Developing genetically modified organisms in containment

ER-AF-N03-4 09/09

BP House

20 Customhouse Quay

PO Box 131, Wellington

Phone: 04-916 2426 Fax: 04-914 0433

Email: [email protected]

Web: www.ermannz.govt.nz

Application for

Developing genetically modified organisms in containment Under section 40(1)(b) of the HSNO Act 1996 (excluding rapid assessment)

of 28

Please note

This application form covers the development of genetically modified organisms that:

1. Do not meet Category A and/or B experiments as defined in the HSNO (Low-

Risk Genetic Modification) Regulations 2003;

2. Occur either in a containment structure (i.e. laboratory) or outdoors within a

containment facility; or

3. Otherwise cannot undergo a rapid assessment for low-risk genetic

modification.

Any extra material that does not fit in the application form must be clearly labelled,

cross-referenced, and included as appendices to the application form.

Commercially sensitive information must be collated in a separate appendix. You

should justify why you consider the material commercially sensitive, and make sure it

is clearly labelled as such.

If technical terms are used in the application form, simply explain these terms in the

Glossary (Section 8 of this application form).

Unless otherwise indicated, all sections of this form must be completed for the

application to progress.

Applicants must sign the application form and enclose the correct application fee

(including GST). The application fee can be found in our published Schedule of Fees

and Charges on the ERMA New Zealand website. We are unable to process

applications that do not contain the correct application fee.

An electronic and paper copy of the final completed form must be submitted.

If you have any queries regarding the information required or would like to discuss

your draft application form, please contact a New Organisms Advisor at ERMA New

Zealand.

This form was approved by the Chief Executive of

ERMA New Zealand on 22 September 2009. This form replaces all previous versions.

of 28

Section 1: Application details

a) Application title

The use of viral vectors and viruses to deliver genes to study their functions in cellular and

tissue physiology.

b) Organisation name

University of Otago

University of Auckland

c) Postal Address

Professor Iain Lamont

Department of Biochemistry

University of Otago

P O Box 56

Dunedin

of 28

Section 2: Summary of application

a) Provide a plain English summary of this application including:

Explain the purpose of your research in the context of your organisation’s history and goals.

The purpose of the application (e.g. what is the research you wish to perform and why do you consider that it is important? what are the benefits of this research?).

If there are any alternative methods to achieve the aims of this research, explain why you wish to perform the research this way.

Describe the project you wish to undertake (section 40(2)(a)(ii) of the HSNO Act).

Are you aware of any possible adverse effects of the organism on the environment? If so, any potential mitigation measures?

Where do you intend to conduct these activities? Are there specific location(s) or are you seeking approval for all of New Zealand?

How do other legislative requirements apply to your proposed activities? (e.g. the Resource Management Act, the Medicines Act.

If this application is for a development outdoors within a containment facility, discuss why your activities are not “field testing” activities for the purpose of the HSNO Act.

If technical terms are used here or elsewhere in the application, add simple explanations for these terms in the Glossary (Section 8 of this application form).

The Universities of Otago and Auckland are research-intensive environments and the

experimentation proposed here contributes to research contracted through local, national and

international funding bodies.

The application seeks approval to enable research into the biology of viruses to be carried out

under appropriate physical containment. Viruses are important pathogens that are responsible for

a range of diseases in humans and animals. The research proposed herein is designed to provide

new knowledge about the biology of virus infection and the link between infection and disease

pertinent to a range of different viruses.

In this proposed research, genetically modified viruses or viral vectors belonging to or derived

from the families of Adenoviridae, Parvoviridae, Baculoviridae, Poxviridae, Papillomaviridae,

Hepadnaviridae and Retroviridae will be used either to study the viral lifecycle or to deliver

genes of interest to cells and animals. Expression or knockdown of target proteins will be studied

in single cells, in an artificial skin culture or in laboratory animals. In vivo experiments will

involve the expression of foreign genes or recombinant viruses in animal models. This research

will be carried out at the Universities of Otago and Auckland in PC2 containment.

Genetically modified organisms will be used here firstly because it is not appropriate to carry out

experiments in humans and secondly, because they provide experimental systems that can be

readily manipulated in a controlled setting. Thus, this application will help us to understand how

viruses and alterations in cellular and tissue physiology contribute to diseases of animals and

humans. This information may lead to the development of therapies, thereby improving animal

and human health and reducing disease-associated costs.

b) Provide a short summary statement of the purpose of this application to be used on ERMA New Zealand’s public register

This statement must be a maximum of 255 characters including spaces and punctuation. If native or human genetic material directly obtained from New Zealanders is to be used, include this information here. Sufficient details must be provided to enable the Authority to provide the information required in the register under section 20(2)(c) of the HSNO Act.

of 28

To genetically modify E.coli, yeast, viruses, cell lines and laboratory animals to study the effects

of viral and cellular sequences and proteins.

of 28

Section 3: The proposed organism(s) to be developed

Section 2(1) of the HSNO Act defines what “identification” is. You must provide sufficient information to fulfill the criteria listed in the HSNO Act to enable the Authority to uniquely identify the organism in the register (as required in section 20(2)(b) of the HSNO Act).

As per sections 40(2)(a)(i)-(iv) of the HSNO Act, you must: Identify the new organism(s) (at the appropriate taxonomic level). Hint — you could start by discussing the characteristics of

the host organism and then how the proposed genetic modifications are expected to alter these characteristics.

Describe the project and the experimental procedures to be used.

Provide details of biological material to be used.

Provide details of the expression of foreign nucleic acid (if relevant).

You must describe the biological characteristics of the new organism(s). The information should be relevant to: The hazardous nature of the organism(s) that you are aware of. For example, is it a bacterium that can cause disease in

plants or humans? Will the modifications enhance the pathogenicity of a microorganism?

Which of its characteristics may enable it to escape from containment? For example, can it produce air-disseminated spores? Can it dig under fences? Can it jump or fly over high fences?

The ability of the organism(s) to form an undesirable self-sustaining population and how easy such a population could be eradicated (section 43(b) of the HSNO Act).

Identification of the host organism

Organisms to be developed:

a. E. coli

b. Baculovirus

c. Poxvirus

d. Papillomavirus

e. Adenovirus

f. Hepatitis B virus

g. Insect cell lines

h. Yeast

i. Mammalian cell lines

j. Whole animals: Rabbit, rat, mouse

This list does not refer to replication-deficient viruses that will be generated in this

research. In accordance with accepted definitions, replication-deficient viruses derived

from Retroviruses, Parvoviruses, Adenoviruses and Papillomavirus are classified here as

vectors and not organisms due to their inability to replicate.

