WO2004111252A1 - Production efficace d'animaux transgeniques - Google Patents

Production efficace d'animaux transgeniques Download PDF

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WO2004111252A1
WO2004111252A1 PCT/EP2004/006496 EP2004006496W WO2004111252A1 WO 2004111252 A1 WO2004111252 A1 WO 2004111252A1 EP 2004006496 W EP2004006496 W EP 2004006496W WO 2004111252 A1 WO2004111252 A1 WO 2004111252A1
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mammal
transgene
cells
human
organs
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PCT/EP2004/006496
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Alexander Pfeifer
Eckhard Wolf
Martin Biel
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Ludwig-Maximilians- Universität München
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • 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
    • 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
    • A01K2227/10—Mammal
    • A01K2227/101—Bovine
    • 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
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • 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 relates to a method for the generation of a mammal carrying at least one transgene, wherein the mammal is a non-human, non-rodent mammal. Furthermore, the invention relates to a method for the preparation of cells, tissue or organs from a mammal carrying at least one transgene, a use of said cells, tissue or organs for a composition for transplantation to a recipient in the need thereof and a method for the transplantation said cells, tissue or organs to a recipient in the need thereof.
  • the invention also relates to a method for the production of one or more compounds produced by a mammal generated by a method according to the invention, the use of corresponding compounds for the preparation of pharmaceutical compositions and methods of treatment of human or animal diseases by administration of said pharmaceutical compositions.
  • retroviral vectors based on Moloney murine leukemia virus have been used for viral transgenesis (2-5). Retroviruses integrate randomly into the host genome and the integrated provirus is transmitted to offspring of infected animals (6). In contrast to pronuclear injection, retroviral vectors efficiently transfer genes into murine and bovine embryos leading to a dramatic increase in transgenesis rates that could have a major impact for the production of transgenic livestock. However, retroviruses are subject to epigenetic modification during development and expression of retrovirally delivered transgenes is shut off during embryogenesis (2-4) or shortly after birth (5).
  • Lentiviruses are complex retroviruses and vectors based on lentiviruses transduce a broad spectrum of dividing and non-dividing cells, including murine and human embryonic stem cells and pre-implantation embryos of mice and rats (7, 8). Although, high transgenic rates were achieved with lentiviral vectors in rodents (7, 8), infection of rhesus monkey embryos with lentiviral vectors resulted in gene transfer only into extraembryonic tissue and transgene expression was confined to the placenta (9), raising the question whether lentiviruses can be used for transgenesis in higher mammals. These results rendered positive results in the generation of transgenic animals using lentiviral systems unlikely.
  • the technical problem underlying the present invention was to provide means and methods for an efficient production of non-human, non-rodent mammals carrying at least one transgene stably integrated into the genome of said mammal.
  • the present invention relates to a method for the generation of a mammal carrying at least one transgene, wherein the mammal is a non-human, non- rodent mammal, the method comprising the steps of:
  • transgene (b) infection of oocytes or preimplantation embryos with recombinant lentiviral vector carrying at least one transgene encoding at least one (poly)peptide of interest or giving rise to at least one anti-sense nucleic acid molecule and/or siRNA; and (c) transfer of the oocytes or preimplantation embryos into hormonally synchronized recipient females.
  • the term "carrying at least one transgene” relates in accordance with the invention to a stable integration of the recited transgene into the genome of the generated mammal.
  • the transgene is integrated into the genome of all cells including germ cells. Animals/mammals carrying at least one transgene stably integrated into cells of the germ line are designated in the context of the invention as transgenic animals/mammals.
  • the term "transgene” defines in accordance with the present invention a "heterologous nucleic acid" which is ectopically expressed in the mammal.
  • the heterologous nucleic acid may be a nucleic acid derived from an animal of the same species or a different species.
  • the nucleic acid may be expressed specifically in only one type of cell, tissue or organ of the mammal, as well as in several or all type of cell, tissue or organ of the mammal.
  • the heterologous nucleic acid is a heterologous gene or a part thereof, encoding a protein.
  • constitutive expression of the transgene is envisaged by the method of the invention.
  • This can be achieved by using, for example, tissue specific, developmental and/or cell regulated and/or inducible promoters which drive the expression of the transgene.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547- 5551) and Gossen et al.
  • the transgene may be expressed alternatively to a homologous gene or coexpressed with said homologous gene.
  • the alternatively expression of the transgene instead of the homologous nucleic acid may require a knock-out of the homologous nucleic acid.
  • RNAi RNA interference
  • Antisense and antisense nucleotides means DNA or RNA constructs which block the expression of the naturally occurring gene product. In principle techniques how to achieve a reduction of gene expression of a homologous gene or the knock-out of such gene are well known to the person skilled in the art.
  • nucleic acid molecule encoding the antisense-RNA is preferably of homologous origin with respect to the mammal's species infected with the recombinant lentiviral vector.
  • nucleic acid molecules which display a high degree of identity to endogenously occurring nucleic acid molecules encoding a protein encoded by a homologous gene.
  • the homology is preferably higher than 80%, particularly higher than 90% and still more preferably higher than 95%.
  • the percentage of identity of two or more given nucleic acid or protein sequences may be calculated by using suitable computer software.
  • suitable computer software examples include BLAST (Altschul et al. (1990), J. Mol. Biol. 215, 403-410), and variants thereof, such as WU-BLAST (Altschul & Gish (1996), Methods Enzymol. 266, 460-480), FASTA (Pearson & Lipman (1988), Proc. Natl. Acad. Sci. USA 85, 2444-2448) or implementations of the Smith-Waterman algorithm (SSEARCH, Smith & Waterman (1981), J. Mol. Biol. 147, 195-197).
