WO2006120455A2 - Promoteur pour vecteur viral - Google Patents

Promoteur pour vecteur viral Download PDF

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Publication number
WO2006120455A2
WO2006120455A2 PCT/GB2006/001735 GB2006001735W WO2006120455A2 WO 2006120455 A2 WO2006120455 A2 WO 2006120455A2 GB 2006001735 W GB2006001735 W GB 2006001735W WO 2006120455 A2 WO2006120455 A2 WO 2006120455A2
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Prior art keywords
promoter
sequence
derived
protein
avian
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PCT/GB2006/001735
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English (en)
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WO2006120455A3 (fr
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Elizabeth Elliot
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Viragen Incorporated
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Priority to GB0712766A priority Critical patent/GB2450688A/en
Application filed by Viragen Incorporated filed Critical Viragen Incorporated
Publication of WO2006120455A2 publication Critical patent/WO2006120455A2/fr
Publication of WO2006120455A3 publication Critical patent/WO2006120455A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3084—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention provides an improved promoter for a lentiviral virus vector which can direct tissue specific expression of a heterologous gene product to a specific tissue of an avian into which the transgene has been incorporated.
  • tissue specific expression will be directed to the oviduct such that the gene product is provided within a laid egg.
  • the transgene may be incorporated into the germline of the avian so that the genotypic characteristic is stably transmitted to the subsequent generations .
  • Tissue specific expression of a heterologous gene is generally achieved through the use of a promoter which drives the expression of a coding sequence for the heterologous gene of interest.
  • the promoter may be selected from any endogenous gene, where the expression of that gene is known to be restricted to a specific tissue of interest in the host.
  • EIAV Anaemia Virus
  • the ability to direct expression of a heterologous gene product to the oviduct of a transgenic hen confers several advantages. For example, the consequences of ubiquitous or ectopic expression of a bioactive gene product in a host animal can be significant, in that the expression of the protein product throughout the avian may be undesirable. Animal welfare will depend upon the degree of cross- species homology between the heterologous gene product and that of the endogenous equivalent .
  • Ectopic expression of a gene product within a transgenic animal could also serve to increase product heterogeneity, with such differences in the produced product being undesirable from a regulatory perspective.
  • Expression of a gene product within a transgenic animal that can be targeted to a particular tissue will facilitate the manufacture and extraction of the protein.
  • tissue specific promoter, and in particular an oviduct-specific promoter would serve to minimise heterogeneity of product produced by a transgenic avian.
  • Tissue specific protein expression in avians By weight, 60% of the egg is composed of albumen.
  • Yolk proteins require specific internal recognition sequences for uptake into the yolk.
  • ovalbumin ovalbumin
  • lysozyme ovotransferrin
  • ovomucoid ovomucoid
  • ovalbumin and lysozyme being the most significant constituents (54% and 12% respectively of the total albumen protein) .
  • lysozyme is also expressed in the macrophage lineage, with a low level of expression in precursors such as myeloblasts to higher levels in mature macrophages.
  • This non-oviduct expression makes the lysozyme promoter a less attractive candidate for expression of an exogenous gene, for example a bioactive peptide with which tight spatio-temporal expression may be critical in the resultant transgenic organism.
  • Ovalbumin is expressed at a high level and with apparent oviduct specificity.
  • the ovalbumin locus spans approximately 16kb and the sequences spanning the entire region are publicly available (see for example Genbank, Accession number J00895) .
  • Work in this field has shown that there are four DNAse I hypersensitive regions (DHRs 1 to 4) within the 7kb 5 '-flanking sequence of the ovalbumin gene chromatin and that the presence of these DHRs is correlated strongly with the expression of the ovalbumin gene in the chicken oviduct (Kaye et al . , 1986).
  • Negative Regulatory Element is located within DHRl (-350 to -100 relative to transcription start site) (Sanders and McKnight, 1988) . Addition of this regulatory region to a homologous promoter lacking it was sufficient to restore repression of expression of a downstream marker gene. Addition of this same element to a heterologous constitutive promoter was insufficient to confer repression of expression.
  • SRE Steroid Regulatory Element
  • DHR3 is located within a 300bp fragment about 3.1kb upstream of the mRNA start site (Kaye et al . , 1986) .
  • This DNase hypersensitivity site partially overlaps with two other subsequently identified functional domains.
  • a more distal 675bp Oestrogen Response Enhancer Element (OREE) DNA sequencing of this element identified four 5'-TGACC-3' half- palindromic motifs.
  • This 675bp fragment confers oestrogen inducibility not only to the proximal 58bp long ovalbumin sequences, but also to heterologous promoter sequences such as HSV tk or rabbit beta- globin promoters (Kato et al . , 1992) .
  • a more proximal 400bp sequence element identified as a tissue-specific silencer-like element during gene gun work (Muramatsu et al., 1998) .
  • lysozyme gene is also active in the oviduct, lysozyme being a constituent of egg white. Both genes are delineated by the presence of 5' and 3' Matrix Attachment Regions (MARs) and have numerous defined positive and negative regulatory regions present within their respective loci .
  • MARs Matrix Attachment Regions
  • the BAC also includes a heterologous gene cloned into the native transcription start site of the ovalbumin locus . Transgenic birds were then produced via cytoplasmic micro-injection of the BAC and ovum transfer.
  • US Patent No 6,825,396 describes a hybrid ovalbumin promoter for use in avian transgenics .
  • This construct includes DHRl, DHR2 , exon 1 and the 5' end of exon 2 from the ovalbumin gene.
  • Intron 1 is not present.
  • An SV40 enhancer and three oestrogen response elements are also present.
  • This promoter was designed for use in a retroviral vector derived from REV-A hence the absence of intron 1.
