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To Obtain Approval for Projects to Develop Genetically Modified Organisms in Containment

To Obtain Approval for Projects to Develop Genetically Modified Organisms in Containment

APPLICATION FORM Containment – GMO Project

To obtain approval for projects to develop genetically modified organisms in containment

Send to Environmental Protection Authority preferably by email ([email protected]) or alternatively by post (Private Bag 63002, Wellington 6140) Payment must accompany final application; see our fees and charges schedule for details.

Application Number

Date

www.epa.govt.nz 2

Application Form Approval for projects to develop genetically modified organisms in containment

Completing this application form

1. This form has been approved under section 42A of the Hazardous Substances and New Organisms (HSNO) Act 1996. It only covers projects for development (production, or regeneration) of genetically modified organisms in containment. This application form may be used to seek approvals for a range of new organisms, if the organisms are part of a defined project and meet the criteria for low risk modifications. Low risk genetic modification is defined in the HSNO (Low Risk Genetic Modification) Regulations: http://www.legislation.govt.nz/regulation/public/2003/0152/latest/DLM195215.html. 2. If you wish to make an application for another type of approval or for another use (such as an emergency, special emergency or release), a different form will have to be used. All forms are available on our website. 3. It is recommended that you contact an Advisor at the Environmental Protection Authority (EPA) as early in the application process as possible. An Advisor can assist you with any questions you have during the preparation of your application. 4. Unless otherwise indicated, all sections of this form must be completed for the application to be formally received and assessed. If a section is not relevant to your application, please provide a comprehensive explanation why this does not apply. If you choose not to provide the specific information, you will need to apply for a waiver under section 59(3)(a)(ii) of the HSNO Act. This can be done by completing the section on the last page of this form. 5. Any extra material that does not fit in the application form must be clearly labelled, cross- referenced, and included with the application form when it is submitted. 6. Please add extra rows/tables where needed. 7. You must sign the final form (the EPA will accept electronically signed forms) and pay the application fee (including GST) unless you are already an approved EPA customer. To be recognised by the EPA as an “approved customer”, you must have submitted more than one application per month over the preceding six months, and have no history of delay in making payments, at the time of presenting an application. 8. Information about application fees is available on the EPA website. 9. All application communications from the EPA will be provided electronically, unless you specifically request otherwise. Commercially sensitive information

10. Commercially sensitive information must be included in an appendix to this form and be identified as confidential. If you consider any information to be commercially sensitive, please show this in the relevant section of this form and cross reference to where that information is located in the confidential appendix. 11. Any information you supply to the EPA prior to formal lodgement of your application will not be publicly released. Following formal lodgement of your application any information in the body of this application form and any non-confidential appendices will become publicly available.

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Application Form Approval for projects to develop genetically modified organisms in containment

12. Once you have formally lodged your application with the EPA, any information you have supplied to the EPA about your application is subject to the Official Information Act 1982 (OIA). If a request is made for the release of information that you consider to be confidential, your view will be considered in a manner consistent with the OIA and with section 57 of the HSNO Act. You may be required to provide further justification for your claim of confidentiality. Definitions

Restricting an organism or substance to a secure location or facility to prevent Containment escape. In respect to genetically modified organisms, this includes field testing and large scale fermentation

Any obligation or restrictions imposed on any new organism, or any person in relation to any new organism, by the HSNO Act or any other Act or any Controls regulations, rules, codes, or other documents made in accordance with the provisions of the HSNO Act or any other Act for the purposes of controlling the adverse effects of that organism on people or the environment

Any organism in which any of the or other genetic material:  Have been modified by techniques, or Genetically Modified  Are inherited or otherwise derived, through any number of replications, from Organism (GMO) any genes or other genetic material which has been modified by in vitro techniques

A new organism is an organism that is any of the following:  An organism belonging to a species that was not present in New Zealand immediately before 29 July 1998;  An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar prescribed as a risk species, where that organism was not present in New Zealand at the time of promulgation of the relevant regulation;  An organism for which a containment approval has been given under the HSNO Act;  An organism for which a conditional release approval has been given under the HSNO Act; New Organism  A qualifying organism approved for release with controls under the HSNO Act;  A genetically modified organism;  An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been eradicated from New Zealand;  An organism present in New Zealand before 29 July 1998 in contravention of the Act 1967 or the Plants Act 1970. This does not apply to the organism known as rabbit haemorrhagic disease virus, or rabbit calicivirus A new organism does not cease to be a new organism because:  It is subject to a conditional release approval; or  It is a qualifying organism approved for release with controls; or  It is an incidentally imported new organism