  • Further commercially available software comprises the MEGALIGN software (DNASTAR, Inc.; Madison, Wis.).
  • step (a) Methods for the collection of embryos according to step (a) are known by the person skilled in the art. Furthermore, corresponding methods are exemplified in the appended examples.
  • preimplantation embryos defines in the context of the present invention all types of embryos prior to their implantation into the uterus of a female.
  • Lentiviral vectors and corresponding vector systems are described herein above. Said vectors are based on human immunodeficiency virus-1 or -2, or other lentiviruses like visna maedi, feline immunodeficiency, simian immunodeficiency virus, equine immunodeficiency virus.
  • lentiviruses are complex retroviruses.
  • Vectors based on lentiviruses transduce a broad spectrum of dividing and non-dividing cells, including murine and human embryonic stem cells and pre-implantation embryos of mice and rats (7, 8).
  • high transgenic rates were achieved with lentiviral vectors in rodents (7, 8)
  • infection of rhesus monkey embryos with lentiviral vectors resulted in gene transfer only into extraembryonic tissue and transgene expression was confined to the placenta (9).
  • the infection of oocytes or preimplantation embryos with lentiviral vectors may be achieved e.g. by injection of lentiviral vectors into the perivitelline space.
  • the perivitelline space lies between the zona pellucida and the cytoplasmic membrane of the oocyte or preimplantation embryo.
  • oocytes or preimplantation embryos can be infected directly with the lentiviral vectors by co-incubation with the vectors after removal of the zona pellucida (denuded oocytes or preimplantation embryos).
  • the timing of the infection may infect the outcome: oocytes can be infected before or after in vitro fertilization.
  • step (c) of the method of the invention Methods for the transfer of embryos according to step (c) of the method of the invention are known in the art. Said methods are specified and exemplified in more detail herein below.
  • the method of the invention is a highly efficient method for the production of animals carrying a transgene.
  • Methods known in the art for the production of transgenic animals are described to have an efficiency of about 2 % for large livestock species (see e.g. Wall, Theriogenology, 45, 57-68 (1996) which result from the technical difficulties to locate the pronuclei in non-rodent mammals in the case of pronuclear DNA microinjection.
  • expression from vectors derived from simple/prototypic retroviruses like Moloney murine leukemia virus is silenced due to epigenetic modifications during embryonic development (2-4) or shortly after birth (5) in the case of retroviral gene transfer.
  • the method of the invention allows production of animals containing stably integrated transgenes with rates of efficiency > 70% of all animals born, and more than 90% of the transgenic animals express the transgene. Due to the high efficiency the method of the present invention is superior to the commonly known methods, since the method requires less animals and material for success.
  • the donor animal, from which the ooctes or embryos are collected according to step (a) of the method is gonadotropin-stimulated prior to the collection of oocytes or embryos
  • the mammal carrying a transgene is a transgenic animal/mammal.
  • transgenic animal and “transgenic mammal have been defined herein above.
  • transgenic animals/mammals in comparison to non- transgenic animals/mammals which carry a transgene in animal breeding is the fact that transgenic animals/mammals are capable of forwarding the transgene to a next generation of animals/mammals due to the present of the transgene in the germ cells of the transgenic animal/mammal.
  • the descendants of a transgenic animal/mammal may also be transgenic animals/mammals.
  • transgenic livestock animal by the method described herein allows the breeding of further transgenic livestock animals, whereas livestock animals known in the art which have been described to carry a transgene not in the germ line cells are not capable of forwarding the transgene to a next generation of animals.
  • the non-human, non-rodent mammal is selected from the group of livestock species.
  • livestock species particularly comprise the species sus, bos, equus, capra, ovis, lepus, felis, canis. It is preferred the livestock species is a sus scorfa or bos taurus. In case of sus scrofa, the infection of preimplantation embryos is particularly preferred, whereas in case of bos taurus the infection of oocytes is preferred.
  • the produced non-human, non- rodent mammal carries more than one transgene.
  • the more than one transgene may be comprised in one vector or, alternatively, in separate vectors. In case of more than one vector the infection of the embryos with the vectors may be affected simultaneously or in subsequent steps.
  • the collection of preimplantation embryos according to step (a) may be carried out after an natural insemination of the donor mammals. It is also envisaged by the invention that the collection of preimplantation embryos according to step (a) may be carried out after an artificial insemination of the donor mammals.
  • Methods for after an artificial insemination of livestock species are know in the art of animal breeding. One possible method is exemplified in the appended examples.
  • the collection of preimplantation embryos according to step (a) is carried out 24 to 36 hours after the artificial insemination of the donor mammals.
  • the preimplantation embryos infected with recombinant lentiviral vector in step (b) are embryos in a cell stadium up to blastocyst stage.
  • the preimplantation embryo may be in line with the invention in a one cell stadium, two cell stadium, four cell stadium, eight cell stadium or 16 cell stadium. Particularly preferred are embryos in a one cell stadium, two cell stadium or four cell stadium.
  • the oocytes or preimplantation embryos are infected in step (b) with recombinant lentiviral vector which is pseudotyped with vesicular stomatisis virus envelope glycoprotein G.
  • lentiviral vector which is pseudotyped with vesicular stomatisis virus envelope glycoprotein G.
  • An example for a corresponding lentiviral vector is described in more detail in the appended examples.
  • the oocytes or preimplantation embryos are infected in step (b) by injection of recombinant lentiviral vector into the perivitelline space.
