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Application Title: Cloning Genes for Expression in AAV Vectors For

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Application Title: Cloning Genes for Expression in AAV Vectors For NO3P Develop in containment a project of low risk genetically ER-AF-NO3P-3 modified organisms by rapid assessment 12/07 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 Yes Appendices enclosed Yes Confidential information identified and enclosed separately NA Copies of references attached NA Application signed and dated Yes Electronic copy of application e-mailed to ERMA New Yes Zealand Signed: Date: 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 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: AgResearch Limited Postal Address: Ruakura Research Center East St Private Bag 3123 Hamilton 3240 Physical Address: Phone: 07 856 2836 Fax: 07 838 5012 Email: Name and details of the key contact person If different from above Name: Ross J. Bland Postal Address: 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: Richard Scott Postal Address: 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 adeno- associated 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. Page 3 of 28 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. Page 4 of 28 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
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