Download Application title: Cloning genes for expression in AAV vectors for

NO3P
ER-AF-NO3P-3
12/07
Develop in containment a project of low risk genetically
modified organisms by rapid assessment
Application title:
Use of recombinant viral vectors for production of
recombinant proteins in animals.
Applicant organisation:
AgResearch Ltd
Considered by:
IBSC
ERMA

Please clearly identify any confidential information and attach as a separate appendix.
Please complete the following before submitting your application:
All sections completed
Appendices enclosed
Confidential information identified and enclosed separately
Copies of references attached
Application signed and dated
Electronic copy of application e-mailed to ERMA New
Zealand
Signed:
20 Customhouse Quay
Cnr Waring Taylor and Customhouse Quay
PO Box 131, Wellington
Phone: 04 916 2426 Fax: 04 914 0433
Email: [email protected]
Website: www.ermanz.govt.nz
Date:
Yes
Yes
NA
NA
Yes
Yes
Develop in containment a project of low risk genetically modified organisms by rapid assessment
Section One – Applicant details
Refer to page 9 of the user guide
Name and details of the organisation making the application:
Name:
Postal Address:
Physical Address:
Phone:
Fax:
Email:
AgResearch Limited
Ruakura Research Center
East St
Private Bag 3123
Hamilton 3240
07 856 2836
07 838 5012
Name and details of the key contact person
If different from above
Name:
Postal Address:
Ross J. Bland
Grasslands Research Centre
Private Bag 11008
Palmerston North 4442
Physical Address:
Phone:
Fax:
Email:
Name and details of a contact person in New Zealand, if the applicant is overseas:
Name:
Postal Address:
Richard Scott
Grasslands Research Centre
Private Bag 11008
Palmerston North 4442
Physical Address:
Phone:
Fax:
Email:
Note: The key contact person should have sufficient knowledge of the application to respond to queries from
ERMA New Zealand staff.
Page 2 of 28
Develop in containment a project of low risk genetically modified organisms by rapid assessment
Section Two: Lay summary and scientific project description
Refer to page 9 of the user guide
Lay summary of the application (approximately 200 words)
Note: This summary should describe the genetically modified organism(s) being developed, the purpose of the
application or what you want to do with the organisms(s). Use simple non-technical language.
This application is for research into the development and use of replication deficient viral
vectors for investigating the production of recombinant proteins (biopharmaceuticals) in
human cells as well as cells and whole animals of the following: mouse, rat, rabbit, cattle,
goat and sheep. The recombinant proteins we want to research are for the prevention,
diagnosis and treatment of the following: endocrine, nutritional and metabolic diseases,
infectious and parasitic diseases, neoplasms, diseases of the blood, diseases of the
respiratory system, diseases of the musculoskeletal system, diseases of the skin, diseases
of the digestive system, diseases of the circulatory system, mental and behavioural
disorders, and diseases of the nervous system. Recombinant viral vectors will be used to
deliver relevant genes-of-interest for expression (gene transfer) or to alter target gene
expression (gene targeting) to mammalian cell lines and various animals in containment for
the expression of). Only non-reproductive cells of whole animals will be altered by the viral
vectors. The resulting genetic modifications cannot be passed on to the progeny or to other
animals. Since the viral vectors are replication deficient, the vectors cannot replicate to
produce further infectious particles that can infect other mammalian cells or whole animals.
Genetic material will not be sourced from humans of Māori descent or from native or valued
flora or fauna. All work will be done in containment.
Scientific project description
Describe the project, including the background, aims and a description of the wider project.
Refer to page 10 of the user guide.
Note: This section is intended to put the genetically modified organism(s) being developed in perspective of the
wider project(s) that they will be used in. You may use more technical language but make sure that any
technical words are included in the Glossary.
Aims
The overall purpose of the project is to investigate the potential of recombinant adenoassociated viral vectors (rAAV) as a tool for investigating the production of high value
recombinant proteins in the bodily fluids of various animals. The recombinant proteins that
may be produced are for use in the prevention, diagnosis and treatment of the following:
endocrine, nutritional and metabolic diseases, infectious and parasitic diseases, neoplasms,
diseases of the blood, diseases of the respiratory system, diseases of the musculoskeletal
system, diseases of the skin, diseases of the digestive system, diseases of the circulatory
system, mental and behavioural disorders, and diseases of the nervous system.
There are four phases for this work:
Development of plasmids, including AAV packaging and expression plasmids in
Escherichia coli.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
The characterisation of these plasmids by transfection of mammalian cell lines for
analysis of gene expression and activity.
The packaging of recombinant AAV (rAAV) vectors in mammalian cell lines.
The genetic modification of mammalian cell lines and animals in containment using
rAAV vectors.
Background
Our aim is to utilise rAAV vectors for gene transfer or gene targeting to various organs for the
production of recombinant proteins in bodily fluids for use in the prevention, diagnosis and
treatment of disease. Gene transfer involves the extra-chromosomal expression of a gene
expression construct that doesn’t interfere with the integrity of the host genome. rAAV can
also be specifically designed for efficient gene targeting - site-specific genetic modification of
chromosomal DNA by homologous recombination. In this case, rAAV vectors have to be
specifically designed to contain large stretches of uninterrupted homologous sequences to an
endogenous genomic locus.
There is potential through the development of transgenic animals to produce
biopharmaceuticals (Pollock et al., 1999). But besides the difficulty in generating transgenic
founders using current techniques, the time taken to then establish a production herd,
approximately 7 years, is prohibitive. The production of recombinant proteins following
transduction of target organs using viral vectors is an alternative approach. Theoretically,
rAAV offers the potential for high levels of continuous recombinant protein production in the
target organ following injection of the vector.
Wildtype AAV can site-specifically integrate into a defined site, AAVS1, on human
chromosome 19. Recombinant AAV has lost this ability, as vectors do not express the rep
gene products required for integration: recombinant AAV vectors only contain the 145 bp
inverted terminal repeat (ITR) sequence from the wildtype virus. While rAAV vectors
predominantly persist in episomal chromatin forms (Penaud-Budloo et al., 2008) there is a
low rate of quasi-random integration (10-3-10-4) (Lin and Ertl, 2008). In dogs, rAAV was
demonstrated to persist episomally for up to 8 years with no evidence of chromosomal DNA
integration (Niemeyer et al., 2009). However, if rAAV vectors are specifically designed to
contain large stretches of uninterrupted sequences homologous to an endogenous genomic
locus then rAAV is able to direct efficient gene targeting and thus mediate site-specific
genetic modification of chromosomal DNA (Russell and Hirata, 1998). The gene targeting
capability could be used to modify a specific gene sequence to increase its commercial value
(e.g. increase stability), target protein production to an endogenous promoter, or to improve
the success of transgenic animal generation.