An individual or collaborative endeavour that is planned to achieve a particular Project aim or research goal

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Application Form Approval for projects to develop genetically modified organisms in containment

1. Applicant details

1.1. Applicant

Company Name: (if applicable) The New Zealand Institute for Plant and Food Research

Contact Name: Georgina Dowd

Job Title: Scientist

Physical Address: 293-297 Akersten Street, Port Nelson, 7010

Postal Address (provide only if not the same as the physical):

Phone (office and/or mobile): 03 989 7596

Fax: 03 546 7049

Email: [email protected]

1.2. New Zealand agent or consultant (if applicable)

Company Name:

Contact Name:

Job Title:

Physical Address:

Postal Address (provide only if not the same as the physical):

Phone (office and/or mobile):

Fax:

Email:

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Application Form Approval for projects to develop genetically modified organisms in containment

2. Information about the application

2.1. Brief application description Approximately 30 words about what you are applying to do

Our work aims to use based immortalisation factors to create continuous lines from various tissues from Oncorhynchus tshawytscha (Chinook salmon).

2.2. Summary of application Provide a plain English, non-technical description of what you are applying to do and why you want to do it

The of mammalian cells in (in vitro) has been used for decades as an important tool for creating an understanding the of cells. Immortalised cell lines, those that continually replicate, have been used for manufacturing , generating biological products such as and and have led to a deeper fundamental understanding of how complex cellular processes occur. Immortalised cells are an extremely useful research tool in that continual sampling from live organisms does not need to happen on a regular basis and the cells can be fully characterised including confirming the species they originated from, their optimal growth conditions and, optimal long and short term storage conditions.

While mammalian has been explored extensively, marine cell culture has been described as enormously neglected. American Type Culture Collection (ATCC), the largest bioresource in the world, currently offers over 2,500 mammalian cell lines. There are just 7 cell lines of fish origin available and each of these originate from freshwater species. Marine cell of NZ-relevant species are almost totally unexplored.

The applications for fish cell lines are numerous including:  Toxicology: the effects of toxicants on individual organisms  Ecotoxicology: the impact of ecotoxicants on ecosystems  Disease control e.g. orthomyxovirus, the causative agent of infectious salmon anemia, Neoparamoeba perurans, the causative agent of amoebic gill disease.  Tissue function including genetic regulation and expression, immunology, and endocrinology.  : technology, artificial gut models for aquaculture feed optimisation We want to develop a platform of fish cell lines from various tissues of Chinook salmon (Oncorhynchus tshawytscha) – one of New Zealands important aquaculture species. While fish cells have been shown to spontaneously immortalise, we wish to explore synthetic means of immortalising the cells by introducing plasmid-based immortalisation factors such as proto-oncogenes, inducers and growth factors. This will allow generation of continuous cell lines of marine origin for use as a contained and sustained research tool.

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Application Form Approval for projects to develop genetically modified organisms in containment

2.3. Technical description

Briefly describe the host organism(s) and the proposed genetic modifications. Please make sure that any technical words used are included in a glossary. Note if any part of this research project is already covered by an existing HSNO Act approval that your organisation holds or uses.

A number of encoding genes of teleosts have be created by synthesis and subcloned into pcDNA3.1-Hyg. These are:

 ssMax  ssNanog  ssHras  ssOct34  ssSox2  drTGFalpha  drEGF (ss = Salmo salar and dr = Danio rerio)

To maintain and propagate each construct, approximately 10ng of the vector will be used to transform a recombination deficient (recA), endonuclease A deficient (endA) E. coli strain. We will use commercially available chemically competent TOP10, TOP10F’ or DH5α strains. Transformants will be selected on LB plates containing an appropriate amount of ampicillin. For long term storage, glycerol stocks of each plasmid containing E. coli strain will be prepared.

Plasmid DNA for into fish cells will be prepared using a commercially available plasmid prep kit (e.g. Purelink Miniprep Kit). Delivery of constructs to cells will be via lipid-mediated transfection reagents such as 2000 or Lipofectamine Stem Reagent. Successful transfection will be monitored via expression of GFP encoded on the construct.

