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APPLICATION SUMMARY
Prepared by applicant
Application code:
Applicant:
GMD02028
Develop in Containment any New Organism under the
Hazardous Substances and New Organisms (HSNO) Act 1996
AgResearch Limited
Applicant contact:
Paul Atkinson
Purpose:
The two broad objectives of this containment application are
to develop transgenic cattle that can express functional
therapeutic foreign proteins in their milk, and to develop
transgenic cattle to study gene function and genetic
performance
Date application received:
1 May 2002
Date application verified
13 May 2002
ERMA New Zealand contact:
Suzanne Lambie
Application category:
SUMMARY
Expression of therapeutic and active proteins by transgenesis
The two broad objectives of this containment application are to develop transgenic cattle that
can express functional therapeutic foreign proteins in their milk, and to develop transgenic
cattle to study gene function and genetic performance.
This application is to develop genetically modified cattle that possess either exogenous genes
controlled to express novel therapeutic proteins in their milk, or modifications of endogenous genes
for altered phenotypic expression of products. Of all systems capable of expressing genetically
modified proteins, the cow mammary gland is necessary because of the high-protein output and the
ability to produce correctly processed functional proteins.
The application is on a “project” rather than single organism basis. We have defined specific
parameters of the project to ensure that risk is managed. These parameters include a single recipient
type (cattle), limited donor species (cattle, sheep, goat, deer, mice, copy human), limited types of
modification (deletion, insertion, deletion and insertion), a restricted number of modifications,
containment of Tg and GMO to experimental sites, a research program to account for all insertions
of Tg DNA and monitoring of genetic modification in microorganisms at disposal sites to ensure no
escape of functional Tg genes. The potential risk for the classes of genes defined does not vary
between individual genes within the class, and we note that genes we will transfer are already in the
environment and fall into the low risk category as defined by the HSNO (Low-Risk Genetic
Modifications) Regulations, 1998. Genes encoding toxins, allergens or human virus receptors will
not be used in transgenesis.
A project based application gives us necessary flexibility to change experimental variables
according to the outcomes of intermediate steps in the experimental process.
The application is for a development and that means genetic modification of any organism but does
not include field testing. In a development program, scientific unknowns are researched and worked
through. The work is not aimed at determining environmental impacts as a field test. A field test
aims to determine environmental impacts of technologies already developed and also to produce a
self-sustaining herd of cows for assessment under controlled field conditions. The development
envisaged in this application in large part will occur in laboratories but whether the transgenesis and
expression in Tg calves is successful will be determined in a highly regulated containment farm.
The application is written sufficiently generally to allow such systematic testing of key experimental
variables as is required in the development of a technology but also to allow the development of Tg
expression/alteration of a number of genes whose products fall into defined classes.
Thus this application seeks approval to develop conditions for controlling expression of genes, of
human and animal pharmaceutical value, by transgenesis and to assess the function of gene
products. Large quantities of many functional proteins require mammalian expression systems for
mammalian proteins and often require tissue systems rather than cultured mammalian cells
(Appendix 10).
We seek approval of this application for the development of new knowledge. Generic issues of the
science will include achievement of significant posttranslational modification, the correctly spliced
isoforms of mRNA and in expression of large amounts of functional human and other therapeutic
and bioactive proteins in milk and the probing of gene function. An example of the importance of
correct splice and posttranslational isoforms is exemplified by human myelin basic protein (hMBP),
which has four splice variants derived from seven exons. It also has a number of charge isoforms
based chiefly on posttranslational de-immination of arginines, protein phosphorylations or
carboxylations. High value biopharmaceutical (see, for example, Appendix 6) protein could also be
a bovine anti-microbial peptide or a mouse monoclonal antibody. Complexities in mammalian
protein expression, also including oligomerization, proteolytic processing and glycosylation all
affect the function of some proteins. As an example, regulatory proteins of the immune system are
especially affected by oligomerisation and the state of terminal sugars in N-linked glycosylation.