Description of GMOs to be developed:
1) Construction of AAV plasmids: E. coli non-pathogenic laboratory strains (e.g., K12 and
B non-conjugative strains) used for constructing AAV plasmids for packaging, expression and
targeting. The AAV packaging plasmids are plasmids that contain the AAV rep and cap
sequences and will include those used in generating serotypes AAV1-12, the serotypes
identified by degenerate PCR in non-human primates (Gao et al., 2004) and other AAV
serotypes yet to be described. The genetic material contained in the AAV expression and
targeting plasmids is described below.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
2) Production of replication deficient AAV viral vectors: Commercially available
mammalian cell lines (e.g. HEK 293) will be used to produce the replication deficient AAV
viral vectors for gene transfer or gene targeting. These cell lines will be modified with AAV
expression plasmids (plasmids that contain the expression construct/transgene flanked by
the AAV ITRs) or AAV targeting plasmids (plasmids that contain a stretch of sequence
homologous to the endogenous target flanked by ITRs), and AAV packaging plasmids
(plasmids that contain the AAV rep and cap sequences). The use of adenoviral helper
plasmids (plasmid containing adenoviral genes (E2A, E4 and VA) that provide the helper
functions necessary for AAV replication) may be included. None of the adenoviral genes will
be packaged into the replication defective AAV vectors. Mammalian cells lines may also be
transfected with the AAV expression plasmid to evaluate transgene expression. The
genetic material contained in the AAV expression and targeting plasmids are described
below.
3) Transducing cell lines and cells in whole animals with replication deficient AAV viral
vectors: Commercially available mammalian cell lines, mammalian primary and embryonic
cells (e.g. bovine fibroblasts; excluding human), and vertebrates (e.g. mice) in containment
will be modified with replication defective rAAV viral vectors (viral vector containing single
stranded DNA genome of either the gene transfer or gene targeting transgene flanked by
ITRs) containing genetic material derived from plants, bacteria, fungi, viruses, invertebrates
and vertebrates consisting of coding, non-coding or regulatory regions of genes for the
elucidation and enhancement of livestock traits. The vectors may also include promoters,
reporter and selection marker genes, expression tags, secretory and targeting signals,
recombination elements, and other gene regulatory elements. All rAAV vectors designed for
gene transfer are expected to persist predominantly episomally for the lifetime of the animal,
although there may be a very low level of quasi-random integration. In contrast, the rAAV
vectors specifically designed for gene targeting by homologous recombination will lead to
efficient and specific chromosomal integration of sequences contained between the ITRs and
the concomitant loss of the ITRs. In whole animals, rAAV vectors for both gene transfer and
gene targeting will only be targeted to the somatic (non-germ line) cells.
Short summary of purpose
Please provide a short summary of the purpose of the application
255 characters or less, including spaces) refer to page 11 of the user guide. This section will be transferred into
the decision document.
To develop genetically modified replication-deficient viral vectors for delivery of transgenes to
cell lines and animals in containment to investigate the production of recombinant proteins for
disease prevention, diagnosis and treatment.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Section Three –Description of the organism(s) to be developed
Refer to page 13 of the user guide
3.1
Identification of the host organism to be modified
Complete this section separately for each host organism to be modified.
Latin binomial
Including full taxonomic authority
Escherichia coli (Migula 1895; Castellani and Chalmers, 1919).
Common name(s)
If any
E. coli
Type of organism
Bacterium
eg bacterium, virus, fungus, plant,
animal, animal cell
Taxonomy
Class, order and family
Gamma Proteobacteria, Enterobacteriales, Enterobacteriaceae
Strain(s)
If relevant
Non-pathogenic laboratory strains (e.g., Genetically
derivatives of Escherichia coli K12 and strain B)
crippled
Other information
There are no known inseparable or associated organisms.
Including
presence
of
any
inseparable or associated organisms
and any related animals present in
New Zealand:
Latin binomial
Mus musculus Linnaeus, 1758
Common name(s)
Mouse
Type of organism
Whole animal and animal cells
Taxonomy
Mammalia, Rodentia, Muridae
Strain(s)
Laboratory strains of mice
Other information
Cell lines will be obtained from reputable commercial suppliers or
research institutes. Laboratory strains of mice will be procured only
from reputable commercial sources. Mice are well characterised and
do not contain inseparable or associated organisms.
Latin binomial
Homo sapiens Linnaeus, 1758
Common name(s)
Human
Type of organism
Human cell lines
Taxonomy
Mammalia, Primates, Hominidae
Strain(s)
Commercially available cell lines (e.g., HEK293, HeLa)
Other information
Human cell lines, excluding human embryonic stem cell lines and
cell lines derived from people of known Māori origin, will be
obtained from reputable commercial suppliers or research institutes.
Only pure cell lines, which do not contain any inseparable or
associated organisms, will be used for viral particle production.
Latin binomial
Rattus norvegicus (Berkenhout, 1769)
Common name(s)
Rat
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Type of organism
Whole animals and animal cells
Taxonomy
Mammalia, Rodentia, Muridae
Strain(s)
Laboratory strains of rats
Other information
Cell lines will be obtained from reputable commercial suppliers or
research institutes. Laboratory rat strains will be procured only from
reputable commercial sources. Rats are well characterised and do
not contain inseparable or associated organisms.
Latin binomial
Oryctolagus cuniculis (Linnaues, 1758)
Common name(s)
Rabbit
Type of organism
Whole animals and animal cells
Taxonomy
Mammalia, Lagomorpha, Leporidae
Strain(s)
See other information
Other information
Cell lines will be obtained from reputable commercial suppliers or
research institutes. Laboratory rabbit strains will be procured only
from reputable commercial sources. Rabbits are well characterised
and do not contain inseparable or associated organisms.
Latin binomial
Bos taurus Linnaeus, 1758
Common name(s)
Cattle, cow
Type of organism
Whole animals and animal cell
Taxonomy
Mammalia, Artiodactyla, Bovidae
Strain(s)
See other information
Other information
Cell lines will be obtained from reputable commercial suppliers or
research institutes. Cattle are well characterised and do not contain
inseparable or associated organisms.
Latin binomial
Capra hircus Linnaeus, 1758
Common name(s)
Domestic goat
Type of organism
Whole animals and animal cells
Taxonomy
Mammalia, Artiodactyla, Bovidae
Strain(s)
See other information
Other information
Cell lines will be obtained from reputable commercial suppliers or
research institutes. Goats are well characterised and do not contain
inseparable or associated organisms.
Latin binomial
Ovis aries Linnaeus, 1758
Common name(s)
Sheep
Type of organism
Whole animals and animal cells
Taxonomy
Mammalia, Artiodactyla, Bovidae
Strain(s)
See other information
Other information
Cell lines will be obtained from reputable commercial suppliers or
research institutes. Sheep are well characterised and do not contain
inseparable or associated organisms.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
3.2
Information on the host organism
Refer to pages 14-19 and pages 33-38 of the user guide for assistance in completing this section
Complete this section separately for each host organism to be modified.
Escherichia coli – non-pathogenic laboratory strains
Yes
1
No