  • the perivitelline space is known in the art as an space between the zona pellucida and the cytoplasmic membrane of the oocyte or preimplantation embryo.
  • the embryos are, preferably, infected in step
  • the embryos are transferred into the recipient in step (c) surgically.
  • the surgical transfer is a microinvasive transfer.
  • Microinvasive transfers particularly comprise endoscopepical transfers.
  • the recipient female mammal is a late puberal gilt or heifer .
  • early pubertal gilt refers to gilts of at least 5 months of age.
  • heifer refers in the context of the invention to animals of at least 14 months of age. Said animals are defined to be capable of carrying the transferred oocytes embryos to term.
  • the transgene in the recombinant lentiviral vector is under the control of a regulatory nucleic acid sequence motive that regulates transgene expression, known in the art as promoters.
  • promoters are incorporated in the lentiviral vector construct to drive transgene expression in the target tissue(s) and organ(s).
  • Either constitutive promoters or promoters that are regulated by small molecules e.g. Tetracycline-inducible system of Bujard et al
  • Constitutive expression in all organs of the transgenic animal can be achieved by choosing an ubiquitously active promoter of mammalian genes.
  • Examples for corresponding promoters comprise, inter alia, the promoter of the murine or human phosphoglycerate kinase gene (pgk) (Adra et al., 1987; Yang et al., 1988). Further examples are compound promoters that contain sequences of the genome(s) of different vertebrate species or viruses, such as the CAG promoter (Niwa et al., 1991) that is derived from sequences of the chicken beta actin promoter and the cytomegalovirus (CMV). The other group of possible constitutive promoters are tissue- or cell-specific promoters.
  • tissue-specific promoters are pancreatic island cell-specific promoters like the rat-insulin-promoter (RIP) (Dandoy-Dron et al., 1991 ; Picarella et al., 1992), heart muscle cell-specific, hepatocytes-specific or promoters that are of interest depending on the use of the transgenic organ, e.g. for xenotransplantation
  • the transgene is at least one gene selected from the group consisting of fluorescent reporter genes, pharmaceutical relevant genes, genes relevant for agriculture improvement of farm animals (gene farming), immunomodulatory genes relevant for xenotransplantation, genes relevant for the production of pig or cattle model of human diseases and genes that interact with cell signaling.
  • the group of fluorescent reporter genes comprises inter alia gene encoding the following proteins and derivatives thereof: eGFP (Clontech, Palo Alto, USA; pEGFP Vector:#632311), eCFP (Clontech; pECFP
  • reporter genes can be incoroporated into the lentiviral vector either alone, or in combination, or as a fusion protein together with another gene of interest, or by incorporating an internal ribosomal entry (IRES) with another gene of interest, to detect the presence of the lentiviral provirus and its expression in the transgenic animals.
  • IRS internal ribosomal entry
  • the group of pharmaceutically or therapeutically relevant genes summarizes genes, the product of which is a drug or a prodrug that can be used for the treatment of human diseases.
  • Examples are expression of clotting factors (Factor VIII and IX) in the milk of transgenic cows or goats driven by mammary-specific promoters.
  • Another example is the expression of human alpha 1 antitrypsin in milk for the treatment of cystic fibrosis.
  • the group of agricultural relevant genes summarizes genes the products of which can be used to improve the value or life-expectancy of the whole animal or the commercial value of organs or the dairy products of the transgenic animal.
  • An example is the introduction of casein genes into farm animals to increase protein and calcium content of the dairy products.
  • Another example is the expression of prochymosin in the mammary epithelium to increase cheese-ripening (reviewed in (Karatzas, 2003)).
  • immunomodulatory genes including those encoding complement regulatory proteins such as CD46, CD55 and CD59 (Platt and Lin, 1998), genes that prevent cellular rejection of xenografted organs or tissues (such as FasL (CD95L) or TRAIL) or other genes such as hemoxygenase (Chen et al. 2003) or inhibitors of the blood clotting cascade (Gaca et al. 2002) which are assessed to improve the survival of grafted organs or tissues.
  • FasL CD95L
  • TRAIL hemoxygenase
  • hemoxygenase Choen et al. 2003
  • inhibitors of the blood clotting cascade Gaca et al. 2002
  • lentiviral gene transfer will be used to achieve overexpression or a functional knock down (e.g. by expression of specific small interfering RNAs) (Tiscornia et al. 2003).
  • Gene transfer using lentiviral vectors will also be applied for the generation of new animal models.
  • Large animal models, particularly pigs are advantageous, since surgical procedures and other interventions can be performed under conditions that very closely mimic the situation of human patients.
  • Important fields, where large animal models are or will be applied include:
  • Transgenic pig models can be used to validate target genes for would healing and to created new models for skin diseases (e.g. for atopic dermatitis by skin-specific expression of IL-4) (Chan et al. 2001)
  • Diabetes e.g. by functional inactivation of the vitamin D receptor (Zeitz et al. 2003) or by expression of a dominant negative mutant of the receptor for glucose-dependent insulin-releasing peptide (Volz et al. 1995)
  • lentiviral gene transfer will be used for animal agriculture.
  • Major applications include: • Growth rate an feed efficiency (e.g. by transferring genes from the growth hormone/insulin-like growth factor family) (Solomon et al., 1994)
  • the recombinant lentiviral vector further comprises a reporter gene under the control of said regulatory nucleic acid sequence motive which is selected from the group consisting of GFP, other fluorescence markers or LacZ.