The application seeks approval to develop transgenic embryos and then transfer those embryos to
conventional recipient cattle housed in a containment facility, better described as an outdoor
laboratory, at Ruakura thus producing transgenic calves. We plan to evaluate expression of the
transgene (or modification) in the facility. The containment facility comprises buildings and
pastures all within a security controlled perimeter, in order to provide for optimal conditions for calf
growth.
Genetic modifications will be introduced in to cattle, and will result in the expression of modified
forms of an endogenous protein or in the expression of exogenous proteins in milk of the animals.
All modifications will be stable genetic changes, being incorporated in to the genome of the animals,
and will be inherited in the normal Mendelian fashion. Genetic modification is expected to occur at
a number of steps (a) to (e) described below. The scope of research for this project based
application will be limited to production of scarce, valuable human and animal therapeutics and
bioactives in the protein, glycolipid and carbohydrate classes of substances to be secreted into cow’s
milk and chemically purified to homogeneity. The proposed research also includes probing gene
function by alteration (e.g. deletion) of limited numbers of genes and their proteins. Some
substances of human therapeutic value can only be expressed in sufficient and functional amounts in
large lactating animals. These complex substances will be increasingly needed as our understanding
of human and animal diseases increases.
Transgenesis will be used for the authentic expression of splice-modified and posttranslationalmodified human therapeutic proteins expressed in cow’s milk in amounts sufficient for purification
of the proteins in useful amounts (Appendix 10). The technology being developed will be generic to
many valuable therapeutic proteins and the outcomes important to new scientific knowledge and to
future useful products. The organisms created by transgenics can be described as genetically
modified cattle that possess modifications to endogenous genes or exogenous genes controlled to
express novel, therapeutic and bioactive proteins and substances in milk for human health therapy
and for improved understanding of animal performance and genetic gain.
The source of the donor nucleic acid material and the purpose of the modification
The donor nucleic acids, in term of genes being studied, will be derived from mammalian sources of
New Zealand (where possible) livestock (cattle, sheep, deer and goats), from murine origin or from
human genes. Genes of ruminant origin will be derived from New Zealand animals of known
pedigree. Transmissible Spongiform Encephalopathys, Bovine Spongiform Encephalopathy (BSE),
sheep scrapie, and Chronic Wasting Disease (CWD) will pose no risk in this experiment.
Transmissible Spongiform Encephalopathy (TSE) infectivity is transmitted by a specifically
misfolded shape of a normal cellular protein, prion protein (see Saborio et al, 2001). The sources of
TSE infectivity (BSE, scrapie, CWD of deer) are not present in NZ (reviewed Atkinson, 2001). In
any event purified genes, copied by bacteria or PCR techniques, will be used for transfections and
preparations will contain no protein as possible sources of TSE infection. In general, such elements
of viruses as structural, non-structural, or polymerase will not be used in the proposed experiment.
No genetic elements from human or mammalian pathogens capable of recreating the viable
pathogen will be used. No toxin genes from any source or genes for biocides of any nature will be
used. Genes for known allergens or human virus receptors will not be used. Where human genes
are used they will be sourced from commercial gene banks overseas and synthetically produced.
None will be sourced from New Zealand. Other donor nucleic acids to be used may include reporter
genes, selectable marker genes, and promoter sequences which may be derived from mammalian
and non-mammalian organisms.
The development of a line (modification) of transgenic cattle will initially span a period of 3-4 years
in which production of the transgenic cattle will occur in the first year and breeding and milking of
the animals would be carried out in the second and third years. Hormonal induction of lactation is
after 16 months and will allow first characterisation of expression. First natural lactation is after
approximately 3 years and determination of milk and protein yields will be year 4. The total length
of the experiment will not exceed 10 years. Successful expression of transgene may lead to a field
test application to expand the herd to test performance in a field test situation.
What the transgenic organism (GM-cows) will be used for and why it has been selected
The genetic modifications will result in either protein products in the milk of the animals or
modifications of the phenotype of the animal for the study of gene function or for genetic
improvement.