Is the organism normally capable of causing disease in humans,
animals, plants or fungi?
If yes, provide details here
2

Is the organism a human cell line?
If yes, provide details here of where the material has been obtained from and whether
approval has been obtained from an Ethics Committee (if required)
3

Is the organism native to New Zealand?
If yes, provide details here for example, from where will this material be obtained?
Be as specific as possible as this information may be needed to determine whether
Māori have been consulted appropriately
4

Does the organism contain infectious agents normally able to cause
disease in humans, animals, plants or fungi?
If yes, provide details here.
5

Does the organism produce desiccation resistant structures (such as
spores or cysts) that can normally be disseminated in the air?
If yes, provide details here.
6
7
Is the organism characterised to the extent that its main biological
characteristics are known?
Does the organism normally infect, colonise or establish in humans?


If yes, provide details here.
8
9
10
If the organism is a whole plant or plant tissue, do you intend to:
a) Allow it to develop reproductive structures If yes, please
provide further information on containment in section 4
b) Keep it in a closed container?
Is the host a Category 1 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
Is the host a Category 2 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
N/A


Note: If the genetic modification does not involve a Category 1 or 2 host organism then the proposed project
does not meet the criteria in section 42A(2)(a) of the HSNO Act for the rapid assessment of projects for low-risk
genetic modification.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Animal cell lines - mouse, rat, rabbit, cattle, goat, and sheep.
Yes
1
No

Is the organism normally capable of causing disease in humans,
animals, plants or fungi?
If yes, provide details here
2

Is the organism a human cell line?
If yes, provide details here of where the material has been obtained from and whether
approval has been obtained from an Ethics Committee (if required)
3

Is the organism native to New Zealand?
If yes, provide details here for example, from where will this material be obtained?
Be as specific as possible as this information may be needed to determine whether
Māori have been consulted appropriately
4

Does the organism contain infectious agents normally able to cause
disease in humans, animals, plants or fungi?
If yes, provide details here.
5

Does the organism produce desiccation resistant structures (such as
spores or cysts) that can normally be disseminated in the air?
If yes, provide details here.
6
7
Is the organism characterised to the extent that its main biological
characteristics are known?
Does the organism normally infect, colonise or establish in humans?


If yes, provide details here.
8
9
10
If the organism is a whole plant or plant tissue, do you intend to:
a) Allow it to develop reproductive structures If yes, please
provide further information on containment in section 4
b) Keep it in a closed container?
Is the host a Category 1 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
Is the host a Category 2 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
N/A


Animals - mouse, rat, rabbit, cattle, goat, and sheep.
Yes
1
Is the organism normally capable of causing disease in humans,
animals, plants or fungi?
No

If yes, provide details here
2

Is the organism a human cell line?
If yes, provide details here of where the material has been obtained from and whether
approval has been obtained from an Ethics Committee (if required)
3
Is the organism native to New Zealand?

If yes, provide details here for example, from where will this material be obtained?
Be as specific as possible as this information may be needed to determine whether
Māori have been consulted appropriately
4
Does the organism contain infectious agents normally able to cause
disease in humans, animals, plants or fungi?
Page 9 of 28
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
If yes, provide details here.
5

Does the organism produce desiccation resistant structures (such as
spores or cysts) that can normally be disseminated in the air?
If yes, provide details here.
6
7
Is the organism characterised to the extent that its main biological
characteristics are known?
Does the organism normally infect, colonise or establish in humans?


If yes, provide details here.
8
9
10
If the organism is a whole plant or plant tissue, do you intend to:
a) Allow it to develop reproductive structures If yes, please
provide further information on containment in section 4
b) Keep it in a closed container?
Is the host a Category 1 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
Is the host a Category 2 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
N/A


Human cell lines (excluding human embryonic stem cell lines and cell lines derived from people
of known Māori origin)
Yes
1
No

Is the organism normally capable of causing disease in humans,
animals, plants or fungi?
If yes, provide details here
2
Is the organism a human cell line?

If yes, provide details here of where the material has been obtained from and whether
approval has been obtained from an Ethics Committee (if required)
Material will be obtained from reputable commercial or academic sources
and will not be obtained directly from human subjects nor be derived from
Maori. Ethics approval is not required.
3

Is the organism native to New Zealand?
If yes, provide details here for example, from where will this material be obtained?
Be as specific as possible as this information may be needed to determine whether
Māori have been consulted appropriately
4

Does the organism contain infectious agents normally able to cause
disease in humans, animals, plants or fungi?
If yes, provide details here.
5

Does the organism produce desiccation resistant structures (such as
spores or cysts) that can normally be disseminated in the air?
If yes, provide details here.
6
7
Is the organism characterised to the extent that its main biological
characteristics are known?
Does the organism normally infect, colonise or establish in humans?


If yes, provide details here.
8
If the organism is a whole plant or plant tissue, do you intend to:
c) Allow it to develop reproductive structures If yes, please
Page 10 of 28
N/A
Develop in containment a project of low risk genetically modified organisms by rapid assessment
9
10
3.3
provide further information on containment in section 4
d) Keep it in a closed container?
Is the host a Category 1 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
Is the host a Category 2 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?