(a) Latin binomial, including full taxonomic authority:

Escherichia coli (Migula, 1895; Castellani and Chalmers, 1919)

Common name(s), if any:

E. coli

Type of organism (eg bacterium, virus, fungus, plant, animal, animal cell):

Bacterium

Taxonomic family:

Enterobacteriaceae

Strain(s) and genotype(s), if relevant:

Genetically crippled derivatives of Escherichia coli K12 and strain B

Other information, including presence of any inseparable or associated organisms, and whether a

prohibited organism is involved:

There are no known inseparable or associated organisms and a prohibited organism is not

involved.

of 28

(b) Latin binomial, including full taxonomic authority:

Autographa californica nucleopolyhedrovirus (AcMNPV)

(ICTVdB - The Universal Virus Database, version 4.

http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/)


Baculovirus


Virus

Taxonomic family:

Baculoviridae


Polyhedrin negative strains such as Baculogold and bac-to-bac not normally able to infect insects.




involved.

(c) Latin binomial, including full taxonomic authority:

Poxviridae




Poxvirus


Virus

Taxonomic family:

Poxviridae


All poxvirus types including risk group II types




involved.

(d) Latin binomial, including full taxonomic authority:

Papillomaviridae




Papillomavirus


Virus

Taxonomic family:

Papillomaviridae


All papillomavirus genotypes



http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/index.htm



of 28


involved.

(e) Latin binomial, including full taxonomic authority:

Adenoviridae




Adenovirus


Virus

Taxonomic family:

Adenoviridae


All serotypes from the Genera Mastadenovirus and Atadenovirus




involved.

(f) Latin binomial, including full taxonomic authority:

Hepadnaviridae




Hepatitis B virus


Virus

Taxonomic family:

Hepadnaviridae


All serotypes




involved.

(g) Latin binomial, including full taxonomic authority:

Spodoptera frugiperda (Smith & Abbot 1797),

Drosophila melanogaster (Meigen, 1830),

Trichoplusia ni (Hubner 1802)


Fall armyworm, fruit fly, cabbage looper


Insect cell lines

Taxonomic family:

Noctuidae, Drosophilidae,




of 28

N/A




involved.

(h) Latin binomial, including full taxonomic authority:

Saccharomyces cerevisiae (Meyen ex E.C. Hansen 1883)


Baker’s yeast


Fungus

Taxonomic family:

Sacchamycetaceae


All laboratory strains such as Y2805, YPH500, HF7




involved.

(i) Latin binomial, including full taxonomic authority:

Mammalian cell lines

Cell lines and primary cultures to include epithelial or fibroblasts originating from:

Mus musculus (Linnaeus, 1758)

Mus spretus (Lataste, 1883)

Rattus rattus (Linnaeus, 1758)

Rattus norvegicus (Berkenhout, 1759)

Homo sapiens (Linnaeus, 1758);

Chlorocebus aethiops (Linnaeus, 1758)

Ovis aries (Linnaeus, 1758)

Bos taurus (Linnaeus, 1758)

Canis familiaris (Linnaeus, 1758)

Oryctolagus cuniculus (Linnaeus, 1758)

Sylvilagus sp (Gray, 1867)

Cricetulus griseus (Milne-Edwards, 1867)

Cricetus cricetus (Linnaeus, 1758)

Cavia porcellus (Linnaeus, 1758)


House mouse, Algerian mouse, black rat, brown rat, human, monkey, sheep, cattle, dog, European

rabbit, cottontail rabbit, Chinese hamster, black-bellied hamster, guinea pig


Animal cells

Taxonomic family:

Muridae, hominidae, cercopithecidae, leporidae, bovidae, canidae, cricetidae, caviidae


N/A


of 28



involved.

Ethical permission will be obtained (human/animal) before primary cells are harvested from

animals or humans.

(j) Latin binomial, including full taxonomic authority:

Mus musculus (Linnaeus, 1758)

Rattus rattus (Linnaeus, 1758)

Oryctolagus cuniculus (Linnaeus, 1758)


Mouse, rat, European rabbit


Animal

Taxonomic family:

Muridae, leporidae, phalangeridae, caviidae, bovidae, cervidae


Laboratory strains of animals, including transgenic animals




involved.

HOW WILL THE NEW ORGANISMS BE DEVELOPED?

1. Production of Replication Defective Viral Particles for Gene Delivery

(a) Production of replication defective recombinant retroviruses:

The self-inactivating (SIN) onco-retroviral vectors used will be derived from the Moloney murine

leukemia virus (MMLV). The parent strain is Moloney murine leukemia virus (MoMuLV),

species: Murine leukemia virus (MuLV), Genus: Gammaretrovirus, Family: Retroviridae.

Transduction vectors will comprise long terminal repeats (LTRs) and packaging sequence from

MMLV (required for packaging of the expression plasmid into recombinant retroviral particles),

the 3‟LTR containing a deletion which results in self-inactivation of the 5‟ LTR following

integration. The viral packaging and envelope proteins will be expressed from accessory plasmids,

co-transfected into HEK 293T cells. Lack of

genes from being included in the packaged virion. Depending on the experimental model to which

they are to be applied, the SIN vectors will be either ecotropic (using plasmid vector pEco),

amphotropic (pAmpho), or pseudotyped by replacing the MMLV envelope gene (env) with a

pantropic envelope.

Lentiviruses have the ability to integrate into non-dividing cells with high efficiency which

makes them useful as the basis for a vector system (Blomer et al., 1997). The lentiviral vector

system (Dull et al., 1998) that we propose to use is derived from the parent species: Human

immunodeficiency virus type 1 (HIV-1), Genus: Lentivirus, Family: Retroviridae. In addition

to the LTRs, genes from HIV-1 are used (gag, pol and rev). This transducing expression vector

into which the candidate gene coding sequence is inserted, contains modified HIV 5‟ and 3‟

LTRs, Rous sarcoma virus enhancer/ promoter for production of viral RNA in producer cells,

the packaging sequence, and antibiotic resistance genes for selection in E. coli and

mammalian cells. The gene of interest is expressed under the control of a heterologous

of 28

promoter. The viral regulatory, packaging and nuclear export genes are supplied on separate

plasmids, which are co-transfected into producer cells.

Retroviral transduction results in integration of viral and transgene sequences into the host cell

genome. Stable transduction is suitable for medium to long-term experiments.

(b) Production of replication defective papillomavirus particles:

Mammalian cell transfection system for generation of papillomavirus pseudovirions will be used.