  • ovalbumin promoter in avian transgenics is also discussed in US Patent Application Publication No 2004/0019922 and a related application also by Avigenics (International Patent Application PCT No WO 99/19472) . These applications describe two possible ovalbumin promoter fragments either 1.4 or 7.4 kb in length although no experimental data is included. The fragments described do not include intron 1 or exon 2 and the smaller of the two is essentially identical to that described in Dierich et al . , 1987 that has already been demonstrated to permit marker gene expression in both oviduct tubular gland cells and embryonic hepatocytes whereas in vivo, the ovalbumin gene is never expressed in the liver.
  • An improved lentiviral vector-compatible promoter for directing oviduct specific expression of a heterologous gene would be highly advantageous in improving protein expression techniques in a transgenic avian.
  • the inventors of the present invention have made the surprising discovery that using a novel, modular approach to promoter function, the regulatory elements of the ovalbumin promoter can be condensed to a size compatible with a replication defective retroviral vector, in particular a viral vector based upon a lentivirus, this permitting the retention of physiologically relevant regulated expression of heterologous gene expression from such a promoter in vivo.
  • a promoter construct for a replication defective retroviral vector comprising, from upstream to downstream, regions which substantially correspond to DHR2 ,
  • the ovalbumin promoter gene is derived from chickens, turkeys, ducks, quail, geese, ostriches, pheasants, peafowl, guinea fowl, pigeons, swans , bantams or penguins .
  • the ovalbumin gene is derived from an avian of the species Gallus gallus.
  • the term 'replication defective retroviral vector' means that the vector itself is incapable of making viral proteins which are required for additional rounds of viral replication and infection.
  • any viral vector system used as a gene transfer vehicle has the ability to package high titres of replication-defective viral genomes .
  • the viral particles are rendered replication defective through the deletion from the viral genome of all but the minimum of viral coding sequences . Sequences which are deleted from the viral genome may be supplied in trans during packaging.
  • the genome of the viral vector which comprises the promoter may further contain at least one heterologous DNA sequence coding for a protein of interest such as a marker gene or for a gene encoding a bioactive protein or peptide fragment of interest.
  • a protein of interest such as a marker gene or for a gene encoding a bioactive protein or peptide fragment of interest.
  • Such viral particles are capable of infecting a target cell (which may be dividing or non-dividing), and integrating into the genome. After integration has occurred, the target cell is effectively stably transduced, with the promoter directing expression of any coding transgene sequences within the viral vector genome.
  • Frame-independent insertion of heterologous coding sequences downstream of the ovalbumin regulatory elements may be achieved by a sequence modification of exon 2 of the chicken ovalbumin gene via PCR- based mutagenesis. After mutagenesis the ovalbumin start codon can be deleted, the flanking Kozak sequence disrupted and a unique restriction enzyme cleavage sequence (CCC/GGG, Sma I) inserted.
  • CCC/GGG, Sma I unique restriction enzyme cleavage sequence
  • the promoter further comprises at least part of a DNA sequence which encodes for the 5 ' exon 2 sequence as derived from an endogenous avian ovalbumin gene, wherein this DNA sequence is located downstream of the intron 1 coding sequence, and wherein the sequence has been modified to remove, or substantially remove the start codon.
  • those sequences flanking the start codon for example a Kozak (translation initiation) sequence, may also be disrupted.
  • the promoter further comprises at least part of the DNA sequence which encodes for a DHR3 region derived from an endogenous avian ovalbumin gene.
  • the sequence is derived from a chicken ovalbumin gene.
  • the DHR3 region can be introduced upstream of DHR2 in a positional relationship not seen in the endogenous ovalbumin gene and therefore unique to the synthetic promoter construct of this aspect of the invention.
  • DHR3 may be positioned directly adjacent to DHR2 with no intervening sequence.
  • DHR3 may be positioned adjacent to DHR2 with approximately 1 kilobase of intervening sequence.
  • the promoter is provided within a vector which is a lentivirus viral vector. Lentivirus-based vectors are suited to gene transfer because of their ability to stably integrate into the genome of both dividing and non-dividing cells and further due to their ability to mediate long- term gene expression.
  • the promoter is provided in a non-primate lentiviral vector that is derived from EIAV.
  • lentiviral vector systems include HIV-I, HIV-2, SIV, FIV, CAEV, BLV (bovine leukaemia virus) or maedi-visna virus (MW) .
  • the lentiviral vector can be introduced ex-vivo or in-vivo.
  • a further aspect of the invention provides a promoter for a replication defective retroviral vector the promoter having a sequence according to SEQ ID NO:1 or 2.
  • SEQ ID NO: 1 is the predicted sequence of the OvaL promoter, while SEQ ID NO : 2 is the actual derived sequence of the OvaL promoter.
  • the promoter includes a nucleotide sequence which is at least 80% homologous to the sequences according to SEQ ID NO : 1 or 2. Further provided are nucleic acid molecules which comprise a sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the sequence of SEQ ID N0:l or 2. Variants and homologues preferably retain the tissue specific promoter activity of the sequence.
  • a yet further aspect of the invention provides a promoter for a replication defective retroviral vector the promoter having a sequence according to SEQ ID NO: 3, 4 or 5.
  • SEQ ID NO: 3 is the proposed sequence for the synthetic chicken derived ovalbumin promoter termed OvaS (S indicating the shorted length of the promoter which compared to the OvaL chicken ovalbumin promoter) .
  • SEQ ID NO : 4 is one variant of the actual derived OvaS promoter, while SEQ ID NO : 5 is an alternative variant of an actual derived sequence of an OvaS promoter.
  • the promoter includes nucleotide sequences which are at least 80% homologous to the sequence according to SEQ ID NO : 3 , 4 or 5. Further provided are nucleic acid molecules which comprise a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the sequence of SEQ ID NO : 3 , 4 or 5. Variants and homologues preferably retain the tissue specific promoter activity of the sequence.
  • a further aspect of the invention provides a promoter for a replication defective retroviral vector the promoter having a sequence according to SEQ ID NO: 6 or 7.
  • SEQ ID NO: 6 shows the 0vaM2 (M2 denotes Middle 2) predicted promoter sequence as derived from a chicken ovalbumin promoter gene.