The mammary gland is capable of very high protein output and therefore production of very high
amounts of transgenic protein can be achieved (e.g. the production of fibrinogen in the udder is
about 1000 fold higher than achieved in cultured cells) see Appendix 10. Also, milk is an easily
harvested fluid and the range of proteins that can be expressed in it is wide, from single chain to
complex proteins requiring significant post-translational modification. Evidence suggests that the
udder can posttranslationally modify complex proteins better than other systems (e.g. transgenic
mammary-produced Fibrinogen and Protein C are both functional/active in vitro).
The ultimate purpose of introducing a novel protein into the milk is the development and production
of pharmaceutical products to meet unmet human health clinical needs. However, these studies are
aimed at development of a functioning transgene and gene products in mammary tissues and have
the outcome of new scientific knowledge on protein expression and gene function.
The risks, costs and benefits and the assessment of these
Risks
•
Physico-biochemical risk in the types of application and the genes to be expressed is not
more than PC1. Apart from selection markers, only mammalian genomic material will be
used to modify the cattle genome in animals. In general, only one mammalian gene at a
time, out of 30,000 to 40,000 total cattle genes will be used in transgenesis. Human
equivalent genes to be used for transgenesis are 85-95% homologous to cattle and the risk of
the modified animal is no greater than the normal genes of existing cows and human beings.
Categories of specific risk genes are excluded. It is very unlikely that transgenic cattle will
escape or be released from the facility, and even less likely that any successful breeding to
produce a sustainable population of introgressed genes would occur without deliberate
human intervention for which this is not an application. Breeds are examples of humancontrived gene introgression and are maintained by ‘human selection pressure’. In the
absence of such selection pressure (selection and culling) breeds rapidly revert to feral types
and original genetic homeostasis of species underscoring that allelic frequencies evolve
which are best suited to the given environments and not just the mere presence of specific
alleles.
There is no conjectural adverse, or positive effects of the animals on the native flora or fauna
that are different from the current conventional farmed cattle spread throughout New
Zealand. No adverse effects are expected on the environment because the transgenes are
unlikely to amplify and survive outside of cattle should they escape the GM animals, and the
animals are not planned for use in normal commercial reproduction. We will likely mate
animals to check gene stability and so some reproduction is likely to occur as part of the
development program described here, especially as in the case of gene knock-outs in order
to achieve homozygosity (see page 15 “Description of Project” and Step (f) page 19.
•
No adverse effects are predicted or anticipated on human health or safety from exposure to,
or consumption of milk or meat from the transgenic cattle expressing therapeutic human
proteins. However, by express intention and execution of AgResearch, no such transgenic
cattle or their products will enter the human food chain. It is relevant to note that these
transgenic animals and their products will have an intrinsic value both experimentally and
therapeutically way beyond any conceivable food value and there will be no incentive to use
them as food. Transgenic animals used for study of genetic gain will likewise not enter any
food chain as a result of this application. However, even if they did inadvertently enter the
environment, genes so released would not be substantially different from those already
present in the environment in their natural hosts in studies on therapeutic protein expression.
In gene heritability studies, mutant genes from outliers in natural variation are also present in
the environment (Galloway et al, 2000) and pose no hazard. Because the Tg cattle subject
of this application will never leave containment or enter any food chain, issues of direct
horizontal gene transfer are not applicable. Background on important HGT issues is given
in Appendix 7 should escape of the animals occur accidentally. In anticipation of the
Hazardous Substances and New Organisms (Genetically Modified Organisms) Amendment
Bill (reported back 28 March 2002) the application includes explicit information on “human
safety and ecological effects” and controls for outdoor containment.
•
There may be adverse effects arising from an affront to spiritual beliefs and cultural values,
the subject of an ongoing assessment of risk in consultation with Maori. Issues of
papatuanuku have been raised for discussion by some members of Ngati Wairere for
example, in relation to ground water, and are part of the ongoing consultation between Ngati
Wairere and AgResearch (see for example, Appendix 4 for evidence of recent consultation;
Appendix 5 for evidence of earlier consulation).