The method utilises transfection of the cell line, 293TT, constructed to express high levels of SV40

large T antigen. The cells are co-transfected with codon-modified papillomavirus capsid genes

(L1 and L2 or L1 alone) along with a plasmid containing the SV40 origin of replication and the

gene of interest. Encapsidation within the capsid is sequence independent and plasmids are

packaged efficiently, provided they are 8 kb or less in size. Non-infectious viral-like particles

(VLPs) containing DNA encoding the gene of interest are produced post-transfection of 293TT

cells. Purification of the VLPs is achieved by utilising standard laboratory techniques such as

Optiprep (iodixanol) density gradient ultracentrifugation.

(c) Production of replication defective recombinant adenoviruses:

We propose to use adenoviral vectors, derived from the Genus: Mastadenovirus, Family:

Adenoviridae. These vectors have been modified to be replication incompetent (Campos and

Barry, 2007). For example, recombinant pAd-DEST vectors supplied with the Invitrogen

ViraPowerTM

adenoviral expression system (Appendix I A) have deletions in the early

transcriptional units (E1 and E3) and contains 28 kb of the 36 kb wild type adenoviral genome.

The vector comprises 5‟ and 3‟ inverted terminal repeats (ITRs), encapsidation signal and

adenoviral late genes. The E1 gene products that are essential for expression of viral late genes are

supplied from the HEK 293A producer cells, which have E1 incorporated into their genome. This

system allows the production of infectious adenoviral particles that are incapable of further

replication (because they lack E1). Recombination sites or multiple restriction sites permit

cloning of the gene of interest from an entry vector into the expression vector. The expression of

the gene of interest is under control of the human cytomegalovirus (CMV) promoter. Adenoviral

vectors enter target cells by binding the CAR, are internalised by integrin-mediated endocytosis,

and transported to the nucleus (Campos and Barry, 2007). Since the virus does not integrate into

the host genome, transcription of the transgene is transient. We anticipate maximal expression

over a period of days to weeks following transduction in our experimental models.

(d) Production of replication defective recombinant adeno-associated adenovirus:

Recombinant adeno-associated virus (rAAV) is a replication deficient virus derived from the

parental virus adeno-associated virus (AAV). To date 12 serotypes and several clones of AAV

have been described and all have been used for the generation of rAAV. rAAV contains a

single stranded DNA genome of ~4.5kbp. The only viral sequence from the parental strain that

is present in rAAV is the inverted terminal repeats (ITRs). The transgene expression cassette is

inserted between the ITRs. In some cells second strand synthesis of the viral genome is a rate-

limiting step in transduction. A “self-complementary” form of rAAV (scAAV) has been

developed by deleting the terminal resolution sequence in one ITR; scAAV has shown

significantly higher rates of transduction in some cells. We propose to use both single stranded

rAAV and scAAV of any of the described serotypes or clones to deliver transgene cassettes.

rAAV is produced in 293 cells by transient transfection. The vector plasmid contains the

transgene cassette inserted between the ITRs of AAV. The transgene cassette contains the

gene of interest driven by a promoter, such as CMV or a tissue-specific promoter, and a

polyadenylation signal. The non-structural (rep) and structural (cap) genes of AAV are

provided in trans on a separate “packaging” plasmid; this plasmid contains no homologous

of 28

sequence with the vector plasmid to prevent homologous recombination. The required helper

functions for rAAV production are also provided in trans, usually on a third plasmid (“helper”

plasmid) but are sometimes combined with the packaging plasmid. The required helper

functions are E1, E2, E4, and VAI from adenovirus; the 293 cell line contains the E1 gene

integrated in its genome. Virus is harvested 48 to 72 hours after transfection by harvesting the

cell monolayer +/- the media. The crude viral preparation is further purified prior to use by

various methods. A baculovirus expression system (described below) has also been used to

produce large amounts of rAAV and may be developed in future.

2. Production of Replication Competent Viruses

(a) Production of replication competent recombinant baculovirus:

Recombinant baculoviruses derived from Autographa californica nucleopolyhedrovirus

(AcMNPV) will be used to express heterologous genes in mammalian cells. Baculovirus

expression vector systems have the capacity for insertion of large DNA fragments and produce

a high yield of recombinant protein. Expression in mammalian cells will be achieved by using

vectors where the insect promoter has been replaced with promoters active in mammalian cells.

Baculovirus is incapable of replication in mammalian cells but has been shown to transduce

cells at high efficiency.

Baculovirus plasmids that contain foreign proteins expressed under the control of heterologous

promoters that are capable of operating in mammalian cells will be used. Baculovirus transfer

vectors, such as pAcUW51-GUS, which contain the polyhedrin promoter controlled GUS

marker gene aligned back to back with the mammalian cell compatible promoter will be used

to transfer the DNA into the AcMNPV genome. For the construction of recombinant

baculovirus, linearised AcMNPV DNA and the transfer vector will be co-transfected into

Spodoptera frugiperda insect cells. Media from the transfected cells will be collected and used

to infect insect cells to expand the recombinant virus.

(b) Production of replication competent recombinant papillomavirus:

Several methods for in vitro production of papillomavirus virions or pseudovirions have been

reported. They include production in yeast system, in keratinocytes organotypic raft culture, in

papillomavirus genomic DNA transfected cells and in cultured monolayers of mammalian cells

after infection with recombinant viruses such as vaccinia expressing L1 and L2 (Roden et al.,

1996; Unckell et al., 1997; Touze and Coursaget, 1998).

(i) Yeast system: The generation of papillomavirus pseudovirions in the yeast system is commonly

used in laboratories to study the biological functions of papillomaviruses. We propose to use

Saccharomyces cerevisiae (yeast) to generate papillomavirus pseudovirions for transduction of

animals.

The propagation of papillomavirus pseudovirions in an in vitro yeast system involves the co-

transformation of 3 individual yeast plasmids carrying genetic information for papillomavirus

replication, amplification and encapsidation. These include firstly, a yeast plasmid containing

target papillomavirus full-length genome (which may include mutants and derivatives thereof) that

is capable of replicating in yeast. Secondly, a yeast plasmid carrying the target papillomavirus E2

ORF, which promotes amplification by acting as a copy number enhancer and also enhances

packaging by interacting with the L2 minor capsid protein, when present. Thirdly, a yeast plasmid

expressing the L1, or L1 and L2 capsid proteins, which are associated with encapsidation of the

target papillomavirus genome.