  • SEQ ID NO: 7 shows the actual 0vaM2 derived promoter sequence.
  • the promoter includes nucleotide sequences which are at least 80% homologous to the sequence according to SEQ ID NO: 6 or 7. Further provided are nucleic acid molecules which comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the sequence of SEQ ID NO: 6 or 7. Variants and homologues preferably retain the tissue specific promoter activity of the sequence.
  • a further aspect of the invention provides a promoter for a replication defective retroviral vector the promoter having a sequence according to SEQ ID NO: 8, 9 or 10.
  • SEQ ID NO: 8 shows the predicted sequence of the OvaMl (Ml denotes the Middle 1 fragment) promoter.
  • SEQ ID NO: 9 shows a first actual sequence of the OvaMl promoter, while
  • SEQ ID NO: 10 shows a second actual sequence of the OvaMl promoter sequence.
  • the promoter includes a nucleotide sequence which is at least 80% homologous to the sequences according to SEQ ID NO: 8, 9 or 10. Further provided are nucleic acid molecules which comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the sequences of SEQ ID NO: 8, 9 or 10. Variants and homologues preferably retain the tissue specific promoter activity of the sequence.
  • a further aspect of the present invention provides an ovalbumin comprising a DNA sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 ,9 or 10 or a DNA sequence which hybridises thereto under moderate, stringent, or highly stringent conditions.
  • a further aspect of the present invention provides a DNA sequence which encodes for a replication defective lentiviral vector for transducing at least one cell in a target animal such that the genomic DNA of the cell contains the integrated vector, the vector comprising at least one heterologous gene and a promoter which drives expression of the gene, the promoter comprising from upstream to downstream, regions which substantially correspond to DHR2 , DHRl, exon 1, intron 1 as derived from an endogenous avian ovalbumin gene.
  • the endogenous avian ovalbumin gene is a chicken ovalbumin gene.
  • the promoter further comprises at least part of the sequence which substantially encodes the exon 2 sequence as derived from the endogenous avian ovalbumin gene, wherein this is located downstream of the intron 1 coding sequence, and wherein the sequence has been modified to remove the start codon.
  • those sequences flanking the start codon for example a
  • the promoter further comprises at least part of the sequence which encodes the DHR3 region derived from an endogenous avian ovalbumin gene.
  • the DHR3 region is derived from chickens, turkeys, ducks, quail, geese, ostriches, pheasants, peafowl, guinea fowl, pigeons, swans, bantams or penguins .
  • the DHR3 region is derived from an avian of the species Gallus gallus.
  • the present invention may, for example, be utilised in the generation of transgenic avians for use in the production of heterologous (recombinant) proteins.
  • viral vectors which contain a modular promoter provided by the present invention can achieve transduction of avian cells with exogenous genetic material.
  • the genetic material is incorporated into the avian genome in such a way that the modification becomes integrated into the germline and results in expression of the encoded protein within the oviduct such that incorporation into the avian egg results.
  • a further aspect of the invention provides a lentivirus viral vector comprising a promoter wherein the promoter comprises, from upstream to downstream, DNA sequences which substantially encodes for regions corresponding to DHR3, DHR2, DHRl, exon 1, intron 1 as derived from an endogenous avian ovalbumin gene.
  • the ovalbumin gene is derived from chickens, turkeys, ducks, quail, geese, ostriches, pheasants, peafowl, guinea fowl, pigeons, swans, bantams or penguins.
  • the ovalbumin gene is derived from an avian of the species Gallus gallus .
  • the promoter further comprises a
  • DNA sequence which substantially encodes for a region corresponding to exon 2 as derived from an endogenous avian ovalbumin gene which is located downstream of the intron 1 coding sequence.
  • the exon 2 DNA sequence is derived from a chicken ovalbumin gene.
  • the promoter further comprises at least part of a sequence which substantially encodes the DHR3 region derived from an endogenous avian ovalbumin gene, said DHR3 region preferably being derived from the endogenous chicken ovalbumin gene.
  • the lentiviral vector preferably contains at least one exogenous DNA.
  • this exogenous DNA comprises a gene which encodes a protein or polypeptide of interest.
  • the present invention further extends to a host cell which has been genetically transformed by the lentiviral virus vector of this aspect of the invention.
  • the present invention further extends to a plasmid comprising at least one gene encoding a protein of interest which is linked to a modified promoter derived from an endogenous avian ovalbumin gene as defined hereinbefore.
  • the protein of interest may be, but is not limited to proteins having a variety of uses including therapeutic and diagnostic applications for human and/or veterinary purposes and may include sequences encoding antibodies , antibody fragments , antibody derivatives, single chain antibody fragments, fusion proteins, peptides, cytokines, chemokines, hormones, growth factors or any recombinant protein.
  • the invention further extends to a method of producing a protein of interest, comprising the steps of making a transgenic avian that expresses the protein of interest in a tissue specific manner such that it is provided in the egg, using a replication defective viral vector containing a promoter in accordance with the first aspect of the invention, obtaining the egg, and purifying the protein of interest from, the egg.
  • production of the protein results in it being a constituent of the albumen.
  • the protein of interest may be, but is not limited to proteins having a variety of uses including therapeutic and diagnostic applications for human and/or veterinary purposes and may include sequences encoding antibodies, antibody fragments, antibody derivatives, single chain antibody fragments, fusion proteins, peptides, cytokines, chemokines , hormones, growth factors or any recombinant protein.
  • the transgenic avian may be, but is not limited to a chicken.
  • a yet further aspect of the invention provides a method for the production of a transgenic avian, the method comprising the step of using a replication defective retroviral vector to deliver exogenous genetic material to avian embryonic cells or cells of the testes, wherein the vector genome comprises a promoter construct, the promoter comprising, from upstream to downstream, regions which substantially correspond to DHR2 , DHRl , exon 1 , intron 1 as derived from an endogenous avian ovalbumin gene.