  • said method further comprises a step (e):
  • transgenicity of the transgene in the generated mammal (e) analysis of the transgenicity of the transgene in the generated mammal.
  • Methods for the analysis of the transgenicity of a transgene in a mammal are known in the art and comprise different approaches.
  • the transgenicity may be analyzed, for example, by biochemical analysis as well as by analysis using a molecular biological or optical approach. Corresponding methods are described in the appended examples.
  • said analysis may be effected by an analysis of the transgenic DNA and/or expressed transgenic RNA.
  • said analysis may be effected by an analysis of the expressed transgene or a transgenic reporter protein.
  • the above defined method of the invention, wherein the transgene encodes a recombinant nutrient, nutrient additive, drug or prodrug is envisaged to further comprise the step of isolation of the compound from cells, tissues, organs or secretions of the mammal.
  • the transgene may be expressed as a cellular polypeptide or as a secreted polypeptide.
  • Dependent form the expression of the transgene which may be affected by the use of specific regulatory nucleic acid sequence motive that regulates transgene expression.
  • Said regulatory nucleic acid sequence motives e.g. may encode leader peptides which affect the secretion of the recombinant polypeptide encoded by the transgene.
  • drug and "prodrug” are known in the art and defined by examples herein above.
  • nutrient and “nutrient additive” define in the context of the present invention compounds which may be added to food of an animal or a human in order to optimize the diet of the animal or human.
  • the invention relates to a method for the preparation of cells, tissue or organs from a mammal carrying at least one transgene, wherein the mammal is a non-human, non-rodent mammal, for the transplantation of said cells, tissue or organs to a recipient in the need thereof comprising the steps of any of the above described embodiments of the method for the generation of a mammal carrying at least one transgene, wherein the mammal is a non-human, non-rodent mammal and further comprising the steps of:
  • said cells are selected from the group consisting of basal keratinocytes of the skin, pancreatic islet cells, liver cells, heart muscle cells and chondrocytes.
  • said tissues or organs are selected from the group consisting of kidney, liver, lung, heart, skin pancreas, eye and blood vessels.
  • said transplantation is a xenogenic transplantation.
  • a further embodiment of the invention is the use of cells, tissue or organs prepared an above disclosed method of the invention for a composition for transplantation to a recipient in the need thereof.
  • the invention also comprises a method for the transplantation of cells, tissue or organs prepared according to an above disclosed method of the invention further comprising the step of transplanting said cells, tissue or organs into a recipient in need thereof. It is also envisaged that said method further comprises administering of pharmaceutical compositions for the prolongation and/or establishment of the acceptance of the transplanted graft by the recipient.
  • said pharmaceutical compositions comprise immuno suppressive pharmaceutical compositions.
  • the invention also relates in an alternative embodiment to a method for the production of one or more compounds produced by mammals carrying at least one transgene, wherein the mammal is a non-human, non-rodent mammal generated by a method according to the present invention, wherein the compound is a recombinant nutrient, nutrient additive, drug or prodrug encoded by the transgene the method comprising the step of isolation of the compound from cells, tissues, organs or secretions of the mammal.
  • a use of a compound produced according to a method of the invention is, in line with the present invention the use of such compound for the preparation of a pharmaceutical composition for the treatment of a human or animal disease.
  • the invention further relates to method for the treatment of a human or animal disease, the method comprising the administration of a pharmaceutical composition comprising a compound produced according to a method of the invention.
  • compositions comprise all kinds of pharmaceutical compositions which comprise recombinant polypeptides as an active ingredient which are produced by a mammal carrying at least one transgene, wherein the mammal is a non-human, non-rodent mammal generated by a method according to the present invention.
  • the compound derived from said mammal may be further formulated prior to its administration.
  • Methods for the further formulation of compounds for the preparation of pharmaceutical compositions, particularly of polypeptide compounds, are known to the person skilled in the art of galenic.
  • compositions recited herein may comprise suitable formulations of carriers, stabilizers and/or excipients
  • Suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers can be formulated by conventional methods.
  • Fig. 1 Southern blot analysis.
  • A Schematic representation of the lentiviral vector used (LV-PGK). LTR, long terminal repeat; ppt, polypurine tract; W, woodchuck hepatitis responsive element; PGK, phosphoglycerate kinase promoter; eGFP, enhanced green fluorescent protein; black triangle, self-inactivating mutation.
  • B Southern blotting of BamH l-digested genomic DNA isolated from skin samples of all piglets born (pregnancies I - IV) and one age-matched control animal (wt).
  • C Analysis of copy number in DNA extracted from spleen, brain, pancreas, muscle and lung of animal #407.
  • Fig. 2 Expression of GFP in pigs derived from the infection of zygotes with lentiviral vector LV-PGK.
  • a - H Analysis of GFP expression by in vivo fluorescence imaging in transgenic and control animals. GFP expression was observed by direct epifluorescence in the skin and claws of #511 (right, A and B), but not in the age- matched control animal (left, A and B). Green fluorescence was also observed in the eye (C and D), gingival tissue and tongue (F and G) of the transgenic animal, but not in the eye (E) and snout (H) of the control.
  • I - P Fluorescence imaging of unfixed internal organs.
  • Kidney (right, I and J), cerebellum (bottom, K and L), pancreas (bottom, M and N), and testis (bottom, O and P) of #511 exhibit green fluorescence. No significant fluorescence is detectable in the control organs (left, I and J; top, K -P). Shown are the bright field photographs (A, C, F, I, K, M, O) and fluorescent images (B. D - H, and J, L, N, P).
  • Fig. 3 Western blot analyses.