In general, the construction of papillomavirus-genome and ORFs vectors for yeast is to insert a

yeast nutritional marker such as Ura3, Trp1, Leu2, or His3 located within the papillomavirus

of 28

genome sequence. The insertion of the nutritional markers into the yeast constructs is sub-cloned

by standard cloning methods. The yeast constructs carrying papillomavirus DNA will be

transformed into yeast by commercially available kit such as Frozen-EZ Yeast Transformation II

kit (Zymo Research, USA), the commonly used yeast mating technology, the standard LiAc and

PEG 8000 method. The papillomavirus pseudovirions will be isolated by centrifugation of the

transformed yeast culture. The characterisation of the pseudovirions will be carried out using

general laboratory methods such as northern blots, southern blots, PCR and viral titration

techniques.

(ii) Organotypic rafts: The organotypic raft culture system has been widely used as an in vitro

system to study papillomaviruses due to its capability of reproducing the complete papillomavirus

life cycle including virion production. This system involves using papillomavirus-containing cells

lines that are derived from biopsies or created by introduction of papillomavirus genomic DNA

into keratinocytes using common laboratory techniques such as transfection, retrovirus-mediated

transduction or electroporation. The expression of papillomavirus genomic DNA in keratinocytes

will be detected using techniques such as western blot, northern blot, RNA isolation, cDNA

synthesis and PCR amplification. The keratinocytes harbouring papillomavirus genomic DNA

will be seeded on a dermal equivalent (collagen matrix) containing feeder cells such as J2 3T3

fibroblast. Once keratinocytes are attached to the collagen matrix, the keratinocytes/collagen

matrix will be lifted to the air-liquid interface that allows for differentiation of the keratinocytes,

which mimics morphological and physiological features of the epithelium in vivo. Raft tissues will

be harvested and used for histology, immunohistochemistry, electron microscopy, biochemical and

molecular biological studies. Viruses will be isolated from the epithelial layer of the raft culture

by using common laboratory technique such centrifugation of ground raft tissues in virus isolation

buffer.

Mammalian cell transfection system for generation of papillomavirus pseudovirions is as described

in Section 3.1(b) above except the cells are co-transfected with codon-modified papillomavirus

capsid genes (L1 and L2 or L1 alone) along with a pseudogenome plasmid containing the SV40

origin of replication. Pseudogenome encapsidation within L1/L2 capsids or L1 alone capsids

occurs and these particles are infectious. Purification of the papillomavirus pseudovirions is as

described in Section 3.1(b) above.

(iii) Recombinant vaccinia virus system: Papillomavirus pseudovirions will be generated by

transducing target cells containing papillomavirus genomic DNA (which may contain reporter

DNA) with recombinant vaccinia virus coding for the papillomavirus L1 or L1 and L2 capsid

sequences. Expression of L1 and L2 capsid proteins by recombinant vaccinia viruses also requires

simultaneous expression of the vaccinia helper virus encoding the phage T7 RNA polymerase.

The resultant pseudovirions will be harvested, purified and characterised by methods such as

density gradient purification and pseudovirus infection assays.

The transfer of papillomavirus genomic DNA into target cells will be carried out by using general

laboratory techniques such as transfection and electroporation. The generation of recombinant

vaccinia viruses will be achieved by homologous recombination. This requires the use of transfer

vectors cloned with the target genes surrounded by vaccinia virus sequence. The transfer vectors

will be expressed in cells such as HuTK-143 B cells together with wild-type vaccinia virus DNA to

allow for homologous recombination. The recombinant vaccinia viruses may be concentrated

under selection in cases where the plasmid contains appropriate genes for selection. Recombinant

vaccinia viruses may be purified by serial plaque purification and characterised by gel

electrophoresis of restriction endonuclease genomic DNA fragments and Southern, northern and

western blotting.

(c) Production of replication competent recombinant poxvirus:

of 28

All modified orf viruses will be constructed in the same general manner by homologous

recombination at a nonessential site within the genome that we have demonstrated is suitable for

the insertion of foreign DNA (Savory et al., 2000).

A DNA plasmid construct will be made in E. coli by firstly cloning the approximately 500 bp

sequences that flank an intergenic insertion site of orf virus into a plasmid such as pSP70. This

manipulation will be followed by the insertion of the coding sequences of the gene to be inserted

under the control of a viral early, early/late or late/synthetic, natural or poxvirus promoter, a

reporter gene and a gene for drug selection. The coding sequence of the inserted DNA may also

fused with DNA encoding an epitope tag, in order to facilitate detection of antigen.

The recombinant virus will be generated by homologous recombination of the plasmid DNA in

wild-type virus-infected, plasmid-transfected cells. The recombinants may be concentrated under

selection, in cases where the plasmid contains drug selection genes. Recombinants may be

purified by serial plaque purification and characterised by gel electrophoresis of restriction

endonuclease genomic DNA fragments and Southern blotting. The expression of the antigen will

be determined by Western blotting.

(d) Production of replication competent recombinant adenovirus:

Vectors based on the ovine adenovirus isolate OAdV287 (OvAd7) a member of the genus

Atadenovirus, will be generated following published procedures for the rescue of recombinant

viruses from ovine cells transfected with a linearised DNA genome in which the transgene is

inserted within the viral inverted terminal repeats (ITRs) Vrati et al, Virology 1996; 220: 200–3.

OAdV7 and recombinant versions rescued from transfected cells are known to replicate efficiently

only in sheep cell lines where it grows to high titre. In a wide range of other animal and human cell

lines replication is abortive. Thus OvAd vectors will exhibit features of replication competent

viruses (when propagated in these cell lines and others derived from sheep) but may be used as

replication-defective viral vectors when used to transduce cells derived from other animals.

(e) Production of replication competent recombinant Hepatitis B virus:

Wild-type HBV genomes and viral genomes harbouring specific deletions/point mutations will be

cloned in plasmid vectors either from existing sources of cloned DNA, or from clinical isolates

obtained in New Zealand (Note: research involving human subjects will require human subjects

Ethics Committee approval). Because of the circular nature of the HBV genome and extensive use

of overlapping reading frames, plasmid vectors will be generated that contain a minimum of 1.5 x

full length HBV genomes to enable viral replication in transfected cells. HBV genes will remain

under transcriptional control of endogenous viral (liver specific) promoter sequences however in

certain vectors HBV sequences will be placed under control of heterologous promoters.

The plasmids thus generated will be introduced by standard transfection techniques into cultured

hepatoma cell lines resulting in transient expression of viral genes and secretion of infectious HBV

into the culture medium. In some cases clones of transfected cells will be isolated by antibiotic

selection (the antibiotic to be used will match the resistance gene encoded in the plasmid with the

HBV genes). Individual clones will be isolated following death of non-transfected cells and

transferred to separate culture plates where they will be maintained under antibiotic selection.