  • the transgenic avian is a chicken (Gallus gallus) .
  • the endogenous ovalbumin gene is derived from a chicken.
  • the vector construct particles are packaged using a commercially available packaging system to produce a vector with an envelope, typically a VSV-G envelope.
  • the exogenous genetic material placed under the control of the synthetic, modular ovalbumin-based promoter as provided by the invention may encode any of a large number of proteins having a variety of uses including therapeutic and diagnostic applications for human and/or veterinary purposes and may include sequences encoding antibodies, antibody fragments, antibody derivatives, single chain antibody fragments, fusion proteins, peptides, cytokines, chemokines , hormones, growth factors or any recombinant protein.
  • the methods of the present invention can be used to generate any transgenic avian, including but not limited to chickens, turkeys, ducks, quail, geese, ostriches, pheasants, peafowl, guinea fowl, pigeons, swans , bantams and penguins .
  • the contributory elements of the promoter of the present invention may be derived from the ovalbumin gene of other avians, such as those listed above. Such ovalbumin genes are likely to have a considerable degree of sequence, organisational and functional homology with the ovalbumin gene of the chicken. Such homology will typically be at least 85% sequence identity at a nucleic acid level . Accordingly the promoter of the invention may be derived, in part or totality from the endogenous ovalbumin gene of other avian species other than chickens .
  • transgenic avian is any member of an avian species, in particular the chicken, wherein at least one of the cells of the avian contains the exogenous genetic material contained in the vector.
  • Transgenic techniques which are suitable for the introduction of such genetic material will be known to the person skilled in the art.
  • the transgenic avian produced by the method of the invention has the exogenous genetic material encoded by the vector incorporated into at least a proportion of germ cells such that the genetic material will be transmitted to at least a proportion of the offspring of the transgenic avian.
  • the present invention further extends to a chimeric avian or a mosaic avian, wherein the exogenous genetic material is found in some, but not all of the cells of the avian.
  • a further aspect of the present invention provides a method for the production of a heterologous protein in an avian, the method comprising the step of delivering genetic material encoding a protein of interest within a replication defective retroviral vector construct to avian embryonic cells so as to create a transgenic avian which expresses the genetic material in specific tissues, wherein the replication defective retroviral vector construct comprises a promoter which controls the expression of the heterologous protein and which comprises, from upstream to downstream, regions which substantially correspond to DHR2 , DHRl, exon 1, intron 1 as derived from an endogenous avian ovalbumin gene .
  • the avian ovalbumin gene is a chicken ovalbumin gene.
  • the promoter further comprises at least part of a sequence that substantially encodes for the exon 2 sequence as derived from an endogenous avian ovalbumin gene, wherein the sequence has been modified to remove the start codon and disrupt the flanking Kozak sequence.
  • the ovalbumin gene is derived from a chicken.
  • the promoter may further comprise at least part of a sequence which substantially encodes the DHR3 region derived from an endogenous avian ovalbumin gene, wherein the DHR3 sequence is introduced upstream of the DHR2 sequence.
  • the ovalbumin gene is derived from a chicken.
  • the transgenic avian expresses the exogenous genetic material in the oviduct such that the expressed genetic material, in the form of a translated protein, becomes incorporated into eggs. It is preferred that the expressed heterologous protein is a component of the albumen.
  • the lentivirus vector construct is used to direct expression of a heterologous protein encoded by the vector in specific tissues (tissue-specific expression) .
  • tissue specific expression is directed such that the heterologous protein is a component of the albumin of an egg.
  • the heterologous protein may be expressed in the egg white or egg yolk, however it is preferable that the protein is present in the egg white.
  • the protein can be isolated from the egg white or yolk by standard methods which will be known to the person skilled in the art .
  • a yet further aspect of the present invention provides the use of a promoter, the promoter comprising from upstream to downstream, regions which substantially correspond to DHR2 , DHRl, exon 1, intron 1 as derived from the endogenous chicken ovalbumin gene in the production of a heterologous protein in a transgenic avian.
  • the promoter further comprises at least part of the sequence which substantially encodes the exon 2 sequence as derived from an endogenous avian ovalbumin gene, wherein this is located downstream of the intron 1 coding sequence, and wherein the sequence has been modified to remove the start codon.
  • those sequences flanking the start codon for example a Kozak sequence, may also be disrupted.
  • the ovalbumin gene is derived from a chicken.
  • the promoter further comprises at least part of the sequence which encodes the DHR3 region derived from an endogenous avian ovalbumin gene.
  • a yet further aspect of the present invention provides the use of a replication defective retroviral vector which comprises a promoter, the promoter comprising, from upstream to downstream, regions which substantially correspond to DHR2 , DHRl , exon 1 , intron 1 as derived from the endogenous avian ovalbumin gene in the production of a heterologous protein in a transgenic avian.
  • the promoter further comprises at least part of the sequence which substantially encodes the exon 2 sequence as derived from the endogenous avian ovalbumin gene, wherein this is located downstream of the intron 1 coding sequence, and wherein the sequence has been modified to remove the start codon.
  • those sequences flanking the start codon for example a Kozak sequence, may also be disrupted.
  • the promoter further comprises at least part of the sequence which encodes the DHR3 region derived from the endogenous avian ovalbumin gene.
  • a yet further aspect of the present invention provides the use of a replication defective retroviral vector which comprises a promoter, the promoter comprising from upstream to downstream, regions which substantially correspond to DHR2 ,
  • DHRl , exon 1 , intron 1 as derived from an endogenous avian ovalbumin gene in the production of a transgenic animal, in particular a transgenic avian.
  • the present invention provides a novel synthetic modular ovalbumin based promoter construct which has been modified to enable its introduction into a viral vector, in particular a lentiviral virus vector, the promoter being able to direct regulated expression of a heterologous protein or other exogenous genetic material to the oviduct of an avian where it can be incorporated into an egg.