  • A Immunoblotting of skin extracts isolated from pigs carrying 3 - 12 copies of LV-PGK, which were diluted (dil.) according to the number of lentiviral integrants.
  • B Correlation between proviral copy number and GFP expression levels quantified by immunoblotting.
  • C Table summarizing the transgenesis rate and GFP expression in the 25 piglets derived from subzonal injection of LV-PGK.
  • D Western blot analyses of tissue extracts of transgenic anima #511 and #407, that carry six and one viral integrant, respectively. Cntr, wild-type kidney; eGFP, 10 ng recombinant eGFP protein.
  • FIG. 4 Histological analysis of GFP expression by direct fluorescence and immunohistochemistry.
  • a - C Cortical sections of transgenic kidney express GFP in glomeruli (arrowheads), proximal tubules (white arrows), and distal tubules (black arrows). The inset shows a higher magnification of the lower left glomerulus.
  • D - F Analysis of GFP expression in the cerebellum. Arrowheads indicate Purkinje cells; asterisk, granule cell layer; inset, border between molecular and granule cell layer.
  • G - I Detection of GFP- and insulin-expressing cells in the pancreas by anti-GFP (brown) and anti-insulin staining (blue). Arrowheads, double-stained cells; inset, islet of Langerhans.
  • Fig. 5 Detection of GFP expression by fluorescence imaging.
  • a and B Direct in vivo epifluorescence of transgenic skin and claws (right) versus an age-matched control animal (left).
  • C - P Fluorescence imaging of isolated organs and tissues. The nostril (C and D), Eye (E and F), fore-brain (G and H), liver (I and J), duodenum (K and L), skeletal muscle (M and N) and heart (O and P) exhibit green fluorescence. No significant fluorescence is detectable in the control organs (right C and D; left E and F; top I - N and right O and P).
  • Fig. 6 Western blot analysis (A) of animals which did not show green fluorescence by direct in vivo imaging and flow cytometer analysis (B) of whole blood of a control animal (cntr) and a transgenic animals (LV-PGK).
  • Fig. 7 Epifluorescent examination of frozen sections isolated from testis (A and B), epidymis (C and D), retina (E and F), fore-brain (G and H), liver (I and J), duodenum (K and L), muscle (M and N) and heart (O and P) of transgenic animal #511 compared to H & E stainings of consecutive sections (A, C, E, G, I, K, M and O).
  • Fig. 8 Expression of GFP in porcine skin in transgenic pigs derived from infection of one- to two-cell stage embryos with lentiviral vector LV-K14
  • a - D Fluorescence imaging of isolated tissues. The skin (A, left) of pig #526 showed green fluorescence, while cerebellum (B, left), kidney (C, left), and pancreas (D, left) did not fluorescence. No fluorescence was detectable in the organs of the age-matched control (A - D, right). Insets, bright filed photographs of the corresponding fluorescence images.
  • E - L Histological analysis of GFP expression in animal #526.
  • E and I Analysis of transgene expression in skin sections. GFP expression was only detected in the basal layer of the epidermis and hair follicles (I, arrows).
  • the inset shows a higher magnigication of a typical junction of epidermis (asterisk) and dermis; the black arrows indicates basal keratinocytes.
  • the cerebellum F and J
  • kidney G and K
  • the pancreas H and L
  • neither green fluorescence nor GFP-specific immunostaining were detectable.
  • Fig. 9 Expression of GFP in porcine germ line stem cells (spermatogonia) in transgenic pigs derived from infection of one- to two-cell stage embryos with lentiviral vector LV-PGK
  • Fig. 10 In vivo fluorescence imaging of a newborn calf derived from injection of lentiviral vectors (LV-PGK) into oocytes
  • lentivirus was injected before in vitro fertilization, to allow for viral integration into the bovine genome before a nuclear membrane encloses the chromatin.
  • Fig. 11 Detection of the transgene in 2- to 4- cell stage embryos derived from a mating of an LV-PGK transgenic male with a wild-type female by nested PCR. Four of the eleven embryos carry the transgen; +, positive control (skin sample of animal #407; 0, empty lane; 1-11 , embryos analyzed.
  • Fig. 12 Generation of transgenic calves by lentiviral infection of oocytes.
  • C) Western blotting of skin samples reveals expression of the transgene in all transgenic animals.
  • Fig. 13 Histological analyses of eGFP expression.
  • A-C transgenic skin
  • D-F pancreas
  • G-l kidney
  • Hematoxylin and eosin staining A, D, G
  • immunohistochemical staining B, E, H
  • direct fluorescence analysis C, F, I
  • Fig. 14 Expression of lentiviral vectors in ductal epithelium of the mammary gland and in the germ line.
  • Fig. 15 Generation of transgenic cattle via nuclear transfer using lentivirally transduced donor cells.
  • BFF bovine fetal fibroblasts
  • Example 1 The invention will now be described by reference to the following biological examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.
  • Example 1 The invention will now be described by reference to the following biological examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.
  • Example 1 Example 1 :
  • pigs are of special interest: they represent the most promising source of tissues and whole organs for xenotransplantation (10).
  • Pig embryos were collected from gonadotropin-stimulated donor animals 24 to 36 hours after artificial insemination.
  • One to two cell embryos were infected with high titer (10 9 - 10 10 infectious units/ml) recombinant lentiviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G (11) by injection into the perivitelline space (5, 7).
  • the lentiviral vector (LV-PGK) carries the phosphoglycerate kinase promoter for expression of the GFP reporter transgene in different cell types, and two enhancer elements to increase transduction efficiency (12, 13) (Fig. 1 A).