Nature and range of the proposed genetic modification(s)
Refer to pages 15-19 and pages 33-38 of the user guide for assistance in completing this section
Provide details on the following
Complete this section separately for each host organism to be modified only if there are significant differences
in the modifications for each of the host organisms listed above.
Information on how the new organism(s) will be developed
Vector system used,
eg cloning or
expression, plasmid,
or viral
Escherichia coli will be developed using standard cloning and
transformation techniques and will employ non-conjugative plasmid
vectors from commercial (e.g. Invitrogen pCR2.1-topo vectors) and
reputable research laboratory sources.
Non-replicative recombinant AAV vectors will be packaged using
calcium phosphate transfection of HEK293 cells with non-conjugative
plasmid DNAs (including AAV expression or targeting plasmids, the
AAV packaging plasmids, and the adenoviral packaging plasmid). The
vector system used is very similar to the three plasmid Stratagene
AAV helper free system (Figure 1) but using proprietary packaging
plasmids.
AAV expression plasmids may be tested for expression in mammalian
cells using commercially available transfection reagents (e.g. Roche’s
Fugene or Invitrogen’s Lipofectamine).
Animals will be developed using rAAV delivered to the somatic target
tissue(s) or organ(s) by injection or infusion. rAAV will not be targeted
to the ovary or testis; hence animals with germ line changes will not be
developed. Vectors may be designed to efficiently integrate into
specifically targeted locations in the chromosome.
Range of elements
that the vectors
may contain
Vectors may contain regulatory elements, coding or non-coding genes,
and regulatory regions of genes.
Regulatory elements:
Promoters
(constitutive,
endogenous
or
(e.g. chicken β-actin promoter, β-tubulin
rapamycin-responsive promoter, U6 promoter).
inducible)
promoter,
Enhancers (e.g. woodchuck post-transcriptional regulatory
element (WPRE)).
Internal ribosome entry site (e.g. encephalomyocarditis virus
IRES).
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Regulatory peptides (e.g. 2A peptide).
Regulatory elements for inducible expression (e.g. rapamycin
system).
Polyadenylation signals (e.g. bovine growth hormone polyA).
Multiple cloning sites.
Origins of replication.
Splice acceptor/donor sites.
Transcriptional activators.
Transcriptional terminator sequences.
Secretory and targeting signals.
Recombination sites and flanking sequences (e.g. CRE/Lox
system).
Selection markers (e.g. zeocin resistance, kanamycin).
Insulators.
Reporter genes such as colourimetric, bioluminescent or
fluorescent genes.
Coding, non-coding or regulatory regions of genes including:
Flanking sequences for targeted homologous recombination.
Variants with nucleotide substitutions or deletions to determine
functional domains or to modify activity.
Truncations or short inverted sequences (RNAi) as inhibitors.
Addition of commercially available protein tags (e.g. his tag) to
determine transgene localisation and/or expression.
Type, source and
function of any
donor genetic
material
Genetic material may include cDNA or genomic DNA sequences and
may be sourced from the Kingdoms Animalia, Planta, Fungi, Protista
and Monera and from viruses or viroids.
The donor genetic material will include coding, non-coding or
regulatory regions of genes coding proteins involved in the prevention,
diagnosis and treatment of endocrine, nutritional and metabolic
diseases, infectious and parasitic diseases, neoplasms, diseases of
the blood, diseases of the respiratory system, diseases of the
musculoskeletal system, diseases of the skin, diseases of the
digestive system, diseases of the circulatory system, mental and
behavioural disorders, and diseases of the nervous system.
Recombinant proteins will include:
Full-length recombinant proteins and variants with nucleotide
substitutions to modify activity or addition of proteins tags or
fusions to aid purification, localisation or expression.
Antibodies, single chain variable fragments, phage display
peptides, nanobodies, and antigens.
Genetic elements and proteins that facilitate inducible gene
expression (e.g. rapamycin system)
Any combination of the above as long as the combination of
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
traits does not fall under the exclusions listed below.
The donor genetic material will exclude:
Genetic material, other than that required for rAAV production,
that increases the pathogenicity, virulence, or infectivity of the
host organism.
Genetic material that results in the modified organism having a
greater ability to escape from containment than the unmodified
host.
Genes that encode
LD50 < 100 µg/kg.
for
vertebrate
toxins
with
an
Genetic material sourced from New Zealand indigenous fauna
and flora, persons of Māori origin, or directly sourced human
genetic material. Human genetic sequences will be
synthesised or obtained from a reputable organisation.
Nucleic acid sequences coding for a product that can lead to
uncontrolled mammalian cellular proliferation.
All genes will be sequenced and characterised prior to cloning into any
E. coli expression vector or AAV expression vector.
Use of special genetic material
Yes
Does the proposed modification use genetic material derived from
organisms capable of causing disease in humans, animals, plants or fungi?
If yes, provide details here including the sequences as well as the species and strains they
were derived from. If the genetic material to be introduced is characterised so that its
sequence and gene function are known, please state this
Page 13 of 28

No
Develop in containment a project of low risk genetically modified organisms by rapid assessment
Adenovirus is commonly associated with respiratory illness in humans. The
adenoviral helper plasmid (pFΔ6) contains approximately 10 kb of the
adenovirus 5 genome (total size of approximately 36 kb) including the E2A
(DNA binding protein), E4 (E2 transactivator) and VA (regulation of gene
expression) genes, which are required for regulation of AAV packaging (Xiao
et al., 1998). pFΔ6 is NOT able to mediate packaging of adenovirus and none
of the adenoviral genes are packaged into rAAV particles.
The chicken β-actin (CBA) promoter used in many of the AAV expression
constructs contains 381 bp of the immediate-early enhancer element from
cytomegalovirus (CMV) (Niwa et al., 1991). Cytomegalovirus rarely causes
disease in humans and this enhancer element has been widely used in human
clinical trials.
The AAV expression cassettes may also contain a 587 bp fragment of a posttranscription enhancing element (WPRE) derived from the woodchuck
hepatitis virus (Paterna et al., 2000). Woodchuck hepatitis virus causes
hepatitis in woodchucks but not in humans, and this WPRE element has been
widely used in human clinical trials.
Other commonly used and/or commercially available regulatory elements,
such as internal ribosome entry sites, may also be derived from organisms
capable of causing disease in humans, animals, plants and fungi. However,
elements will not be utilized unless they have been well characterised; if their
use would result in increased pathogenicity, infectivity (not related to rAAV
production), or virulence in the host organism: if they are toxic or have LD 50 of
greater than 100 micrograms/kg in vertebrates; or if they would increase the
ability of the host organism to escape containment.