  • the heterologous protein can be expressed in a spatially and temporally restricted manner under the control of the promoter in vivo.
  • An advantage of the promoter of the present invention is that there is no requirement for MARs (matrix-attachment regions) . Historically, MARs have been utilised in transgenics to insulate the integrated transgene against position effect- mediated silencing.
  • lentiviral vectors precludes this requirement as transgene silencing has not been reported or observed.
  • the utilisation of the native transcription start site is also suboptimal as it can confer strict frame requirements on subcloning.
  • CCATGG commonly Ncol
  • the amino acids directly adjacent to the ATG start codon usually constitute a signal peptide, necessary for secretion of the heterologous peptide.
  • Signal peptides sequences display a strong preference for specific types of amino acids at key positions and the presence of a non-native amino acid may affect the efficiency of signal peptide recognition and therefore secretion.
  • a further advantage of the promoter of the present invention is that it would not permit ectopic expression, a phenomenon with possible regulatory and animal welfare implications .
  • Particularly preferred promoter sequences of the invention is the modified chicken ovalbumin promoter sequences as defined in any one of SEQ ID N0:l, 2, 3, 4, 5, 6, 7 ,8, 9, or 10.
  • the invention provides isolated DNA sequences encoding promoters of the invention, selected from: (a) DNA having the sequence of SEQ ID N0:l, 2, 3, 4, 5, 6, 7 ,8, 9, or 10 (b) DNA capable of hybridization to a DNA of (a) under conditions of moderate stringency and which encodes polypeptides of the invention; (c) DNA capable of hybridization to a DNA of (a) under conditions of high stringency and which encodes polypeptides of the invention, and (d) DNA which is degenerate as a result of the genetic code to a DNA defined in (a) , (b) , or (c) and which provides for a functional promoter which can control expression of a gene.
  • Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used.
  • “Stringent conditions” or “highly stringency conditions”, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 degrees C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 degrees C; or (3) employ 50% formamide, 5*SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5* Denhardt ' s solution, sonicated salmon sperm DNA (50 [mu]g/ml), 0.1% SDS, and 10% dextran
  • Modely stringent conditions may be identified as described by Sambrook et al . , Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
  • An example of moderately stringent conditions is overnight incubation at 37 degrees C.
  • nucleic acid has a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the sequences of SEQ ID NO:1, 2, 3, 4, 5, 6, 7 ,8, 9, or 10.
  • the percent identity may be determined by visual inspection and mathematical calculation.
  • the percent identity of two nucleic acid sequences can be determined by comparing sequence information using a computer programme. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST or ALIGN. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferred features and embodiments of each aspect of the invention are as for each other aspect, mutatis mutandis, unless the context demands otherwise.
  • Frame-independent insertion of heterologous coding sequences downstream of the ovalbumin regulatory elements was achieved by a sequence modification of exon 2 of the chicken ovalbumin gene via PCR-based mutagenesis (see Example 1) . After mutagenesis the ovalbumin start codon was deleted, the flanking Kozak sequence disrupted and a unique restriction enzyme cleavage sequence (CCC/GGG, Sma I) inserted. This restriction site was then used for frame- independent insertion of any heterologous coding sequence downstream of the modified ovalbumin exon 2. This modification is present in all of the modular ovalbumin-based promoters described herein.
  • the modular promoters were provided within a retroviral vector of limited packaging capacity (about 10kb) and as such it was not possible to incorporate the entire native ovalbumin promoter region encompassing DHRs 1 to 4 ( about 8kb) .
  • introns most preferably- first introns .
  • ovalbumin intron 1 has been demonstrated to contain signals that direct nucleosome alignment over the ovalbumin gene in a way conducive to its regulation (Lauderdale & Stein, 1992) .
  • intron sequences poses particular problems in retroviral vector delivery systems that utilise an essential RNA intermediate.
  • Replication-defective viral vectors are packaged after a three or four plasmid transient transfection of a producer cell line such as HEK293TS. Plasmids used include those that encode all the necessary viral gene products as well as the viral vector genome plasmid.
  • RNA molecules and virus replication enzymes contained within a viral protein core and surrounded by an envelope. After infection, the viral core is released into the cytoplasm, viral RNA is reverse transcribed into double-stranded DNA and transported into the cell nucleus whereupon it is permanently integrated into host chromosomal DNA.
  • Host RNA polymerase II will initially transcribe the full-length viral genome RNA from the viral genome plasmid. Any internal intronic sequences within the genome RNA will be processed as normal and as such are liable to excision by splicing. This would have significant effects on the level of coding sequence expression; integrated genomes derived from viral vectors that retain the intron would be expected to yield significantly higher levels of expression.
  • the majority, preferably all, of the integrated genomes of the vector retain intron 1.
  • EIAV causes a self-limiting, lifelong but rarely fatal infection of all Equidae. Transmission is as a result of insect bites . There have been no reported cases of EIAV infection in humans suggesting that it is an intrinsically safe virus on which to base a vector system for use in the clinic. EIAV cannot replicate in human cells, either because of the inability of the envelope to mediate entry into human cells, or the low transcriptional activity of the long terminal repeat (LTR) in human cells .
  • LTR long terminal repeat
  • the EIAV LTR is shorter than other retroviral LTRs and the sequence of the U3 region of different EIAV strains is highly variable compared to those of other lentiviruses . The sequence variation results in the presence of different transcription factor binding sites in the LTRs and is almost certainly a contributing factor in determining the tropism and pathology of the different strains .
  • EIAV does not contain a vif open reading frame between the end of pol and the start of env.
  • EIAV has the shortest pol-env intergenic region (190bp) . This together with the short long terminal repeats (320bp) , accounts for the small size of the EIAV genome (8kb) relative to other lentiviruses of which many are closer to 10kb.
