outline Overview of GM food safety assessment Emergence of new technologies/new breeding techniques for modifying genomes The current problem and the FSANZ review Genome editing as an example of issues and questions

The problem Many of the newer techniques do not fit neatly into the current definitions for gene technology Generates uncertainty about whether foods derived from these techniques currently come within the scope of the GM food standard Because of their similarity to conventionally-bred foods, there is debate about whether foods derived using genome editing (in particular) should be subject to pre-market approval Some argue the risk from these foods is no different to that of conventionally-bred foods, therefore pre-market scrutiny is not warranted

FSANZ Review FSANZ commenced a review in June 2017 to consider http://www.foodstandards.gov.au/consumer/gmfood/Pages/Review-of-new-breeding-technologies-.aspx FSANZ commenced a review in June 2017 to consider the extent to which food derived from various NBTs should be captured for pre-market approval under Standard 1.5.2, and whether the definitions for ‘food produced using gene technology’ and ‘gene technology’ should be changed to improve clarity about which foods require pre-market approval An Expert Advisory Group on New Breeding Techniques (EAG NBT) has been established to assist with the review There is no intent to review any other aspect of the standard or our approach to foods unambiguously captured by the standard

Review Process First Meeting with EAG  Second meeting with EAG Targeted consultation with key stakeholders Second meeting with EAG Public consultation on issues paper Final review report Aug – Oct 17 Early 2018 Jun 17 Dec 17 Jun 18

Outline Overview of GM food safety assessment Emergence of new technologies/new breeding techniques for modifying genomes The current problem and the FSANZ review Genome editing as an example of issues and questions

Genome editing Most attention in the debate has focused on the genome editing techniques These techniques can be applied in a variety of ways to produce different types of outcomes Some derived food products may be similar to, or indistinguishable from, those that may be produced through conventional breeding There are two main approaches to facilitate genome editing - Use of site-directed nucleases - Oligonucleotide-directed mutagenesis

Site directed nucleases (SDNs) – 3 classes There are two key components to each SDN: A DNA recognition code (protein-based or RNA) that finds a specific site in the genome A nuclease (protein) that cuts the double stranded DNA at that specific site These components can be added to the host : directly as the proteins or RNA via a transgenic step If by transgenesis, once the component genes have done their job, the idea is that they are segregated away during subsequent breeding

Site directed nucleases (SDNs) There are three ways in which each SDN technique can be used

Genome editing Most attention in the debate has focused on the genome editing techniques These techniques can be applied in a variety of ways to produce different types of outcomes Some derived food products may be similar to, or indistinguishable from, those that may be produced through conventional breeding There are two main approaches to facilitate genome editing - Use of site-directed nucleases - Oligonucleotide-directed mutagenesis

Oligonucleotide-directed mutagenesis (ODM) The chemically synthesised oligonucleotide (20 – 100 bp) has homology with the target gene except for the nucleotide(s) to be changed. Oligonucleotide Mismatch oligonucleotides are added directly and hence do not involve a transgenic stage oligonucleotides themselves are not “heritable material“ oligonucleotides act like a mutagen, but are sequence specific - the obtained mutations cannot be distinguished from spontaneous mutations (natural variation) or mutations induced by classical mutagenesis Modified from Songstad et al (2017) Critical Reviews in Plant Sciences

Changes from genome editing mainly achieved using site-directed nuclease techniques equivalent to transgenesis - food from transgenic organisms already subject to pre-market assessment and approval may involve stable insertion of the nuclease gene which would be segregated away in final breeding steps (null segregant) – no new DNA or proteins in final food producing line safety concerns would primarily arise from expression of new proteins or other substances in a food (potential allergenicity/toxicity), but also potential for off-targeting/unintended changes DNA insertions safety concerns would primarily arise from potential for off-targeting/unintended changes Deletions small nucleotide changes typically but could be more extensive can be achieved using site-directed nuclease techniques as well as oligo-directed mutagenesis oligo-directed mutagenesis does not involve a gene insertion step safety concerns would primarily arise from altered protein structure/function, altered expression of existing gene products, as well as potential for off-targeting/unintended effects Nucleotide changes

Questions generated by genome editing Do all food products derived from genome edited organisms warrant pre-market assessment and approval? What have we learned from 20 years of GM food safety assessment, and can this be applied to the current problem? Can the presumption of safety applied to conventional foods also be applied to foods from genome edited organisms? How do the genome-editing changes compare to those arising from conventional methods? Are off-target effects different (nature and frequency)?

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