  • Transgene expression was assayed first in whole animals by in vivo fluorescence imaging (14). Twelve animals expressed GFP at levels detectable by direct fluorescence, whereas the age matched control animals did not exhibit green fluorescence (Fig. 2A and B, Fig. 5). In the GFP-positive animals, all tissues accessible to this non-invasive technique - skin, claws, eye, tongue, gingiva, and teeth (Fig. 2A - H) -exhibited green fluorescence. Expression of GFP was also analyzed by direct fluorescence imaging of internal organs and tissues samples freshly isolated from animal #511 that carried 6 copies of LV-PGK (see also Fig. 1).
  • Transgene expression was quantified by Western blot analyses of skin biopsies. In contrast to in vivo imaging, GFP expression was detected by immunoblotting in 30 (94%o) out of 32 transgenic animals (Fig. 3A - C, Fig. 6).
  • Figure 3B shows the relationship between transgene expression levels and provirus number. Interestingly, the relationship between copy number and GFP concentration was almost identical in low (e.g. three integrants) and high (e.g. 12) copy animals.
  • Transgene expression levels increased over a broad range almost linearly with increasing lentiviral vector number (Fig. 3B). Similar transgene expression levels were detected in derivatives of all three germ layers (kidney, cerebellum, pancreas) of different animals (Fig. 3C).
  • Transgene expression was analyzed at a cellular level by direct fluorescence imaging of histological sections and immunohistochemistry using monoclonal antibodies against GFP. Direct GFP fluorescence was observed in all histological sections of organs from animal #511 (Fig. 4B, E, and H and Fig. 7), while no fluorescence was observed in the control sections (Fig. 4K).
  • Fig. 4B, E, and H and Fig. 7 For detailed histological analyses, we focused on relevant organs derived from the three germ layers: kidney, cerebellum and pancreas. The cortex and medulla of the kidney exhibited GFP expression (Fig. 2J). Analyses of GFP expression in cortical sections revealed GFP-positive cells in glomeruli and distal tubules (Fig.
  • Pigs are the most promising donor species for xenotransplantation.
  • several important obstacles have to be overcome, including the efficient transfer of foreign (human) genes into the pig genome.
  • transgenesis rates are the major determinant of production costs of transgenic livestock
  • the inefficiencies of pronuclear DNA microinjection and retroviral transgenesis result in immense costs of transgenic pigs and other livestock species produced by these techniques (15, 16).
  • Lentiviral gene transfer into early preimplantation pig embryos resulted in an unprecedented efficiency of transgenesis in pigs and could, therefore, allow for the high throughput and low cost production of transgenic pigs.
  • the estimated costs for transgenic pigs might be reduced to 1/10 th - 1/100 th of the present costs (16).
  • the homogenous distribution of the lentiviral integrants throughout the organism indicates that the embryos were transduced shortly after virus injection at the one- to two-cell stage.
  • animals derived from infection of embryos at later stages e.g. 4- to 30-cell stage with retro- or lentiviral vectors (4, 8, 17) invariably exhibit somatic mosaicism, which is based on differences in the number of viral particles that infect individual blastomeres.
  • lentiviral vectors can be used for the delivery of short interfering RNA (siRNA) to mouse preimplantation embryos and embryonic stem cells, resulting in the functional inactivation of the target gene in the living animal (18, 19), could have an impact for lentiviral transgenesis in livestock.
  • siRNA short interfering RNA
  • lentiviral vectors By delivering siRNA to pig embryos through lentiviral vectors, one may be able to knock-down pig genes that are of relevance for xenotransplant rejection.
  • siRNA short interfering RNA
  • Recombinant lentivirus was produced as described (20).
  • 293T cells were transfected with the vector (LV-PGK (21)); and packaging plasmids.
  • Lentiviral particles were pseudotyped with the G glycoprotein of the vesicular stomatitis virus.
  • Virus was harvested 48 and 72 h after transfection and concentrated by ultracentrifugation. The titers were determined by HIV-1 p24 ELISA (Alliance, NEN, K ⁇ ln, Germany) and correlated with green fluorescence of infected 293T cells.
  • Example 3 Embryo collection and virus injection
  • Embryos were collected from 6-month-old crossbred gilts after slaughter. These donors were superovulated with 1200 IE pregnant mare serum gonadotropin (PMSG; Intergonan ® , Intervet, Unterschleissheim, Germany), and ovulation was stimulated with 750 IE human chorionic gonadotropin (HCG; Ovogest ® , Intervet) three days later. During the following 24 to 36 hours donor animals were artificially inseminated twice, and slaughtered one and a half day after first insemination.
  • PMSG pregnant mare serum gonadotropin
  • HCG human chorionic gonadotropin
  • Oviducts were flushed with 38°C flush-media [phosphate-buffered saline (PBS) supplemented with 20% heat-inactivated lamb Serum (Invitrogen, Düsseldorf, Germany) and 50 mg gentamicin sulfate (Sigma, Steinheim, Gemany)]. Embryos were collected in flush- media and directly used for subzonal virus injection with glass capillaries containing concentrated virus.
  • PBS phosphate-buffered saline
  • gentamicin sulfate Sigma, Steinheim, Gemany
  • Prepuberal gilts of six to seven months of age were used as recipients.
  • Recipients were synchronized by oral administration of altrenogest (Regumate ® , Serum-Werk Bernburg AG, Bemburg, Germany) over a 15 days period, followed by administration of 750 IE PMSG (Intergonan ® ) one day after the last gestagen feeding. Ovulation was induced three days later with 750 IE HCG (Ovogest ® ). During the following 24 to 36 hours estrus behavior of the recipients was recorded. Only animals with clear signs of estrus were used as recipients.