  • VSV-G vesicular stomatitis virus G protein
  • Figure IA shows the sequences of the PCR primers used to amplify the promoter fragments contained in pLE7 and pLElO,
  • Figure IB shows ovalbumin exon 2 sequence modifications
  • Figure 2 shows a schematic illustrating the pLE7 plasmid map
  • Figure 3a shows the predicted nucleotide sequence of the pLE7 promoter (SEQ ID N0:l),
  • Figure 3b shows the actual nucleotide sequence of the pLE7 promoter (SEQ ID NO : 2 ) ,
  • Figure 4 shows a schematic illustrating the pLElO plasmid map
  • Figure 5a shows the predicted nucleotide sequence of the pLElO promoter (SEQ ID NO:3)
  • Figure 5b shows the actual nucleotide sequence of the OvaS promoter in the plasmid pLE45 (SEQ ID NO : 4 )
  • Figure 5c shows the actual nucleotide sequence of the OvaS promoter in a non-pLE45 plasmid (SEQ ID NO : 5 )
  • SEQ ID NO : 5 shows the actual nucleotide sequence of the OvaS promoter in a non-pLE45 plasmid
  • Figure 6 shows a schematic detailing the pRl40 plasmid map
  • Figure 7a shows the predicted nucleotide sequence of the pRl40 promoter (SEQ ID N0:6)
  • Figure 7b shows the actual nucleotide sequence of the pRl40 promoter (SEQ ID NO : 7 ) .
  • Figure 8 shows a schematic detailing the pLE21 plasmid map
  • Figure 9a shows the predicted nucleotide sequence of the pLE21 promoter (SEQ ID N0:8)
  • Figure 9b shows the actual nucleotide sequence of the ovaMl promoter in the plasmid pLE46 (SEQ ID NO : 9 ) ,
  • Figure 9c shows the actual nucleotide sequence of the OvaMl promoter in a non-pLE4 ⁇ plasmid (SEQ ID NO: 10) ,
  • Figure 10 (a) shows a matrix detailing the identity of all relevant GFP expression plasmids given in the construction of ovalbumin-based lentiviral vectors
  • Figure 10 (b) shows a summary of the mosaic GO founder birds that contain pRl50-derived transgenes
  • Figure 11 (a) shows titre of the control genome pONY8.45NCZ following co-packaging with a ubiquitous interferon expression genome
  • Figure 11 (b) shows titre of the control genome pONY8.45NCZ following co-packaging with a modular ovalbumin interferon expression genome
  • Figure 12 shows PCR analysis of two Gl birds from the OVR 2-4 line demonstrating retention of intron 1 in ovalbumin-containing lentiviral vectors ,
  • Figure 13 is a table showing the identity of various human interferon beta 1 expressing birds and their intron retention status
  • Figure 14 (a) is a picture showing GFP expression in the oviduct of a sexually mature mosaic GO hen transgenic for pRl44 (short ova-GFP) ,
  • Figure 14 (b) is a picture showing GFP expression in the oviduct of a sexually mature mosaic GO hen transgenic for pRI50 (ovaMl-GFP) ,
  • Figure 15 is a picture showing ovalbumin GFP expression in oviduct sections harvested from mosaic GO hen transgenic for pRI50 (ovaMl-GFP) .
  • GFP expression is absent from the surface epithelial layer and present only in the underlying cell layer, the known location of secretory tubular gland cells,
  • Figure 16 is a table summarising the integration event identity of birds in the OVR2-4 transgenic line
  • Figure 17 is a graph to illustrate the expression trends for R24 in the eggs of bird OVR2-4:l ⁇ ,
  • Figure 18 is a collation of both Northern blot data and ELISA data from a Gl OVR2-4 bird.
  • Northern blot results show the expression patterns for R24 mRNA in various tissues of two separate transgenic birds .
  • ELISA data shows the detectable level of R24 protein in the same tissues of the two transgenic birds,
  • Figure 19 is an equivalent figure except that the data was obtained from a G2 OVR2-4 bird.
  • Figure 20 shows a section through the oviduct of a G2 0VR2-4 transgenic bird visualised with Hoescht staining only and also with a FITC- conjugated anti-human IgGl constant domain antibody
  • Figure 21 shows the mean levels of interferon beta from the OvaMl promoter in several lines of transgenic hens,
  • Figure 22 shows trend data demonstrating the persistence of Interferon beta expression in the eggs of one bird, Mli3-ll:649.
  • the shared 3 ' primer also removes the endogenous ovalbumin start codon and Kozak sequence and as such frame- independent cloning of any downstream sequence is possible ( Figure IB) .
  • the sequence modifications at the 3' end of ovalbumin were also designed to retain a hairpin structure in any fusion mKNAs expressed under the control of the various promoter fragments . This was because these hairpins comprise a trigonal stem loop structure. Hairpin 3 lies within the ovalbumin coding sequence so will inevitably be lost when a heterologous gene product is expressed from the ovalbumin promoter. However, hairpin 1 and 2 are retained in mRNAs that include the modified exon 2.
  • hairpin 1 positively affects the rate of ovalbumin mRNA translation in vitro and is part of a high affinity binding site for eukaryotic initiation factor 2(eIF2), an initiation factor involved in the high efficiency translation of the mRNA (Kopper efc al . , 1994).
  • Example 2 Modular construction of synthetic ovalbumin promoters
  • the ovalbumin promoter is a candidate to achieve oviduct-specific expression of a heterologous gene.
  • Literature exists in which large (up to 195kb) segments of the ovalbumin gene were used in delivery vectors such as BACs, in contrast small regions of the gene (about 1.3kb) have also been used in both viral and non- viral delivery systems .
  • BACs delivery vectors
  • small regions of the gene about 1.3kb
  • ovalbumin promoter to be condensed and inserted into a lentiviral vector backbone permitting the successful generation of transgenic animals .
  • MlG line established with birds transgenic for the pRI50 (OvaMl-GFP) expression construct (see Figure 10 (b) ) .
  • a PCR strategy was used to amplify key promoter fragments from the chicken genome using unique 5' primers and a shared 3' primer.