For creation of stable cell lines, the constructs will firstly be linearised to decrease the likelihood of the vector integrating into the genome in a way that disrupts the gene of interest. Selection of stable transformants will be achieved through addition of hygromycin.

3. Information about the new organism(s)

3.1. Identity of the host organism(s)

For each host organism:  Provide its taxonomic name and describe what type of organism it is.  Provide a description of the strain(s) being applied for (if relevant).  If the host organism is derived from humans (eg, cell lines) or may have cultural significance (e.g. sourced from native biota), provide details of its source.  State the category (Category 1 or Category 2) of the host organism (as per the HSNO (Low Risk Genetic Modification) Regulations).

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Application Form Approval for projects to develop genetically modified organisms in containment

Primary cultures will be established from the following finfish species and immortalised via plasmid- based factors :

o Chinook salmon Oncorhynchus tshawytscha Category 1 host organism

Escherichia coli (Category 1 host organism) will be used as vector for amplifying the plasmid constructs.

Chinook salmon and the E. coli strain will be sourced from commercial suppliers.

3.2. Regulatory status of the organism

Is the organism that is the subject of this application also the subject of:

An innovative application as defined in section 23A of the Act 1981?

☐ Yes ☒ No

An innovative agricultural compound application as defined in Part 6 of the Agricultural Compounds and Veterinary Medicines Act 1997?

☐ Yes ☒ No

4. Information about the project

4.1. Describe the nature and range of the proposed genetic modifications

 Describe the nature and range of the proposed genetic modifications (e.g. the range of elements that the vectors or gene constructs may contain, and the type, source and function of the donor genetic material).  State the category (Category A or Category B) of the genetic modifications (as per the HSNO (Low Risk Genetic Modification) Regulations).

Each of the constructs described here has been created by Dr Bertrand Collet (French National Institute for Agricultural Research) and Dr Catherine Collins (Marine Scotland Science).

 Construct 1: pcDNA3.1-Hyg.ssMax encodes the transcriptional regulator gene, Max, of Salmo salar (Atlantic salmon). This gene is expressed at relatively constant levels up to the embryonic stage of with particularly high expression in the , gills and uterus. It is also expressed in the brain and .

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Application Form Approval for projects to develop genetically modified organisms in containment

 Construct 2: pcDNA3.1-Hyg.ssNanog encodes a DNA binding homeobox factor, nanog, of Atlantic salmon. Nanog is involved in proliferation, renewal and pluripotency. It is expressed in 22 organs with the highest expression level in blastula.  Construct 3: pcDNA3.1-Hyg.ssHras encodes the Atlantic salmon proto-oncogene and GTPase hras. Defects in this gene are implicated in a variety of cancers.  Construct 4: pcDNA3.1-Hyg.ssOct34 encodes the octamer transcription factor-3/4 of Atlantic salmon. This factor is specifically expressed in cells with pluripotent capacity (e.g. embryonic stem cells).  Construct 5: pcDNA3.1-Hyg.ssSox2 encodes the transcriptional activator, sox2, of Atlantic salmon. The product of this gene is required for stem-cell maintenance.  Construct 6: pcDNA3.1-Hyg.drTGFalpha encodes for the transforming growth factor alpha of Danio rerio (zebrafish). The receptor of this growth factor, EGFR, activates signalling pathways responsible for cell proliferation, differentiation and development.  Construct 7: pcDNA3.1-Hyg.drEGF encodes for epidermal growth factor (EGF) of zebrafish. The product of this gene is a potent mitogenic factor that plays an important role in the growth, proliferation and differentiation of numerous cell types.

The proposed modifications are Category A genetic modifications. They

1. Involve category 1 host organisms (Chinook salmon cells, E. coli Top10, Top10F’, DH5α). The hosts are identifiable, and classifiable according to the genus, species and strain. The whole organism or cells thereof are not known to normally cause disease in humans, animals, plants or fungi.

2. Are carried out under a minimum of PC1 containment (Note; the proposed work will be carried out under PC2 containment).

3. Do not increase the pathogenicity, virulence , or infectivity of the host organism to the personnel, the community, or the environment

4. Do not result in the genetically modified organism having a greater ability to escape from containment than the unmodified host organism.