  • Transfers were performed by laparoscopy under general anesthesia with a combination of 1.2 ml/10 kg ketamine hydrochloride (Ursotamin ® , Serumwerk Bernburg, Bernburg, Germany) and 0.5 ml/10 kg xylazine (Xylazin 2%, WDT, Germany) injected intravenously. To each recipient, 30 to 40 injected or control embryos were transferred in one oviduct.
  • Genomic DNA was digested with the restriction enzymes BamHI. DNA fragments were separated by electrophoresis through a 0.7% agarose gel and transferred to Gene Screen Plus Hybridization Transfer Membranes (PerkinElmer Life Sciences, Boston, USA). The blot was hybridized with a full-length 32 P-labeled EGFP cDNA probe in Church-buffer (1 % BSA, 387 mM, Na 2 HP0 4 , 113 mM NaH 2 P0 4 , 7% SDS, 1 mM EDTA, 100 ⁇ g/ml ssDNA) at 60°C overnight.
  • Church-buffer (1 % BSA, 387 mM, Na 2 HP0 4 , 113 mM NaH 2 P0 4 , 7% SDS, 1 mM EDTA, 100 ⁇ g/ml ssDNA
  • Example 6 In vivo fluorescence imaging
  • Excitation of green fluorescence was achieved using a Schott 2500 light source (Zeiss, Jena, Germany) and a 485 nm filter (Zeiss).
  • the emitted fluorescence was visualized using a long-pass filter (HQ 500, Zeiss) and images were acquired either using a digital camera (Canon) or using a STEMI SV6 (Zeiss) stereomicroscope with a HRc cooled charged-coupled device camera (Zeiss) attached.
  • Tissues were fixed with 4% paraformaldehyde overnight at 4 °C, and then saturated with 20% sucrose at 4 °C for 2 days. After embedding in Tissue-Tek ® O.C.T. Compound (Sakura Finetek, Zoeterwoude, Netherlands), tissues were cut at 10 ⁇ m on a microtome and stored at -20 °C.
  • the immunohistochemical procedure was performed based on the avidin-biotin- peroxidase complex (ABC) method. Endogenous peroxidase was blocked by incubation with methanol containing 3.3% H 2 O 2 at room temperature for 30 minutes. After incubating with blocking solution [2% normal chicken serum, 1 % BSA in TBS (50 mM Tris, 150 mM NaCl, pH 7.4)] for 30 minutes, the sections were incubated overnight with the primary antibody against EGFP (Living Colours ® A. v. Monoclonal Antibody, JL-8, Clontech Laboratories, Palo Alto, USA) diluted 1 :200 in blocking solution.
  • ABS avidin-biotin- peroxidase complex
  • the secondary biotinylated antibody polyclonal goat anti-mouse IgG, H+L, Dianova, Hamburg, Germany
  • blocking solution was applied for 1 hour at room temperature.
  • the sections were incubated with ABC solution (Vector Laboratories, Burlingame, CA, USA) for 30 minutes, washed in TBS, and stained with the peroxidase substrate 3 ⁇ 3- diaminobenzidine (DAB; 270 ⁇ g/ml; Sigma-Aldrich, Taufkirchen, Germany) in 0.5 M Tris containing 0.02% H 2 O 2 .
  • DAB peroxidase substrate 3 ⁇ 3- diaminobenzidine
  • pancreas islet cells Staining of pancreas islet cells was performed using polyclonal guinea pig anti-insulin antibodies (BioTrend Chemikalien GmbH, Koln, Germany) at a 1 :1000 dilution and incubated for 1 hour at room temperature. After washing in TBS sections were incubated for 1 hour at room temperature with goat anti-guinea pig IgG (H+L; Dianova) conjugated with alkaline phosphatase diluted 1 :200 and again washed in TBS.
  • polyclonal guinea pig anti-insulin antibodies BioTrend Chemikalien GmbH, Koln, Germany
  • Insulin was visualized by 5-bromo-4-chloro-3-indoyl phosphate (BCIP, Sigma- Aldrich) and nitrotetrazolium blue chloride (NBT, Sigma-Aldrich) in Tris-buffered solution (1.21 g/l Tris, 5.84 g/l NaCI, 1.01 g/l MgCI 2 , 100mg/l BCIP, 200 mg/l NBT, pH
  • Tissue samples were minced in lysis buffer (0.5% Triton X-100, 150 mM NaCI, 2 mM CaCI 2 and protease inhibitors) by ULTRA-TURRAX ® T 8 (IKA Laboratory Equipment, Staufen, Germany) and centrifuged at 13.400 rpm at 4 °C for 15 minutes. The soluble phase was dissolved in SDS-sample buffer and proteins were separated by 15% SDS-PAGE.
  • TBSTm After washing with TBSTm, the secondary antibody, a polyclonal peroxidase-conjugated goat anti-mouse IgG (H+L; Dianova) diluted 1 :3000 in TBSTm was added and incubated at room temperature for 1 hour. After washing with TBSTm and TBST, proteins were visualized using an enhanced chemoluminescent (ECL) detection kit and Hyperfilm ECL (Amersham Pharmacia Biotech).
  • ECL enhanced chemoluminescent
  • GFP concentration was calculated by comparing individual bands with the band intensity of a recombinant EGFP standard (BD Biosciences Clontech, Palo Alto, CA) and given as ng/ ⁇ g total protein.