  • the first such promoter fragment was designed to encompass the promoter region including three DNase hypersensitivity sites (DHRs 1-3) and the overlapping Oestrogen Response Enhancer Element (OREE) ; so-called Long Ovalbumin promoter (OvaL) .
  • the OvaL PCR product was amplified using primers 675Le and 3Sma ( Figure IA) and cloned into the plasmid pGEMTeasy (Promega) to generate the plasmid pLE7 ( Figure 2) .
  • the sequence of the OvaL promoter was predicted based on publicly available information and this sequence is given in Figure 3a (SEQ ID NO:1).
  • the actual sequence of the OvaL promoter in pLE7 is shown in Figure 3b (SEQ ID NO:1).
  • the second promoter fragment was designed to encompass the promoter region including two DNase hypersensitivity sites (DHRs 1 and 2) only, so- called Short Ovalbumin promoter (OvaS) .
  • the OvaS PCR product was amplified using primers 5DHR2 and 3Sma ( Figure IA) and cloned into the plasmid pGEMTeasy (Promega) to generate the plasmid pLElO ( Figure 4) .
  • the sequence of the OvaS promoter was predicted based on publicly available information and this sequence is given in Figure 5a (SEQ ID NO : 2 ).
  • Middle 1 and 2 ovalbumin fragments were created via a modular approach in which known 5' regulatory sequences have been directly juxtaposed with intervening sequence of unknown function simultaneously deleted.
  • the 0vaM2 variant was created by the demethylation and double restriction digestion of pLE7 with CIaI and AfIII .
  • the linearised vector was then treated with T4 DNA polymerase and the blunt ends re-ligated to generate the plasmid pRl40.
  • the map of pRl40 is given in Figure 6.
  • the sequence of the 0vaM2 promoter of pRI40 was predicted based on publicly available information and this sequence is given in Figure 7a (SEQ ID NO: 6).
  • the actual sequence of the 0vaM2 promoter in pRl40 is shown in Figure 7b (SEQ ID N0:7) . Note, the level of homology was assigned (T-COFFEE, www.chembnet.org) and SEQ ID NO : 7 is greater than 99% homologous to SEQ ID NO: 6, this high conservation demonstrates that all such sequences are functionally equivalent.
  • the OvaMl ovalbumin promoter was constructed from two components .
  • a region corresponding to the Oestrogen Response Enhancer Element and overlapping partial sequences from both DHR3 and potential tissue-specific silencer element was PCR amplified and cloned into pLE20A.
  • This OREE/DHR3 /silencer DNA sequence element was excised from pLE20A by digestion with the restriction enzyme EcoRI .
  • pLElO was then linearised immediately upstream of DHR2 , the 5' extent of the ovalbumin regulatory regions present in this plasmid, by digestion with the restriction enzyme Ncol and blunt ends generated by treatment with T4 DNA polymerase. The excised
  • OREE/DHR3 /silencer DNA sequence element was then ligated into pLElO to generate the plasmid pLE21 (see Figure 8) .
  • Correct orientation of the introduced DNA element was confirmed by PCR, the full sequence of the OvaMl promoter present in pLE21 was predicted based on publicly available information and this sequence is given in Figure 9a (SEQ ID NO: 8) .
  • Two variants were obtained for this promoter sequence, the first was shown to be present in a plasmid (pLE4 ⁇ ) and is given in Figure 9b (SEQ ID NO: 9), the second was shown to be present in a non-pLE4 ⁇ plasmid and is given in Figure 9c (SEQ ID NO:10).
  • SEQ ID NO : 9 is 99% homologous to SEQ ID NO : 8
  • SEQ ID NO: 10 is greater than 99% homologous to SEQ ID NO : 8. All such sequences are functionally equivalent.
  • ovalbumin-based lentiviral vectors were performed in two stages; first, the insertion of a coding sequence downstream of the ovalbumin promoter, second, the subcloning of the ovalbumin-coding sequence expression cassette into a lentiviral backbone.
  • a lentiviral backbone Preferably an EIAV lentiviral vector such as that from Oxford Biomedica UK Ltd. is used.
  • a matrix detailing the identity of all relevant GFP expression plasmids is given in Figure 10(a) .
  • EIAV lentiviral vector plasmids that contained the human Interferon Beta (IFN-B) coding sequence under the control of one of several ubiquitous promoters were constructed. All virus packagings were completed as normal and while adequate control virus titres were obtained high titre preparations of the ubiquitous IFN-B genomes were not. The most probable explanation for this observation is the expression of IFN-B from the vector genomes during packaging. The anti-viral effects of IFN-B are well documented (reviewed in Malmgaard, 2004) . The negative influence of IFN-B expression during packaging was confirmed by a mixing experiment . As shown in Figure HA a significant reduction in the titre of the control genome pONY8.45NCZ occurs following co-packaging with a ubiquitous IFN expression genome.
  • IFN-B Interferon Beta
  • ovalbumin intron 1 and a retroviral vector was expected to result in the splicing out of intron 1 from the viral genome KNAs such that the integrated genomes represented in transgenic birds would lack this intron.
  • a PCR assay was developed to assess intron status in Gl birds.
  • the first line analysed was transgenic for a lentiviral vector construct containing the Short ovalbumin promoter driving the expression of a recombinant antibody (OVR2-4) .
  • OVR2-4 Short ovalbumin promoter driving the expression of a recombinant antibody
  • intron 1 was shown to have been retained in the integrated transgenes present in these birds ( Figure 12) .
  • Intron retention frequency was also analysed in two further types of transgenic birds by a related PCR assay. First, those that contain an integrated proviral transgene in which human Interferon beta 1 expression is regulated by the OvaS promoter (OVI birds) .
  • OvaS promoter OvaS promoter
  • Example 6 - In vivo Reporter Gene Expression Data As shown in Figure 14A, three lentiviral vectors were constructed in which the GFP reporter gene was placed under the control of an ovalbumin promoter fragment (referred to as Short, s; Middle 1, Ml; and Middle 2 , M2 ) .