Does the proposed modification use genetic material from native biota?
If yes, provide details here including where this material will be obtained from. Be as
specific as possible as this information may be needed to determine whether Māori have
been consulted appropriately
Does the proposed modification involve human genetic material?
Answer yes if human genetic material in any form is used, i.e. whether it is obtained
directly from humans, from a gene bank, synthesised, copied and so on.
If yes, provide details here including where the material is obtained from, and whether
approval has been obtained from an Ethics Committee (if required). Also complete
section 5 of this form.
Donor genetic material may include human genes or regulatory elements
derived from human DNA. Human genetic material will be obtained from
reputable commercial suppliers or research institutes and will not be derived
from persons of Māori origin. Approval from an Ethics Committee is not
required.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Other details of the modification
Including any unusual manipulations, if the foreign genetic material is to be expressed, where it is expected to
be expressed and what techniques will be used in the modification.
Background
Recombinant adeno-associated viral vectors (rAAV)
Originally isolated as a contaminant of adenovirus preparations, wild type adeno-associated virus
(AAV) is replication deficient – it requires the presence of a helper virus, usually adenovirus of
herpes simplex virus, for its replication – hence the Dependovirus classification (Carter and
Laughlin, 1984). AAV is not associated with any human disease (Lin and Ertl, 2008) and has
been graded as a Biosafety Level 1 (BL1) organism by the United States National Institutes of
Health (NIH), the same level as Escherichia coli. Over 50 clinical trials using rAAV vectors are
currently underway in the US including for Parkinson’s disease (Kaplitt et al., 2007).
Wildtype AAV can site-specifically integrate into a defined site, AAVS1, on human chromosome
19. Recombinant AAV has lost this ability, as vectors do not express the rep gene products
required for integration. While rAAV vectors predominantly persist in episomal chromatin forms
(Penaud-Budloo et al., 2008) there is a very low rate of quasi-random integration (Lin and Ertl,
2008) that is only detectable using positive selection. In dogs, liver-directed rAAV was
demonstrated to persist episomally for up to 8 years with no evidence of chromosomal DNA
integration (Niemeyer et al., 2009). However, rAAV vectors can be specifically designed to direct
site-specific genetic modification of chromosomal DNA by homologous recombination by
including significant stretches of homologous sequence for the target locus (Russell and Hirata,
1998).
The AAV genome is a ~4.7 kb single-stranded DNA consisting of the rep and cap genes flanked
by the inverted terminal repeats (ITRs), the only cis-sequence required for packaging. In rAAV
vectors, the rep and cap genes are replaced by the gene-of-interest and regulatory elements. For
packaging of rAAV, the rep and cap gene products are supplied in trans. The rep genes produce
the Rep proteins which are required for replication, integration and packaging. The cap genes
produce the capsid proteins VP1, VP2 and VP3 that constitute the viral particle coat. The
various serotypes of AAV, over 100 at present (Gao et al., 2004), differ in the sequence of the
capsid proteins. The variation in capsid sequence accounts for the ability of the various
serotypes to transduce different cell types.
Generation of rAAV vectors
There are a number of different strategies for the production of recombinant AAV vectors (Aucoin
et al., 2008). We utilize a three plasmid cell culture-based transfection vector packaging system
that eliminates the use of helper viruses (Figure 1). The three plasmids are the AAV expression
plasmid (contains the transgene flanked by AAV ITRs; for gene transfer) or the AAV targeting
plasmid (contains targeting construct flanked by AAV ITRs; for gene targeting), the AAV
packaging plasmid (contains AAV rep and cap), and the adenoviral helper plasmid (provides
the adenovirus helper functions). The rep and cap and genes in the AAV helper plasmid contain
artificial introns that increase their size beyond the AAV packaging limit, thus eliminating the
potential for the generation of replication competent viral particles by non-homologous
recombination (Cao et al., 2000). This system has been used for the production of vector for the
treatment of Parkinson’s disease (Kaplitt et al., 2007).
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Figure 1: Overview of rAAV production and transduction of target cells. Reproduced from
Stratagene.
The AAV expression plasmid contains the gene-of-interest under the control of a promoter
and/or other regulatory elements flanked by the 145 bp AAV ITRs. The ITRs are the only cis
sequences required for packaging and are the only AAV-derived sequences that are contained in
the rAAV vectors. Any sequence lying between the ITR’s, up to the packaging limit of ~4.7 kb,
will be packaged into the rAAV vectors. The ITRs are derived from the AAV2 serotype.
In the specifically designed AAV targeting plasmids, the sequence in-between the ITRs
contains stretches of sequence homologous to an endogenous genomic locus that may flank a
selection and/or reporter cassette or specific mutations directed at the target locus.
The AAV packaging plasmid (e.g. pH21) contains the AAV rep and cap viral sequences which
can be supplied in trans to provide the necessary viral proteins required for replication,
packaging and the viral capsid coat. There is a different AAV helper plasmid for each available
AAV serotype which contains the specific cap gene and the AAV2 rep gene. Neither the rep or
cap genes will be packaged into the rAAV vectors. The rep and cap and genes in the AAV helper
plasmid contain artificial introns that increase their size beyond the AAV packaging limit, thus
eliminating the potential for the generation of replication competent viral particles by nonhomologous recombination (Cao et al., 2000).
Wild type AAV belongs to the dependovirus genus and it relies on the presence of another virus
(typically adenovirus) to provide additional functions to allow it to replicate. The adenoviral
helper plasmid (pFΔ6) contains the adenoviral genes (E2A, E4 and VA) that provide the helper
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
functions necessary for AAV replication and hence production of rAAV (Xiao et al., 1998). None
of the adenoviral helper genes will be packaged into the rAAV vectors.
The three plasmid system is used to generate all rAAV serotype vectors and there are 3 types of
vectors that can be developed by varying the AAV-derived components of the helper plasmid:
a. “True” rAAV serotype vectors: AAV serotype vectors are generated using the ITR’s and
capsid proteins derived from the same serotype, e.g. the traditional rAAV2 vector has
all AAV2-derived elements.
b. Pseudotyped rAAV vectors: these vectors contain the ITRs from one serotype (AAV2)
but express capsid proteins from a different serotype.
c. Chimeric AAV vectors: Chimeric vectors have a capsid shell containing a mix of capsid
proteins from two serotypes e.g. AAV1 and AAV2. This can be produced by inclusion of
two AAV helper plasmids expressing two different cap genes (therefore a four plasmid
transfection). This strategy can be used to facilitate purification and also to broaden the
selectivity of rAAV for certain cell populations.
In vitro and in vivo studies
For in vitro studies, mammalian cells will be transduced with rAAV vectors to examine transgene
expression or for targeted homologous recombination. All in vitro work is conducted under sterile
conditions and under stringent culturing conditions in order to maintain cell viability.
For in vivo studies, rAAV vectors may be administered topically (e.g. intranasal), enternally
(e.g. orally) or by parenteral injection or infusion (e.g. intravenously) to target specific adult
organ(s) and/or tissue(s) using standard surgical procedures. When rAAV vectors transduce a
cell, the vector is unable to replicate and spread without the presence of with both wild-type AAV
and adenovirus. Germ line cells will not be targeted.
rAAV vectors expressing reporter genes have demonstrated high and sustained levels of
transgene expression. In dogs, rAAV-mediated gene transfer of factor IX was recently
demonstrated to persist episomally for up to 8 years, with no evidence of chromosomal DNA
integration, and with high levels of factor IX expression (Niemeyer et al., 2009). Only one surgical
intervention is required to maintain prolonged gene expression.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
3.4
Identify the category of experiments as described in the HSNO (Low-Risk Genetic
Modification) Regulations, 2003.
Refer to pages 17-19 and pages 33-38 of the user guide for assistance in completing this section.
Escherichia coli – non pathogenic laboratory strains
Yes
No

1
Is the proposed modification to a Category 1 host organism?
2
Is the proposed modification to a Category 2 host organism?

