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.

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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.

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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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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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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 Escherichia coli (Migula 1895; Castellani and Chalmers, 1919). Including full taxonomic authority Common name(s) E. coli If any Type of organism Bacterium eg bacterium, virus, fungus, plant, animal, animal cell Taxonomy Gamma Proteobacteria, Enterobacteriales, Enterobacteriaceae Class, order and family Strain(s) Non-pathogenic laboratory strains (e.g., Genetically crippled If relevant derivatives of Escherichia coli K12 and strain B) 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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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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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 No 1 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 Is the organism characterised to the extent that its main biological  characteristics are known? 7 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: N/A 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? 9 Is the host a Category 1 organism (as defined in the HSNO (Low-Risk  Genetic Modification) Regulations 2003)? 10 Is the host a Category 2 organism (as defined in the HSNO (Low-Risk  Genetic Modification) Regulations 2003)? 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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Animal cell lines - mouse, rat, rabbit, cattle, goat, and sheep. Yes No 1 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 Is the organism characterised to the extent that its main biological  characteristics are known? 7 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: N/A 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? 9 Is the host a Category 1 organism (as defined in the HSNO (Low-Risk  Genetic Modification) Regulations 2003)? 10 Is the host a Category 2 organism (as defined in the HSNO (Low-Risk  Genetic Modification) Regulations 2003)?

Animals - mouse, rat, rabbit, cattle, goat, and sheep. Yes No 1 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?

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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 Is the organism characterised to the extent that its main biological  characteristics are known? 7 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: N/A 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? 9 Is the host a Category 1 organism (as defined in the HSNO (Low-Risk  Genetic Modification) Regulations 2003)? 10 Is the host a Category 2 organism (as defined in the HSNO (Low-Risk  Genetic Modification) Regulations 2003)?

Human cell lines (excluding human embryonic stem cell lines and cell lines derived from people of known Māori origin) Yes No 1 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 Is the organism characterised to the extent that its main biological  characteristics are known? 7 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: N/A c) Allow it to develop reproductive structures If yes, please

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provide further information on containment in section 4 d) Keep it in a closed container? 9 Is the host a Category 1 organism (as defined in the HSNO (Low-Risk  Genetic Modification) Regulations 2003)? 10 Is the host a Category 2 organism (as defined in the HSNO (Low-Risk  Genetic Modification) Regulations 2003)?

3.3 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, Escherichia coli will be developed using standard cloning and eg cloning or transformation techniques and will employ non-conjugative plasmid expression, plasmid, vectors from commercial (e.g. Invitrogen pCR2.1-topo vectors) and or viral 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 Vectors may contain regulatory elements, coding or non-coding genes, that the vectors and regulatory regions of genes. may contain Regulatory elements: Promoters (constitutive, endogenous or inducible) (e.g. chicken β-actin promoter, β-tubulin promoter, rapamycin-responsive promoter, U6 promoter). Enhancers (e.g. woodchuck post-transcriptional regulatory element (WPRE)). Internal ribosome entry site (e.g. encephalomyocarditis virus IRES).

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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 Genetic material may include cDNA or genomic DNA sequences and function of any may be sourced from the Kingdoms Animalia, Planta, Fungi, Protista donor genetic and Monera and from viruses or viroids. material 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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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 for vertebrate toxins with an

LD50 < 100 µg/kg. 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 No 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

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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 post- transcription 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 LD50 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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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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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 non- homologous 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

Page 16 of 28 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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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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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.

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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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? 

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.

Page 20 of 28 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? X

2 Is the proposed modification to a Category 2 host organism? 

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?

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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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 AgResearch operates laboratory containment facilities at the level (AS/NZS 2243.3:2002) Palmerston North (Grasslands and Hopkirk Research Institute) is this containment facility and Hamilton (Ruakura) campuses which are approved to PC1 approved to operate at (where and PC2 levels. Work carried out in laboratory containment will relevant)? 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 MAF-approved transfer of organisms between laboratory and do you propose to use to animal containment facilities will be in double containment and contain this organism? carried out according to the relevant Standards.

What procedural or All procedural and operational measures to contain, or dispose operational measures do you of, the genetically modified organisms will be conducted in MAF propose to use to contain this Biosecurity approved containment facilities in compliance with organism? 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”. Any other information There is no other information relevant to the containment of the relevant to the containment of organisms. the organism.

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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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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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Are there any other potential adverse effects?

Not that we are aware of.

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Section Six – Additional information Refer to page 31 of the user guide for assistance in completing this section Additional Information Y/N If yes, explain

Do any of the organism(s) need Y Modification of whole animals will also approvals under any other New require approval under the Animal Welfare Zealand legislation? Act 1999.

Does New Zealand have any N international obligations relating to (any of) the organism(s)?

Have any of the new organism(s) in Y ERMA application GMD03096: To develop in this application previously been containment for use in biomedical research considered in New Zealand or recombinant adeno-associated viral vectors elsewhere? 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 N that you consider relevant to this application that has not already been included?

Following the development of this rAAV vectors will be used to transduce organism what will the genetically various animals in containment. The animals modified organism be used for? eg will be then experimentally investigated for will experimental animals or plants transgene related effects in containment. be exposed to this organism?

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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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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List of references attached:

Aucoin, M.G., Perrier, M., and Kamen, A.A. (2008). Critical assessment of current adeno- associated 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 inhibitor- prone 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 adeno- associated virus vectors in the absence of helper adenovirus. Journal of virology 72, 2224- 2232.

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