Costs
•
“Cost” is defined in Reg 2 of the Methodology Order as “the value of a particular adverse
effect expressed in monetary or non-monetary terms”. There is one potential adverse effect
which generates a greater than negligible risk. This is the adverse effect (or effects) arising
from affront to spiritual beliefs and values of Ngati Wairere (and maybe other Maori/other
cultures). This is assessed as non-monetary. Its magnitude is high for those who hold the
spiritual beliefs and values.
Benefits
•
A major benefit from the work described in this application will be increased scientific
knowledge, in a number of areas including the following: complex protein expression and
appropriate posttranslational modification, potential of the mammary gland to express
authentic functional proteins coming from multiple splice isoforms and a range of
posttranslational modifications, functional characterisation of bovine genes (by overexpression of authentic product), development of large animal sources of human proteins for
research into human diseases namely the provision of therapeutic and research reagents,
improvement in the efficiencies of cloning technologies by an increased understanding of
nuclear reprogramming that occurs in nuclear transfer (NT), and in vitro embryonic
development and transplant technologies in livestock animals. Somatic cells from day 60
foetuses will mostly be the source of cloning genetics but adult somatic cells may be used
also and genetic reprogramming of the donor cell nucleus during nuclear reprogramming
will be analysed providing another scientific benefit. A further benefit will be to improve
the potential for rate of genetic gain by understanding through transgenesis of the genetic
basis of outliers, namely the genes involved in beneficial natural mutations identified
through techniques of quantitative genetics. We will not attempt direct use of Tg for genetic
gain, which is unlikely to be an economic way of accelerating genetic gain in herds.
Summary of assessment of risks, costs and benefits
The risks have been assessed as low and the track record of the containment facility indicates that
the likelihood of escape is very low. In any event, the transgenic animals generated will have high
intrinsic value because of the sophistication and cost of their development, but also because of the
therapeutic and biologically active substances they may be producing. Such animals will not enter
the food chain. AgResearch recognises that the risk to the relationship of Maori (particularly Ngati
Wairere) with their taonga is likely to be significant. AgResearch does not believe that this risk
outweighs the benefits of the research. The benefits of the programme are considerable particularly
the advancement of scientific knowledge. AgResearch believes the benefits potentially far outweigh
the conjectural or potential adverse effects.
The containment systems proposed
Molecular work with bacterial and animal cell cultures will be done in laboratories meeting PC1 and
PC2 criteria.
The outdoor containment facility is based on MAF Biosecurity Authority guidelines for low security
quarantine facilities. The facility design and management has been based on MAFBA Standard
154.03.06. The containment facility was approved with application GMF98009. The containment
facility has the characteristics of an outdoor laboratory in order to provide animals with healthy
living conditions whilst the transgenes of interest are studied.
The perimeter fencing of the facility completely encloses the site within two, 2 metre high netting
fences. This standard of fencing exceeds the low security quarantine standards. A two-metre
spacing separates the two fences. The area between the fences will be sprayed to control grass
growth and will remain free of livestock animals so as to act as an early warning of possible
breakout of stock if cattle enter the area. In order to avoid any possibility of trees falling across the
perimeter fence during a storm, either the fences have been positioned so as to avoid large old trees
or the trees have been removed from the site. Likewise, no fences have been placed over or near
waterways, which may also be difficult to ensure complete containment.
Within the enclosed containment facility, we have also located the buildings and yards that will be
used for animal handling and care. The transgenic animals, or recipient cattle carrying transgenic
embryos will not leave or be taken out of this containment facility. Furthermore, embryos
reconstructed in the laboratory will only be transferred to recipient animals within the containment
area.
This application is comparable to AgResearch’s previous cattle application GMF98009 that
included modifying milk proteins in cloned transgenic cattle. This application was approved
by ERMA and we refer to the application and decision. Practices will be the same as in the
previously approved application.