3
Will the proposed modification increase the pathogenicity, virulence,
or infectivity of the host organism to laboratory personnel, the
community, or the environment? If you answer yes to this question,
please confirm with an ERMA advisor that the modification is low risk.

4
Will the proposed modification result in a genetically modified
organism with a greater ability to escape from containment than the
unmodified host? If you answer yes to these questions, please confirm
with an ERMA advisor that the modification is low risk.

5
Is the proposed modification to be carried out under a minimum of
PC1 containment?
6
Is the proposed modification to be carried out under a minimum of
PC2 containment?
7
Does the proposed modification conform to the requirements of a
Category A genetic modification?
8
Does the proposed modification conform to the requirements of a
Category B genetic modification?




Explanation of categorisation, if necessary: This is particularly important for work involving pathogenic
microorganisms and viral vectors.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
HEK293 cell line – the production of the rAAV viral vectors
Yes
No

1
Is the proposed modification to a Category 1 host organism?
2
Is the proposed modification to a Category 2 host organism?
3
Will the proposed modification increase the pathogenicity, virulence,
or infectivity of the host organism to laboratory personnel, the
community, or the environment? If you answer yes to this question,
please confirm with an ERMA advisor that the modification is low risk.
4
Will the proposed modification result in a genetically modified
organism with a greater ability to escape from containment than the
unmodified host? If you answer yes to these questions, please confirm
with an ERMA advisor that the modification is low risk.

5
Is the proposed modification to be carried out under a minimum of
PC1 containment?

6
Is the proposed modification to be carried out under a minimum of
PC2 containment?
7
Does the proposed modification conform to the requirements of a
Category A genetic modification?
8
Does the proposed modification conform to the requirements of a
Category B genetic modification?





The HEK293 cell line is classified as Category 1 host organism. HEK293 cells can be identified
to the appropriate taxonomic level, do not normally cause disease, are free of infectious and
pathogenic agents, do not produce cysts, and is biologically well characterised. The production
of rAAV vectors using cell lines such as HEK293 and rAAV packaging plasmids is a Category B
genetic modification as this modification increases the infectivity of the host as infectious
particles are produced. Due to the genetic material exclusions, this research will fall under the
Low Risk Regulations.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Transduction of mammalian cell lines with the replication-deficient rAAV viral vectors
Yes
No

1
Is the proposed modification to a Category 1 host organism?
2
Is the proposed modification to a Category 2 host organism?

3
Will the proposed modification increase the pathogenicity, virulence,
or infectivity of the host organism to laboratory personnel, the
community, or the environment? If you answer yes to this question,
please confirm with an ERMA advisor that the modification is low risk.

4
Will the proposed modification result in a genetically modified
organism with a greater ability to escape from containment than the
unmodified host? If you answer yes to these questions, please confirm
with an ERMA advisor that the modification is low risk.

5
Is the proposed modification to be carried out under a minimum of
PC1 containment?
6
Is the proposed modification to be carried out under a minimum of
PC2 containment?
7
Does the proposed modification conform to the requirements of a
Category A genetic modification?
8
Does the proposed modification conform to the requirements of a
Category B genetic modification?




The mammalian cell lines are classified as Category 1 host organisms. The mammalian cells can
be identified to the appropriate taxonomic level, do not normally cause disease, are free of
infectious and pathogenic agents, do not produce cysts, and are biologically well characterised.
The transduction of the mammalian cell lines with replication-defective rAAV is a Category A
genetic modification as this modification will not increase pathogenicity, infectivity (infectious
particles will not be produced) or virulence of the host and will not result in a host with a greater
ability to escape from containment. Due to the genetic material exclusions, this research will fall
under the Low Risk Regulations.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
rAAV transduction of animal organs and tissues in vivo
Yes
No
1
Is the proposed modification to a Category 1 host organism?
2
Is the proposed modification to a Category 2 host organism?
3
Will the proposed modification increase the pathogenicity, virulence,
or infectivity of the host organism to laboratory personnel, the
community, or the environment? If you answer yes to this question,
please confirm with an ERMA advisor that the modification is low risk.

4
Will the proposed modification result in a genetically modified
organism with a greater ability to escape from containment than the
unmodified host? If you answer yes to these questions, please confirm
with an ERMA advisor that the modification is low risk.

5
Is the proposed modification to be carried out under a minimum of
PC1 containment?

6
Is the proposed modification to be carried out under a minimum of
PC2 containment?
7
Does the proposed modification conform to the requirements of a
Category A genetic modification?
8
Does the proposed modification conform to the requirements of a
Category B genetic modification?
X