  • the final lentiviral vectors were named pRl44, pRI50 and pRl47 respectively and all were used in early embryo injections in an attempt to generate transgenic founder birds .
  • a PCR specific for the EIAV packaging signal was used to screen for the presence of each transgene in the resultant birds . Only cockerels were required for breeding to generate Gl progeny. Therefore GO hens were analysed for GFP expression in the oviduct upon reaching sexual maturity ( Figure 14 (a) and 14 (b)) .
  • GFP expression was observed in hens transgenic for pRl44 (Short ovalbumin promoter, Figure 14 (a) ) and pRI50 (Ml ovalbumin promoter, Figure 14 (b) ) . This data is sufficient to allow us to conclude that both promoters are able to direct GFP expression to the oviduct. Furthermore, the GFP expression in the section shown in Figure 15 demonstrates that GFP expression is absent from the surface epithelial cell layer and present only in the underlying layer, known to be the location of the secretory tubular gland cells.
  • Example 7 In vivo therapeutic gene expression data from the OvaS promoter
  • the coding sequence of therapeutic genes can also be operably linked to the modular ovalbumin-based promoters described here.
  • the coding sequence of the chimaeric R24 minibody is a recombinant single chain antibody molecule with antigen specificity for the tumour- associated GD3 ganglioside.
  • the R24 coding sequence was inserted into the Smal site introduced in exon 2 of the Short ovalbumin promoter fragment. This ovalbumin promoter-R24 expression cassette was then subcloned into an EIAV lentiviral vector genome plasmid.
  • the final EIAV lentiviral vector was named pLE38 and was used in early embryo injections in an attempt to generate transgenic founder birds .
  • Two founder mosaic cockerels were generated, OVR2-4 and 0VR2-7 (also referred to in Example 5) . Both cockerels gave rise to transgenic Gl progeny in which all cells of each animal contain one copy of the integrated proviral genome. All protein expression analysis was undertaken on Gl progeny from the OVR2-4 line. Southern blots demonstrated that two types of integration event were represented in the OVR2-4 GIs (called A and B) indicating that two separate viral transduction and integrations had occurred within the germline of the GO bird (0VR2- 4) .
  • Figure 16 shows a summary of the sex and integration event (A or B) of numerous OVR2-4 Gl progeny.
  • RNA samples were analysed by Northern blot, sequentially probed for the presence of R24 mRNA, ovalbumin mRNA and 18S RNA as a loading control . Protein extracts were analysed by ELISA. Figure 18 shows the results.
  • Example 8 In vivo therapeutic gene expression data from the OvaMl promoter
  • the coding sequence of therapeutic genes can also be operably linked to the modular ovalbumin-based promoters described here.
  • the coding sequence of a human Interferon beta (IFNB) was inserted into the Smal site introduced in exon 2 of the Ml ovalbumin promoter fragment.
  • This ovalbumin Ml promoter-IFNB expression cassette was then subcloned into an EIAV lentiviral vector genome plasmid.
  • the final EIAV lentiviral vector was named pLE4 ⁇ and was used in early embryo injections in an attempt to generate transgenic founder birds .
  • FIG. 21 shows the average expression of rhIFN- ⁇ in eggs from the individual transgenic birds described above.
  • Figure 22 shows the trend data for line M1I3-11 : 649. In general, the expression of hlFN ⁇ la protein in each egg is fairly consistent given the variability in egg mass from day-to-day.
  • Example 9 In vivo therapeutic gene expression data from the 0vaM2 promoter
  • the coding sequence of therapeutic genes can also be operably linked to the modular ovalbumin-based promoters described here.
  • the coding sequence of a human interferon alpha was inserted into the Smal site introduced in exon 2 of the 0vaM2 ovalbumin promoter fragment.
  • This ovalbumin promoter-IFN expression cassette was then subcloned into an EIAV lentiviral vector genome plasmid.
  • the final EIAV lentiviral vector was named pLEl34 and was used in early embryo injections in an attempt to generate transgenic founder birds .
  • Two founder mosaic cockerels were generated, M2A2-3 and M2A2-12 and shown by PCR to carry transgenic semen at a high frequency.
  • a PCR strategy to assess intron 1 retention in the proviral integrants in these birds demonstrated that both contained unspliced 0vaM2-containing transgenes .
  • Comparative analysis of data from the OvaS and OvaMl promoter contained within this document indicates that higher expression levels were obtained from the OvaMl promoter. This is exactly as expected as the OvaMl promoter contains a 675bp DNA sequence predicted to correspond to an Oestrogen Response Enhancer Element (OREE) . This same element is present within the 0vaM2 promoter and as such expression levels of heterologous proteins directed by this promoter are expected to be high. Good correlation between the spatio-temporal expression patterns from the 0vaM2 promoter and the endogenous ovalbumin gene is expected due to the retention of an intact sequence element identified as a tissue-specific silencer-like element during gene gun work

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Abstract

L'invention se rapporte à un promoteur d'ovalbumine modifié pouvant être utilisé dans l'expression spécifique d'un tissu de protéines hétérologues dans un organisme aviaire transgénique. Ce promoteur d'ovalbumine modulaire permet l'expression du produit protéique hétérologue à l'intérieur de l'oeuf, après quoi la protéine peut être purifiée. Les éléments régulateurs du promoteur d'ovalbumine peuvent être condensés jusqu'à une taille compatible avec un vecteur rétroviral défaillant s'agissant de sa réplication, notamment un vecteur viral basé sur un lentivirus, ceci permettant la conservation de l'expression régulée physiologiquement adaptée de l'expression génique hétérologue à partir d'un tel promoteur in vivo.
PCT/GB2006/001735 2005-05-11 2006-05-11 Promoteur pour vecteur viral WO2006120455A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2126071A1 (fr) * 2007-01-26 2009-12-02 Synageva BioPharma Corp. Expression transgenique chez des oiseaux
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