3. Transfection, transduction or infection of mammalian cell lines or animals

Replication competent or defective viral particles will be transduced or infected into cells or

animals. Cell lines that have been transfected, transduced or infected may be inoculated into

animals. DNA also will be directly delivered into animals.

Types and sources of additional genetic material:

of 28

Reporter gene inserts

Transgene expression in experimental models will be monitored using reporter genes in place of,

or in addition to candidate gene inserts. Also, measurement of transcriptional regulation by

coupling the expression of a reporter gene may also be used to monitor various physiological or

molecular events in target cells such as receptor activity, signal transduction, expression of

transcription factors, or protein-protein interactions.

Candidate gene inserts

DNA for the coding regions of candidate genes and mutants thereof will be sourced commercially,

from reputable scientific research laboratories or cloned by digestion of the genome with

restriction enzymes, PCR or RT-PCR. The genes of interest will comprise of viral, eukaryote and

prokaryotic genes and mutants thereof (e.g. deletion, substitution and chimeric mutants).

Additional vector elements

Expression constructs might also include:

eukaryotic, prokaryotic and viral enhancers/promoters

silencing elements (short interfering RNA, short hairpin RNA

recombination sites

internal ribosomal entry sites (IRES)

sequences for fusion protein tags

polyadenylation signals

genes for antibiotic resistance

other regulatory elements that are components of existing or new commercially

available vectors

Use of special genetic material: please complete this table by marking the correct box

Yes No

Does this application use native flora or fauna as host organism(s)?

If Yes, provide additional details below. X

Does this application use genetic material from native flora and

fauna? If Yes, provide additional details below. X

Does this application involve human cell lines? Answer Yes if

human cell lines in any form are used, ie obtained directly from

humans (either Māori or non-Māori) or from a commercial supplier

etc. Please provide additional details below.

X

Does this application use cell lines obtained directly from human

beings? X

Does this application involve human genetic material? Answer Yes

if human genetic material in any form is used, ie obtained directly

from humans (either Māori or non-Māori), from a gene bank,

synthesised, copied and so on. Please provide additional details

below.

X

Does this application use genetic material obtained directly from

human beings? X

End of development

The functional screening phase of the proposed work will conclude with either the destruction of

transduced cells or organisms, or frozen storage. Primary cells (epithelial or fibroblast derived)

and host animals with genetically modified somatic cells all have limited life spans, and will be

autoclaved (cultured systems) or humanely culled (animals) at the conclusion of the experiment for

which they were developed according to the MAF /ERMA New Zealand Standard “Facilities for

of 28

Microorganisms and Cell Cultures: 2007a”. Some stably transduced cell lines will be stored

frozen in liquid nitrogen and a register kept as per section 8.4 of the MAF Biosecurity New

Zealand and ERMA New Zealand Standard “Facilities for Microorganisms and Cell Cultures:

2007a”.

of 28

Section 4: The proposed containment system (section 40(2) of the HSNO Act)

In this section you should outline how you propose to adequately contain the new organism(s) and manage any hazards associated with the organism(s), i.e. discuss the method of containment (based on the characteristics of the organism). For example, bagging plants to prevent pollen escape or requiring spore-producing bacteria to be handled within class II biosafety cabinet. Hint—refer to the appropriate MAF/ERMA Standards and AS/NZS 2243.3:2002 (or any updated version) requirements and your facility’s containment manual where appropriate.

Are you aware of any possible adverse effects of the organism on the health and safety of the person people working the containment facility? If so, what risk mitigation strategies do you propose? For example, requiring pathogenic bacteria to be handled only by personnel using the appropriate safety gear.

If this application is for development within an outdoor containment facility: Discuss whether controls are required for inspection and monitoring before, during and after a development outdoors

within a containment facility.

Section 45A(2)(a) and (b) of the HSNO Act requires that at the completion of an outdoor development the organism and any heritable material from the organism (along with some or all of the remaining genetic elements) are removed or destroyed. Describe how you would achieve these objectives.

The adequacy of the containment regime is a principal consideration for the Authority so you need

to provide comprehensive information on the containment system and the containment structure. A

containment structure is a vehicle, room building, or other structure set aside and equipped for the

development of GM organisms. Your containment structure must be registered by MAF, and you

should provide documentary evidence of this.

The experiments described in this application will be carried out in an approved containment

facility in accordance with MAF Biosecurity Authority and ERMA Standard „Facilities for

Microorganisms and Cell Cultures: 2007a‟, and/or MAF/ERMA Standard 154.03.03 „Containment

Facilities for Vertebrate Laboratory Animals‟. These laboratories comprise PC1 and PC2

containment areas, which meet regulatory requirements, including the AS/NZS 2243.3:2002

standard (Safety in laboratories Part 3: Microbiological aspects and containment facilities). A

register is maintained of all GMOs held within the transitional/ containment facility. Entry to

containment facilities is controlled by electronic identity card readers, both from outside the

building and to the specialised laboratory and animal facilities within. Visitor access is via a

staffed reception area. After normal working hours the Containment Facility is locked, alarmed,

and is patrolled and monitored by security services. Those who might carry out work on the

genetically modified organisms that may be developed in this proposal might include Senior

Scientists, Post-doctoral Fellows, Technical Staff and Students. All researchers are required to

undergo formal safety training, plus special training appropriate to their work area.

Experimentation by researchers on this approval would be restricted until they are sufficiently

trained by experienced Staff. Personal protective clothing is required to be worn. The building,

operating procedures, and records are subject to regular internal and independent external audits

for health and safety and regulatory compliance.

(i) PC2 laboratory

The described experimentation will be carried out in PC2 containment in accordance with

AS/NZS2243.3:2002. Safety in laboratories Part 3: Microbiological aspects and containment

facilities. Access is restricted to staff trained in the specific protocols of the PC2 laboratory, and

there is clear signage outlining the entry restrictions, biohazards and containment status within the

servicing area.

The PC2 containment facility is equipped with dedicated labware, consumables and instruments

including:

class II Biological Safety Cabinet

CO2 incubator

clearly labelled cold storage (fridge/freezer, liquid nitrogen dewer)

electric operated or hands-free wash basin and eye wash

of 28

All plastic-ware, glassware and waste associated with bacterial culture, plasmid propagation and

virally transduced material will be autoclaved according to MAF-approved protocols.