Whole animals are classified as Category 2 hosts. The transduction of the somatic animal
organs and tissues with rAAV is a Category B genetic modification. Genetic modification of
somatic animal organs and tissues with replication-defective rAAV will not increase
pathogenicity, infectivity (infectious particles will not be produced) or virulence of the host, will not
result in a host with a greater ability to escape from containment, and the introduced sequences
will be well characterised with respect to gene sequence and function. Due to the genetic
material exclusions, this research will fall under the Low Risk Regulations.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Section Four – The proposed containment system
Refer to page 20 of the user guide for assistance in completing this section
Describe the containment facility and the proposed containment system (physical and operational)
Question
Answer
Which MAF/ERMA Standard The work will be carried out in AgResearch facilities at
is this containment facility
Palmerston North and Hamilton approved to the required
approved under?
standard for the specific aspects of the work. MAF/ERMA New
Zealand Standard Facilities for Microorganisms and Cell
Cultures: 2007a for work requiring laboratory containment
involving bacteria and cultured cells; MAF/ERMA New Zealand
Standard Containment Facilities for Vertebrate Laboratory
Animals for work requiring animal containment involving live
animals.
What physical containment
level (AS/NZS 2243.3:2002)
is this containment facility
approved to operate at (where
relevant)?
AgResearch operates laboratory containment facilities at the
Palmerston North (Grasslands and Hopkirk Research Institute)
and Hamilton (Ruakura) campuses which are approved to PC1
and PC2 levels. Work carried out in laboratory containment will
be undertaken in facilities approved to at least the required
physical containment level. The work with animals will be carried
out in animal facilities approved at PC2 level.
What other physical measures
do you propose to use to
contain this organism?
MAF-approved transfer of organisms between laboratory and
animal containment facilities will be in double containment and
carried out according to the relevant Standards.
What procedural or
operational measures do you
propose to use to contain this
organism?
All procedural and operational measures to contain, or dispose
of, the genetically modified organisms will be conducted in MAF
Biosecurity approved containment facilities in compliance with
MAF/ERMA
New
Zealand
Standard
Facilities
for
Microorganisms and Cell Cultures: 2007, MAF/ERMA New
Zealand Standard Containment Facilities for Vertebrate
Laboratory Animals and AS/NZS 2243.3:2002.
All processing of products from genetically modified organisms
shall be performed within a containment facility.
Production of rAAV will be restricted to trained personnel and
will be performed in Class II Biological Safety Cabinets. In
addition, injection of rAAV into animals will be restricted to
trained personnel to mitigate the risk of “self inoculation”.
There is no other information relevant to the containment of the
Any other information
relevant to the containment of organisms.
the organism.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Section Five – Identification and assessment of adverse effects
Refer to page 21 of the user guide for assistance in completing this section
This section should only be completed in detail if pathogenic microorganisms, human cells, native or valued
flora and fauna were identified as host or sources of donor genetic material in section 3. It is expected that
organisms meeting the low-risk regulations will not normally have any significant biological risks associated
with them. However, there may still be some adverse effects that need to be identified and assessed. This might
include economic, social and cultural adverse effects and other risks not addressed by the HSNO (Low-Risk
Genetic Modification Regulations) 2003
What adverse effects could this organism have on the environment?
For all stages of the life cycle
All organisms that will be developed in this project meet the low-risk regulations and are kept
in physical containment. Thus, it is expected that these organisms will have minimal
environmental risks associated with them. Even in the unlikely event of escape into the
environment, environmental risks are minimal as further outlined below.
The attenuated strains of E. coli used to propagate the vector packaging plasmids and for
cloning are highly unlikely to survive outside of laboratory conditions, thus any adverse effect
on the environment is highly improbable.
The mammalian cells lines transduced with rAAV vectors or transfected with AAV targeting or
expression plasmids require stringent culture conditions and will therefore not survive outside
of laboratory conditions, thus any adverse effect on the environment is highly improbable.
The potential for rAAV to survive outside of containment and to transduce animals in the
environment is extremely low; therefore, inadvertent release of rAAV would have negligible
environmental impact. Deliberate inoculation of high titer rAAV to animals outside of
containment would have negligible environment impact considering that 1) rAAV is replication
defective and no further infectious particles will be produced, and 2) the packaged genetic
material will not result in increased pathogenicity, infectivity, virulence or toxicity.
Animals genetically modified with rAAV will be kept in containment. In the unlikely event that
GMOs escaped, or were released, from MAF-approved containment facilities it is highly
unlikely that the GMO would have any more adverse effect on the environment than an
unmodified host. Contact or mating of rAAV modified animals with “wild” populations would
not enable transmission of the rAAV vector or the genetic modifications that have been
developed as rAAV is replication defective and therefore cannot spread either within the host
organism or be passed from one animal to another. While rAAV vectors used for gene
transfer predominantly persist in episomal chromatin forms (Penaud-Budloo et al., 2008)
there is a very low rate of quasi-random integration (Lin and Ertl, 2008). In addition, rAAV
vectors used for gene targeting are able to direct efficient site-specific genetic modification of
chromosomal DNA (Russell and Hirata, 1998). Therefore, rAAV will only be targeted to
somatic cells and hence released GMOs would not be capable of passing on chromosomal
genetic modifications to progeny.
The ability to produce a replication competent vector by non-homologous recombination is
eliminated by the design of the AAV helper plasmid. The rep and cap genes in the AAV
helper plasmid contain artificial introns that increase their size beyond the AAV packaging
limit, thus eliminating the potential for the generation of replication competent viral particles by
non-homologous recombination (Cao et al., 2000).
The generation of replication competent vector through super infection of rAAV transduced
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
cells with both wild type AAV and helper virus (adenovirus or herpes simplex virus) is a
theoretical possibility however the chance of getting both wild type AAV and a helper virus
infecting the transduced cells is extremely remote. In the unlikely event that replication
competent rAAV was generated it would have negligible environmental impact since (1) still
require escape or deliberate release of animals from containment and (2) as per the
exclusions, packaged genetic material will not result in increased pathogenicity, infectivity,
virulence or toxicity.
The animals to be used in this application are already common in New Zealand and hence
there is no risk of introducing a new species to the environment.
What adverse effects could this organism have on human health and safety?
Wild-type AAV causes no known human disease and has been widely used for human gene
therapy applications.
The greatest risk of occupational exposure to staff would be during the preparation of viral
particles or during injection of animals. The use of a Class II Biological Safety Cabinet during
production will minimize this possibility. Even with deliberate or inadvertent injection of high
titer rAAV vectors the exclusion of genetic elements that would result in increased
pathogenicity, infectivity, virulence or toxicity this would minimize the possibility of any
adverse effects on human health.