  • Tissue-specific expression of transgenes by use of specific promoters driving transgene expression in the context of recombinant lentiviral vectors used for subzonal injection.
  • Skin-specific expression in animals derived from injection of a lentiviral vector carrying the GFP expression cassette under the control of the human keratin K14 gene promoter (Munz et al., 1999).
  • LV-K14 was constructed by replacing the PGK promoter of LV-PGK with the promoter of the human K14 gene (Munz et al., 1999). Results are shown in figure 8.
  • Bovine cumulus oocyte complexes were collected by aspirating ovarian follicles obtained from slaughtered animals. The in vitro production of bovine embryos was performed as previously described (Stojkovic et al., 2001). Briefly, oocytes were matured in vitro for 22 hours in modified TCM 199 (Invitrogen) at 39°C in 5 % C0 2 .
  • COCs were cocultured with frozen-thawed semen (10 6 spermatozoa/ml; capacitated in a swim-up procedure) for 18 hours. Presumptive zygotes were then denuded by vortexing and injected subzonally with LV-PGK.
  • oocytes were stripped free from cumulus cells, injected subzonally with LV-PGK, followed by in vitro fertilization as described above.
  • Embryos were cultured in modified synthetic oviduct fluid (SOF) supplemented with 10 % (v/v) estrous cow serum (ECS) (Stojkovic et al., 2001) at 39°C in a humidified atmosphere of 5% C0 2 , 5% 0 2 and 90% N 2 .
  • SOF modified synthetic oviduct fluid
  • ECS estrous cow serum
  • a corresponding newborn calf carrying and expressing a transgene for GFP is shown in figure 10.
  • Transgenic pig #507 (see also Fig. 1) was used to inseminate one wild-type female pig. The female pig was sacrificed and 2- to 4-cell stage embryos were isolated. Using nested PCR, the transgene was detected in 4 out of 11 embryos, clearly showing that the transgene/lentiviral provirus is transmitted through the germ-line. Nested PCR: 2- to 4-cell stage porcine embryos were isolated, and incubated with Protease K (Roche).
  • bovine oocytes were infected with lentiviral vectors, followed by IVF (in vitro fertilization).
  • Metastase II oocytes lack a nuclear envelope, which might hamper lentiviral integration.
  • a total of 48 oocytes were infected by subzonal injection with LV-GFP (lentiviral vector carrying the transgene eGFP), and within 7 days after IVF 12 blastocytes developed. Fluorescence microscopy revealed eGFP expression in 10 (83%) of these blastocytes. Five of the GFP-positive blostocytes were transferred into four heifers.
  • eGFP-specifc antibodies demonstrated transgene expxession in skin (Fig. 13, A-C), pancreas (Fig. 13, D-F) and kidney (Fig. 13, G-l), which are derived from different primary germ layers: ectoderm, endoderm, and mesoderm, respectively.
  • Expression of foreign genes in the bovine mammary gland is the basis for production of large quantities of recombinant proteins in the milk.
  • histological analysis revealed strong eGFP expression in ductal epithelial cells of the mammary gland (Fig. 14, A and B). Germ line transmission is another central aspect of transgenic studies in cattle.
  • eGFP expression in the testis of a newborn transgenic bull derived from subzonal injection of LV-GFP (no. 581) was analyzed. eGFP-positive cells were detected by immunohistochemistry in the seminiferous tubules (Fig. 14C).
  • a specific marker for spermatogonia DBA
  • DBA has a specific affinity for bovine gonocytes and spermatogonia in the testis during the first weeks after birth. Staining of testis sections with fluorescence-labeled DBA followed by direct fluorescence analysis revealed eGFP expression in DBA- positive cells (fig. 14D and 14E).
  • Another route to produce transgenic cattle by lentiviral gene transfer occording to the invention is the infection of donor cells followed by nuclear transfer NT.
  • Infection of bovine fetal fibroblasts (BFF) with LV-GFP resulted in more than 85% transduction (Fig. 15, A and B).
  • 214 nuclear transfer embryos were produced by transfer of infected fibroblast nuclei into enucleated bovine oocytes without further selection. After a week in culture, 76 (36%) blastocysts were obtained; 28 of the blastocysts were transferred into 16 synchronized recipients, resulting in five pregnancies. Although four pregnancies were lost, one calf (no. 991) was born naturally.

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Abstract

L'invention concerne un procédé de production de mammifère porteur d'au moins un transgène, non humain, non rongeur, et un procédé de préparation de cellules, de tissu ou d'organes de mammifère porteur d'au moins un transgène, l'utilisation des cellules, du tissu ou des organes en question pour une composition destinée à la transplantation vers un receveur, et un procédé de transplantation des cellules, du tissu ou des organes en question vers un donneur. L'invention concerne également un procédé de production d'un ou plusieurs composés venant d'un mammifère ainsi produit, l'utilisation de ces composés pour la préparation de compositions pharmaceutiques, et des procédés de traitement de maladies animales ou humaines par l'administration des compositions en question.
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WO2008106985A1 (fr) * 2007-03-07 2008-09-12 Aarhus Universitet Cochon utilisé comme modèle

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WO2003022040A2 (fr) * 2001-09-13 2003-03-20 California Institute Of Technology Procede de production d'animaux transgeniques

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008106985A1 (fr) * 2007-03-07 2008-09-12 Aarhus Universitet Cochon utilisé comme modèle
US8581021B2 (en) 2007-03-07 2013-11-12 Aarhus Universitet Pig whose genome comprises a heterologous site-specific recombination site and a transpohon tag

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