(ii) PC2 Animal Facility

Live animals infected with recombinant viruses or viral vector will be maintained in a MAF-

approved animal containment facility in accordance with MAF Regulatory Authority Standard

154.03.03. Containment Facilities for Vertebrate Laboratory Animals have been approved as

providing the level of containment deemed appropriate for the proposed work in line with MAF

Regulatory Standard, „Facilities for Microorganisms and Cell Cultures: 2007a‟. The facilities meet

the requirements of animal house containment level PC2 as defined in AS/NZS 2243.3.

Access to these facilities is restricted by electronic swipe card to authorised investigators and

animal maintenance staff.

Mice and Rats

Mice and rats will be genetically modified by foreign genes following transduction with replication

defective virus or infection with pseudovirions or viruses, which have been delivered in a Class II

Biological Safety Cabinet or a dedicated Biobubble with 80-100 Air changes per hour (ACH) via

HEPA filter held in a PC2 containment facility. Mice and rats infected with recombinant viruses

will be maintained in a MAF-approved animal containment facility and in microisolator cages

when mammalian-infectious, replication-competent virus is used. Microisolator cages are solid

polycarbonate with filtered-top lids designed to prevent transmission of microorganisms including

viruses in and out of the boxes. Animal carcasses, used bedding litter and other waste will be

collected and disposed of following sterilization.

It should be noted that the integration of replication defective retroviral vector and decay of

circulating viral vector appears to be quite rapid in small animals. A study by Karlen and Zuffery

(2007) indicates that rats injected intravenously with lentiviral vector, no longer shed vector after 3

days and can be downgraded to BSL 1. The authors speculate that these quarantine times should

be considerably shorter for intracranial injections.

Rabbits

Rabbits will be genetically modified by using a virus, pseudovirions or viral vector(s) delivered in

a Class II Biological Safety Cabinet or a dedicated Biobubble with 80-100 Air changes per hour

(ACH) via HEPA filter held in a PC2 containment facility. Following transduction, infection or

foreign gene delivery, rabbits will be individually housed in rabbit racks and isolated in a separate

room. The racks have perforations on the sides and back of the cages for ventilation. The floors

have drainage to allow urine and faecal material to drop through onto the plastic collection trays.

Waste in the trays will be transferred to biohazard bags to be sterilized and disposed of. The trays

will be changed once each week and old trays will be disinfected with 1% virkon or 1% trigene.

On completion of the experiment, the room will be either be fumigated with formaldehyde or

disinfected with 1% virkon or 1% trigene. Animal carcases will be disposed of following

sterilization.

Access to the room will be restricted to approved personnel and only trained staff will handle

animals. All staff will undergo compliance training in accordance with the AS/NZS 2243.3

Standard and additional training in accordance with the dedicated 'Viral vector Standard Operating

Procedure' based on the approval controls. All personnel working in these facilities will be

required to wear protective clothing such as a disposable gown, gloves, mask, shoe covers and hat.

All used disposable clothing will be discarded in biohazard waste at the room exit.

of 28

Section 5: Details of consultation (if applicable)

Discuss the consultation process and summarise the outcomes. Attach specific details of the consultation process (such as copies of written responses) as a separate Appendix. Discuss any adverse or beneficial effects identified during consultation in more detail in Section 6.

No native flora or fauna will be used.

Genetic material and cells derived from humans will be obtained from reputable commercial

suppliers or sourced from research institutes. Ethical permission will be obtained before primary

cells are harvested from animals or humans. Any human genetic material or cells used will be

derived from non-Maori donors.

Otago region

Consultation with the Ngai Tahu representative on the IBSC with regard to this application has

been undertaken and no issues were raised. Any human material used in experimental models

(DNA or cells) will be derived from non-Maori donors. Should there be a breach of containment,

the possible risks are outlined above under the sections headed „(a) Potential adverse effects on the

environment and (b) Potential adverse effects on public health‟.

Auckland and greater Auckland region

This application in its final form was considered by representatives of Ngati Whatua and iwi of the

greater Auckland region who are members of the University of Auckland Biological Safety

Committee. The broad nature of the research permitted under this application was noted by the

representative from Ngati Whatua. It was agreed by all members of the IBSC that while the

application seeks approval to generate a very wide range of GMOs in containment, it does not

permit the genetic manipulation or analysis of any native species. Similarly, the research permitted

under this approval will not involve Maori donors of genetic material (DNA) or of cells that might

be genetically manipulated without the additional consent of an approved Human Subjects Ethics

Committee. Finally, the representatives of Ngati Whatua and iwi of the greater Auckland region

who are members of the University of Auckland Biological Safety Committee acknowledged that

research for which approval is sought in this application has the potential to benefit Maori. For

example, the propagation of hepatitis B virus in genetically-engineered cells in culture might

contribute to new knowledge with the potential to translate to improved health outcomes for

Maori, who have a disproportionately high incidence of chronic hepatitis B disease.

of 28

Section 6: Identification of risks, costs and benefits

This section must include information on the beneficial and adverse effects, risks, costs and benefits referred to in the HSNO Act and the HSNO (Methodology) Order 1998. It is easier to regard risks and costs as being adverse (or negative) effects and benefits as beneficial (or positive) effects. You should consider both non-monetary and monetary (dollar value) costs and benefits, the distribution of their occurrence as well as who and what might be affected.

Provide a description of where the information in the application has been sourced from e.g. from in-house research, independent research, technical literature, community or other consultation. Please attach copies of all reference material cited in the application.

a) What are the nature of the adverse effects and the costs of the organism(s) that you are aware of?

i. On the environment (section 40(2)(a)(v)of the HSNO Act)

For example, could the organism adversely affect the environment while in containment? If the organism were to escape could it have an adverse effect on the environment?

No native flora or fauna or valued introduced species are involved in the proposed work. All

work will be conducted in laboratory containment with both physical and procedural barriers

to prevent release (section 4.1). Should viral vectors, transduced cells or genetically

modified animals escape from containment, no adverse effects on natural ecosystems,

agriculture, or urban environments are envisaged.

ii. Adverse effects of occupational exposure (section 40(2)(a)(v) of the HSNO Act)

For example, could the organism adversely affect the health and safety on any person exposed in the workplace environment while in containment?

The proposed project has a risk associated with it because it involves the development of

new organisms containing hybrid genes. The described experimentation will be carried out

in PC2 containment in accordance with appendix 5 of Genetic Manipulation Advisory

Committee „Guidelines for work involving genetically-modified viruses for gene transfer

into animal and human cells‟.

Replication defective viral vectors

Replication defective viral vectors are by their nature less pathogenic than the parent

organism. There is a risk of occupational exposure to staff. Exposure to replication defective

viral particles in containment laboratories is most likely to occur through stab injuries, spills,

or aerosol. Procedures will be performed according to strict standards of operation, and

conducted within a Class II Biological Safety Cabinet within PC2 category laboratories, as

described above in Section 4.