What adverse economic effects could this organism have?
To the best of our knowledge, there are no economic effects that inadvertent or deliberate
release of the genetically modified organisms described in this application would entail.
What adverse effects could this organism have on the relationship of Māori and their culture
and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna
and other taonga (taking into account the principles of the Treaty of Waitangi)?
Include details of any consultation that you have undertaken.
To the best of our knowledge, there are no adverse effects of the GMOs on the relationship of
Māori and their culture and traditions, Genetic material will not be sourced from New Zealand
indigenous fauna and flora and human genetic material will be obtained from reputable
commercial suppliers or research institutes and will not be derived from Māori donors. In
addition, the work is undertaken in physical containment minimising any potential
environmental impact.
The research covered in this application has been reviewed by local iwi at both the
Palmerston North and Hamilton sites. No areas of concern for Māori were identified.
This application was reviewed by Jonathan Procter of Tanenuiarangi o Manawatu Inc. A letter
from Jonathan is attached as Appendix 1.
This application was reviewed by Wiremu Puke of Ngati Wairere. A letter from Wiremu has
been submitted directly to ERMA.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Are there any other potential adverse effects?
Not that we are aware of.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Section
Six
–
Additional
Refer to page 31 of the user guide for assistance in completing this section
Additional Information
information
Y/N
If yes, explain
Do any of the organism(s) need
approvals under any other New
Zealand legislation?
Y
Modification of whole animals will also
require approval under the Animal Welfare
Act 1999.
Does New Zealand have any
international obligations relating to
(any of) the organism(s)?
N
Have any of the new organism(s) in
this application previously been
considered in New Zealand or
elsewhere?
Y
ERMA application GMD03096: To develop in
containment for use in biomedical research
recombinant adeno-associated viral vectors
and genetically modified rodents and
mammalian cell lines expressing transgenes
that have roles or potential roles in regulating
mammalian cell growth.
GMD09011: Development of recombinant
adeno-associated viral vectors (rAAV) for
delivery of transgenes to cell lines and
animals in containment to investigate animal
health and disease.
Is there any additional information
that you consider relevant to this
application that has not already been
included?
Following the development of this
organism what will the genetically
modified organism be used for? eg
will experimental animals or plants
be exposed to this organism?
N
rAAV vectors will be used to transduce
various animals in containment. The animals
will be then experimentally investigated for
transgene related effects in containment.
Provide a glossary of scientific and technical terms used in the application
AAV – wild type adeno-associated virus
rAAV – recombinant adeno-associated virus. AAV that is genetically modified so that it has no
ability to replicate but functions as a gene delivery vehicle to deliver a gene to a host cell.
Capsid – protein shell of a virus.
Cis – genetics elements contained on the same DNA molecule.
Concomitant – two or more things occurring simultaneously.
Biopharmaceuticals – medical drugs manufactured using biotechnology.
Bioreactor - vessel for the production of biological products.
Biotechnology – any technological application that uses biological systems, living organisms,
or derivatives thereof, to make of modify products or processes for specific use.
Episomal – DNA that is not integrated into the chromosome.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Expression construct – a DNA molecule containing a combination of coding and regulatory
sequences necessary to produce expression of a transgene
Gene targeting – site-specific genetic modification of chromosomal DNA by homologous
recombination
Gene transfer - the extra-chromosomal expression of a gene expression construct that doesn’t
interfere with the integrity of the host genome
Homologous recombination – genetic recombination between two identical strands of DNA.
ITR – inverted terminal repeat
Neoplasm – abnormal mass of tissue as a result of abnormal multiplication of cells
Non-conjugative bacterial strain – strains that are unable to mediate the transfer of genetic
material (plasmids) to other bacteria.
Nutraceutical – food extracts with medicinal effect on health.
Promoter – piece of DNA that drives the expression of a transgene.
Pseudotyped - the process in which the cellular specificity of the vector is changed by the
replacement of its capsid proteins from a related virus that infects a different or broader
range of cell types.
Reporter gene – a gene with a product that can be readily detected or that catalyses a reaction
that can be readily detected
RNAi – various methods of down regulating RNA expression by small RNA molecules.
Serotype – classification of viruses based on capsid proteins of AAV.
Somatic – non-germline tissues.
Trans – a genetic element produced on one DNA molecule that acts on another DNA molecule.
Transduction – the transfer of the genetic material into a target cell by a recombinant viral
vector.
Transfection – transfer of genetic material into a host cell, usually in a plasmid vector.
Transgene – the selected gene-of-interest or genetic material.
Transgenic – genetically modified organism. Organism whose genome has been altered by the
inclusion of foreign genetic material.
List of appendices attached:
Appendix 1
Letter of support from Jonathan Procter of Tanenuiarangi o Manawatu Inc.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
List of references attached:
Aucoin, M.G., Perrier, M., and Kamen, A.A. (2008). Critical assessment of current adenoassociated viral vector production and quantification methods. Biotechnology advances 26,
73-88.
Cao, L., Liu, Y., During, M.J., and Xiao, W. (2000). High-titer, wild-type free recombinant
adeno-associated virus vector production using intron-containing helper plasmids. Journal of
virology 74, 11456-11463.
Carter, B.J., and Laughlin, C.A. (1984). Adeno-associated virus defectiveness and the nature
of the adenovirus helper function. In The parvoviruses, K.I. Berns, ed. (New York, Plenum
Press), pp. 67-128.
Gao, G., Vandenberghe, L.H., Alvira, M.R., Lu, Y., Calcedo, R., Zhou, X., and Wilson, J.M.
(2004). Clades of Adeno-associated viruses are widely disseminated in human tissues.
Journal of virology 78, 6381-6388.
Kaplitt, M.G., Feigin, A., Tang, C., Fitzsimons, H.L., Mattis, P., Lawlor, P.A., Bland, R.J.,
Young, D., Strybing, K., Eidelberg, D., et al. (2007). Safety and tolerability of gene therapy
with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open
label, phase I trial. Lancet 369, 2097-2105.
Lin, S.W., and Ertl, H.C.J. (2008). Safety of adeno-associated viral vectors. Future Virology
3, 491-503.
Niemeyer, G.P., Herzog, R.W., Mount, J., Arruda, V.R., Tillson, D.M., Hathcock, J., Van
Ginkel, F.W., High, K.A., and Lothrop Jr, C.D. (2009). Long-term correction of inhibitorprone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy.
Blood 113, 797-806.
Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression
transfectants with a novel eukaryotic vector. Gene 108, 193-200.
Paterna, J.C., Moccetti, T., Mura, A., Feldon, J., and Bueler, H. (2000). Influence of promoter
and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in
the rat brain. Gene therapy 7, 1304-1311.
Penaud-Budloo, M., Le Guiner, C., Nowrouzi, A., Toromanoff, A., Cherel, Y., Chenuaud, P.,
Schmidt, M., von Kalle, C., Rolling, F., Moullier, P., et al. (2008). Adeno-associated virus
vector genomes persist as episomal chromatin in primate muscle. Journal of virology 82,
7875-7885.
Pollock, D.P., Kutzko, J.P., Birck-Wilson, E., Williams, J.L., Echelard, Y., and Meade, H.M.
(1999). Transgenic milk as a method for the production of recombinant antibodies. Journal of
immunological methods 231, 147-157.
Russell, D.W., and Hirata, R.K. (1998). Human gene targeting by viral vectors. Nature
genetics 18, 325-330.
Xiao, X., Li, J., and Samulski, R.J. (1998). Production of high-titer recombinant adenoassociated virus vectors in the absence of helper adenovirus. Journal of virology 72, 22242232.
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