4.2. Proposed containment of the new organism(s) (physical and operational)

 State which Containment Standard(s) your facility is approved to.  State the minimum containment level (PC1 or PC2 as per AS/NZS2243.3:2002) required to contain the GMOs (as per the HSNO (Low Risk Genetic Modification) Regulations).  Discuss whether controls in addition to the requirements listed in the Standard(s) are necessary to adequately contain the GMOs.

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Application Form Approval for projects to develop genetically modified organisms in containment

The proposed work will be carried out in the MPI-approved PC2 laboratory (Facility Number 29779) at Plant and Food Research’s Nelson site. This lab has been approved as a containment and transitional facility in accordance with MPI Standard 154.02.17: Transitional Facilities for Biological Products and MPI and EPA Standard 154.03.02: Facilities for and Cell Cultures 2007a.

The minimum containment level required for this work is PC1 but will be carried out in a PC2-certified laboratory.

Additional controls are not required in this instance.

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Application Form Approval for projects to develop genetically modified organisms in containment

5. Risks, costs and benefits

Provide information of the risks, costs and benefits of the GMOs in the following areas of impact:  The environment.  Human health and safety.  The economy (e.g. the ability of people and communities to provide for their economic wellbeing).  The relationship of Māori and their culture and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna and other taonga, and the principles of the Treaty of Waitangi (The details of any engagement or consultations with Māori that you have undertaken in relation to this application should be discussed here).  Society and community.  New Zealand’s international obligations.

We do not envisage any risk associated with the proposed work. The genetically modified cells would be unable to survive without human intervention, and are not a risk of transmissible disease.

The benefits of immortalised cell lines from Chinook salmon are numerous. As the species is an important export commodity for New Zealand, having a tool which can be used as part of an integrated health management strategy may prove to be invaluable to the industry. Cells lines can be used as a method of monitoring water quality which may have implications for the wider New Zealand community.

We have discussed the applications and possible uses of fish cell lines with Te Tau Ihu Fisheries Forum and have received positive feedback on the work. Should this proposal result in successful generation of immortal cell lines, the possibility of immortalising cells of taonga species will be discussed further.

6. Other information

Add here any further information you wish to include in this application including if there are any ethical considerations that you are aware of in relation to your application.

By generating cell lines of this nature, we hope to reduce the need for using whole animals in testing models. As the trend towards in vitro models continues, we hope to generate cell lines which are useful and relevant to New Zealand.

Animal ethics approval for this work have been granted by the MPI-approved Nelson Marlborough Institute of Technology Ethics Committee under the Good Practice Guide for the use of animals in research, testing and teaching (Protocol Number AEC2019-PFR-01).

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Application Form Approval for projects to develop genetically modified organisms in containment

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Application Form Approval for projects to develop genetically modified organisms in containment

7. Checklist This checklist is to be completed by the applicant

Application Comments/justifications All sections of the application form completed ☒ Yes ☐ No or you have requested an information waiver (If No, please discuss with an under section 59 of the HSNO Act Advisor to enable your application to be further processed)

Confidential data as part of a separate, ☐ Yes ☒ No identified appendix

Supplementary optional information attached:

 Copies of additional references ☐ Yes ☒ No

 Relevant correspondence ☐ Yes ☒ No

Administration Are you an approved EPA customer? ☐ Yes ☐ No If Yes are you an: Applicant: ☐ Agent: ☐

If you are not an approved customer, payment of fee will be by:  Direct credit made to the EPA bank ☐ Yes ☐ No account (preferred method of payment) ☐ Payment to follow Date of direct credit:

 Cheque for application fee enclosed ☐ Yes ☐ No ☐ Payment to follow

Electronic, signed copy of application e- ☐ Yes mailed to the EPA

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Application Form Approval for projects to develop genetically modified organisms in containment

Signature of applicant or person authorised to sign on behalf of applicant

☒ I am making this application, or am authorised to sign on behalf of the applicant or applicant organisation.

☒ I have completed this application to the best of my ability and, as far as I am aware, the information I have provided in this application form is correct.

16th December 2019

Signature Date

Request for information waiver under section 59 of the HSNO Act

I request for the Authority to waive any legislative information requirements (i.e. concerning ☐ the information that has been supplied in my application) that my application does not meet (tick if applicable).

Please list below which section(s) of this form are relevant to the information waiver request:

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Application Form Approval for projects to develop genetically modified organisms in containment

Appendices and referenced material (if any) and glossary (if required)

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