If transduction of an individual were to occur, foreign genes could be expressed in cells that

came in direct contact with the replication defective viral vector. No viraemia would result

because the vectors are replication defective. The harmful effects that could result from the

expression of foreign genes in human cells include carcinogenesis following expression of

multiple oncogenes. This risk is acknowledged but is mitigated by the containment

procedures that will be used. The risks are well documented in Evaluation and Review

Reports for Applications to the Authority for use of replication defective retroviral and AAV

vectors – Approvals GMD03091 and GMD 03096 respectively

Papillomavirus and Adenovirus

Humans and animals are frequently infected with adenoviruses and papillomaviruses, which

are the organisms from which the majority of the inserted genes are derived. The most

common effects following infection with these viruses are colds from adenovirus and benign

warts from papillomavirus. High-risk papillomaviruses such as HPV type 16 can cause

of 28

cancer of the cervix and head and neck cancers. All viruses will be handled under strict

standards of operation.

Replication competent Ovine Adenovirus

This virus and cells harbouring infectious virus will be handled only in PC2 containment

following standard operating procedures. Recombinant OAdV7 is known to replicate

efficiently only in fetal lung (CSL503) and skin (HVO156) cell lines where it grows to high

titre. In a wide range of other animal and human cell lines replication is abortive, with the

replication cycle being blocked at different stages, depending on the cell type, due to the lack

of viral promoter function. Because adenoviruses do not integrate their genomes with great

efficiency, OAdV7 vectors are considered to be safe and have been approved for gene

therapy use in humans. Viruses of this type are distributed across pastoral environments

worldwide including New Zealand but are not generally recognized as pathogenic under

field conditions.

Hepatitis B virus

Cultured cell lines that are transfected with plasmids containing full-length HBV genomes

secrete infectious virus particles. HBV is a NIH Risk Group 2 pathogen. The virus is

transmitted mainly through blood contact. In adults exposure to HBV results in mild to

moderate acute hepatitis in 90-95% of cases but can also lead to chronic infection. These

risks can be effectively mitigated by handling of the transfected cells and culture media

containing virus within a Class II biohazard hood located within a PC2 laboratory. Adequate

protection to laboratory workers is provided by the wearing of gloves and lab coats (which

will not leave the PC2 lab except in sealed bags for autoclaving) and by avoidance of the use

of sharps. A safe and effective prophylactic HBV vaccine exists and has been included in

the NZ immunization schedule for more than 15 years. Research staff involved in HBV-

related work or who operate in the PC2 laboratory in which HBV work is carried out must

be able to demonstrate evidence of vaccination and possess an anti-HBsAg titre> 100 U/ml.

Workers will be tested annually for antibody levels and where these are below this titre will

be given a booster dose of vaccine.

Thus although the transfected cells will produce a human pathogen, the risk to researchers

exposed is no greater than to hospital laboratory workers handling human blood and is

effectively mitigated by the procedures described above.

iii. On the relationship of Māori to the environment and the principles of the Treaty of Waitangi (section 6(d), 8 and 40(2)(b)(v)of the HSNO Act)

For example, if the organism were to escape could it have an adverse effect of potential specific importance to Māori. When identifying potential effects you should consider effects to environmental (e.g. physical impacts on native flora and fauna, water bodies, traditional food resources etc), cultural (e.g. the recognised kaitiakitanga role of Māori), health and wellbeing (e.g. specific physical and spiritual health effects), economic (e.g. the ability of Māori to develop economically) and Treaty of Waitangi (e.g. the ongoing management by Māori of their cultural or natural resources). Include any relevant issues raised or information obtained through consultation.

Otago region

Consultation with the Ngai Tahu representative on the IBSC with regard to this application

has been undertaken and no issues were raised. Any human material used in experimental

models (DNA or cells) will be derived from non-Maori donors. Should there be a breach of

containment, the possible risks are outlined above under the sections headed „(a) Potential

adverse effects on the environment and (b) Potential adverse effects on public health‟.

Auckland and greater Auckland region

Consultation with the Ngati Whatua and iwi of the greater Auckland region representatives

on the Auckland Biological Safety Committee with regard to this application has been

of 28

undertaken. No objections to the proposed research were raised.

iv. On society and the community including public health (section 40(2)(a)(v) of the HSNO Act)

For example, could the organism in containment adversely affect individuals or communities? If the organism were to escape could it have an adverse effect on society or on people’s wellbeing?

We are unaware of any risk of the genetically modified organisms described in this

application to individuals or communities while they are in containment. If the organisms

were to escape from containment, there is a risk of cell transformation for any individual

who is directly infected with a replication defective or competent virus containing multiple

oncogenes. Replication defective viral vectors are unlikely to have any broad adverse effect

on communities because they are not able to spread from one individual to another. There is

a risk if genetically modified hepatitis B virus, papillomavirus, adenovirus or poxvirus were

to escape from containment, as these viruses are replication competent. The genetic

modifications of these viruses generally result in loss of function therefore reduced

pathogenicity is anticipated. There is no risk to the community from baculovirus, as it is not

able to infect humans and additionally is a crippled strain that is extremely sensitive to UV

inactivation.

v. On the market economy (section 40(2)(a)(v) of the HSNO Act)

For example, could there be any adverse effects on the New Zealand economy at a local, regional or national level? Are there any public commercial risks or costs?

No adverse effects on the NZ economy have been identified.

vi. Are there other potential adverse effects that do not fall under sections (i) – (v)?

No other potential adverse effects have been identified.

of 28

b) What is the nature of the potential beneficial effects associated with the organism(s) that you are aware of?

i. Beneficial effects on the environment and ecosystems

For example, could the organism beneficially affect the environment while in containment? If the organism were to escape could it have a beneficial effect on the environment?

None that we are aware of.

ii. Beneficial effects on the relationship of Māori to the environment and the principles of the Treaty of Waitangi

For example, if the organism were to escape could it have a beneficial effect of potential specific importance to Māori. As for the identification of adverse effects, you should consider effects to environmental, cultural, health and wellbeing, economic and Treaty of Waitangi. Include any relevant issues raised or information obtained through consultation.

The aim of this research is to advance our scientific knowledge in how genes regulate the

cellular and tissue physiology that may lead to the development of therapeutics strategies

for the treatment of diseases. There is an appreciation by the Maori representatives

consulted that this research may provide benefits for all members of society.

The research on hepatitis B has special significance to Maori given that chronic hepatitis B

is more prevalent among Maori than non-Maori New Zealanders.

iii. Beneficial effects on public health, society and community

For example, if the organism were to escape could it have a beneficial effect on society or on people’s health and wellbeing? Could the organism in containment have benefits for individuals or communities? This might include increased knowledge.

The proposed research will generate increased scientific knowledge in the fields of

molecular physiology and genetics, which are likely to lead to high value applications. The

results of the work will have beneficial affects for public health by increasing scientific

capability for the Universities of Otago and Auckland and New Zealand health research.

Results will be published in the public domain and the findings that arise from this work

may ultimately benefit all New Zealanders and the international scientific community.

Positive outcomes also include greater research opportunities in papillomaviruses and other

viruses, and maintaining New Zealand‟s international standing in science by enabling New

Zealand scientists to carry out innovative research projects. Furthermore, advancing the

understanding of how viruses modulate their host may lead to the development of

therapeutic strategies for the treatment of diseases such as cancer.

of 28

This research will allow our research teams to deliver scientific knowledge and invent new

biotechnology applications to enhance our understanding of the mechanisms used by

viruses to modulate their host. In addition, this research is expected to bring benefits to the

researchers of the Universities of Otago and Auckland, through collaborations with

scientists within New Zealand and overseas and the ability to attract funding both within

New Zealand and internationally.

The proposed research is expected to produce high-level internationally recognised

research data with publications in top level scientific publications. The results that arise

from this work may ultimately benefit all New Zealanders through the development of new

therapeutic strategies for the treatment of virus-associated diseases.

iv. Beneficial effects on the market economy

For example, could there be any beneficial effects on the New Zealand economy at a local, regional or national level? Are there any public commercial benefits?

The treatment of diseases will have economic positive effects by reducing the costs to

health system.

The organisms described here provide tools for New Zealand scientists to develop

innovative research projects to generate increased scientific knowledge. This research is

expected to advance our understanding in how genes such as viral genes regulate the

cellular and tissue functions, based on identifying the key genes and mechanisms

regulating tissue physiology. The results arising from this study may provide opportunities

to develop high value biotechnologies in therapeutics for the treatment of viral infections

and virus-associated disease such as cancer.

v. Are there other potential beneficial effects that do not fall under sections (i) – (iv)?

None that we are aware of.

Section 7: Is there any other information relevant to the consideration of this application that has not been mentioned earlier?

of 28

This application has significant overlap with the University of Otago approval ERMA 200041,

both in purpose and in the organisms to be developed. The Universities of Auckland and Otago

are co-applicants on this current application. The proposed work conforms to current and

widespread research practices of screening for gene function, and other projects with similar aims

and utilising recombinant viruses the viral vector technologies that have previously received

ERMA approval (Application codes GMD01085, GMD03091, GMD03096, GMD03105,

GMD99002, GMD02131, GMD01171, GMD01067, GMD05036).

The risks to the environment and public health are extremely low because the research will be

conducted entirely in containment with strict operational controls. Effects of genetic

manipulations will be, where practicable, first observed in vitro, and tested in animal models after

effects on cultured cells are established. Given this cautious approach, we submit that the benefits

of this functional genomics research, in regard to the knowledge gained and the potential

applications outweigh the risks and costs, including the very low risk of occupational exposure.

A significant consideration is that multiple, unspecified genetic modifications are proposed.

However, the risk of unforeseen and undesirable biological events is mitigated by the progressive

nature of the functional tests, starting with cell culture screens and proceeding to whole animal

models after an understanding of the possible role of the candidate gene has been developed.

of 28

Section 8: List of appendices, referenced material and Glossary (if applicable)

a) List of appendices attached

Appendix Number Title

b) List of references used

Author Title and Journal

Blomer, U., Naldini,

L., Kafri, T., Trono,

D., Verma, I.M. and

Gage, F.H.

Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol

71, 6641-9. 1997

Campos, S.K. and

Barry, M.A.

Current advances and future challenges in Adenoviral vector biology and targeting. Curr

Gene Ther 7, 189-204, 2007

Dull, T., Zufferey, R.,

Kelly, M., Mandel,

R.J., Nguyen, M.,

Trono, D. and

Naldini, L.

A third-generation lentivirus vector with a conditional packaging system. J Virol 72, 8463-71,

1998

Karlen, S and R

Zufferey

Declassification of Rodents exposed to Third-Generation HIV-based Vectors into Class 1

Animals.” Applied Biosafety 12(2) pp 93-99, 2007

Roden, R.B.,

Greenstone, H.L.,

Kirnbauer, R., Booy,

F.P., Jessie, J., Lowy,

D.R. and Schiller, J.T

In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion

pseudotype. J Virol 70, 5875-83, 1996

Savory, L.J., Stacker,

S.A., Fleming, S.B.,

Niven, B.E. and

Mercer, A.A.

Viral vascular endothelial growth factor plays a critical role in orf virus infection. J Virol 74,

10699-706, 2000

Touze, A. and

Coursaget, P.

In vitro gene transfer using human papillomavirus-like particles. Nucleic Acids Res 26, 1317-

23, 1998

Unckell, F., Streeck, Generation and neutralization of pseudovirions of human papillomavirus type 33. J Virol 71,

of 28

R.E. and Sapp, M. 2934-9, 1997

Vrati S, Macavoy ES,

Xu ZZ, Smole C,

Boyle DB and Both

GW

Construction and transfection of ovine adenovirus genomic clones to rescue modified viruses

Virology220(1):200-203, 1996

c) Glossary

Term Definition

of 28

Section 9: Declaration and signing the application form

In preparing this application I have: Taken into account the ethical principles and standards described in the ERMA New Zealand Ethics Framework

Protocol (http://www.ermanz.govt.nz/resources/publications/pdfs/ER-PR-05-1.pdf);

Identified any ethical considerations relevant to this application;

Ensured that this application contains an appropriate level of information about any ethical considerations identified, and provided information about how these have been anticipated or might be mitigated; and

Contacted ERMA New Zealand staff for advice if in doubt about any ethical considerations.

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.

Signed

Date

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

Before submitting your application you must ensure that: All sections are completed.

Appendices (if any) are attached.

Copies of references (if any) are attached.

Any confidential information identified and enclosed separately.

The application is signed and dated.

An electronic copy of the final application is e-mailed to ERMA New Zealand.

http://www.ermanz.govt.nz/resources/publications/pdfs/ER-PR-05-1.pdf

Developing genetically modified organisms in containment

Documents

Transcript of Developing genetically modified organisms in containment