WO1993005817A1 - Mammiferes transgeniques depourvus des antigenes des classes mhc i et ii - Google Patents

Mammiferes transgeniques depourvus des antigenes des classes mhc i et ii Download PDF

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WO1993005817A1
WO1993005817A1 PCT/US1992/007992 US9207992W WO9305817A1 WO 1993005817 A1 WO1993005817 A1 WO 1993005817A1 US 9207992 W US9207992 W US 9207992W WO 9305817 A1 WO9305817 A1 WO 9305817A1
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mhc
cells
class
deficient
mhc class
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PCT/US1992/007992
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Laurie H. Glimcher
Virginia E. Papaioannou
Michael J. Grusby
Randall S. Johnson
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President And Fellows Of Harvard College
Tufts University
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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
    • C12N15/63—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
    • 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
    • A01K67/0276—Knock-out vertebrates
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A01K2227/105—Murine
    • 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
    • A01K2267/03—Animal model, e.g. for test or diseases
    • 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
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0381—Animal model for diseases of the hematopoietic system
    • 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

Definitions

  • the invention relates to transgenic animals.
  • MHC Major histocompatibility complex
  • T lymphocyte antigen receptors T lymphocyte antigen receptors. This tripartite interaction triggers antigen-specific responses in the immune system to invading organisms such as bacteria and viruses.
  • the class II antigens also act as immunogenic determinants, allowing an organism to distinguish self from non-self; it is this recognition which leads, e.g. , to graft rejection.
  • the invention features a transgenic non-human mammal, the surface of whose cells are deficient in all ' MHC class II antigens and one or more MHC class I antigens.
  • a chromosome of the mammal includes a null mutation in an MHC class II antigen gene and a null mutation in the ⁇ 2- microglobulin gene; the jS2-microglobulin gene and class II antigen genes including null mutations replace the endogenous jS2-microglobulin and MHC antigen genes; the mammal is homozygous for the mutant genes; and the mammal's cell surfaces are deficient in all MHC antigens.
  • the invention features a transgenic mouse, the surface of whose cells are deficient in MHC class I and class II antigens; a chromosome of the mouse includes a null mutation in the Aj0 b gene; a chromosome of the mouse includes a null mutation in the E ⁇ b gene; the chromosome of the mouse includes a null mutation in the /3 2 -microglobulin gene; and the mouse's cell surfaces are.deficient in all MHC- surface antigens.
  • the invention features a method of testing a substance for efficacy in the treatment of an MHC antigen deficiency. The method involves exposing a transgenic animal of the invention to the substance and then examining the animal's cell surfaces for the presence of the MHC antigen, the presence of the antigen being an indication that the substance is useful for the treatment of the deficiency.
  • the invention features use of the isolated cells from the transgenic animal as a medicament of wound healing.
  • the cell used as a medicament is an epithelial cell, for example, a keratinocyte; the cell produces a recombinant growth factor; and the isolated cell is cultured in vitro prior to use for wound healing.
  • the invention features use of the transgenic tissue as a medicament for diseases requiring transplantation of tissue into a human.
  • the tissue used as a medicament is a whole organ, for example, a heart, a kidney, or a liver; or the method further involves introducing into the donor tissue of a gene which expresses a recombinant protein, for example, Factor VIII.
  • the invention features a mammalian cell whose cell surface is deficient in all MHC class II antigens and an MHC class I antigen; the mammalian cell is isolated from a transgenic animal of the invention; the mammalian cell is a human cell; and the mammalian cell expresses a recombinant protein, e.g., Factor VIII.
  • Transgenic as used herein means a mammal which includes a DNA sequence which is inserted by artifice into a cell and becomes a part of the genome of the animal which develops from that cell. Such a transgene may be partly or entirely heterologous to the transgenic animal.
  • the DNA sequence is used to insertionally inactivate a gene encoding an MHC class II antigen.
  • the inactivated class II antigen gene is homologously recombined into the mammal's chromosome, replacing all or most of the "endogenous" gene.
  • the transgenic animals thus produced are "deficient" in MHC class II antigens, meaning that the animals exhibit fewer than wild-type numbers of such antigens on their cell surfaces; preferably, the animals • exhibit no class II antigens on their cell surfaces or exhibit so few class II antigens that they are undetectable by standard techniques of immunochemistry.
  • Any non-human mammal which may be produced by transgenic technology is included in the invention; preferred mammals include, in addition to mice, cows, pigs, sheep, goats, rabbits, guinea pigs, hamsters, and horses.
  • MHC class II antigens any protein normally presented on the surface of immune cells which, in combination with a foreign antigen, form a complex which is recognized by T lymphocyte antigen receptors.
  • MHC class II surface antigens include MHC class II I-A and MHC class II I-E molecules.
  • MHC class II surface antigens include the products of the HLA-DP, HLA-DQ, and HLA-DR genes.
  • null mutation is meant a mutation which renders a gene incapable of producing a functional protein product. As used herein, it includes mutations which destroy an MHC antigen gene's ability to express any product at all; MHC genes containing such a mutation are identified, e.g., using the immunohistochemical methods described herein. Null mutations also include those mutations which permit the production of some protein or protein fragment, but the protein or protein fragment does not function (as used herein) as an MHC antigen; MHC antigen functionality is tested, e.g., using methods described in Fundamental Immunology (ed. William E. Paul, Raven Press, New York, 1989) .
  • a null mutation includes a mutation which leads to the production of a mutant protein or protein fragment which is degraded by the host cell before it is localized to the cell surface.
  • a null mutation is a functional null allele comprising a targeted alteration that interferes with the efficient expression of a functional gene product from the allele.
  • cells deficient in MHC class I and MHC class II antigens is meant cells of a mammal which are nucleated somatic cells and germ cells.
  • the invention provides useful model systems for MHC antigen immunodeficiencies, such as Bare Lymphocyte Syndrome (characterized by the absence of MHC class II surface antigens) .
  • MHC class II antigens are substantially absent, cells from such animals are less prone to rejection by recipient animals; the transgenic animals thus provide a universal source of donor tissue for transplant.
  • tissue may be used to promote wound healing (e.g., using epithelial cells, e.g., keratinocytes) , or it may be used for whole organ transplantation.
  • somatic cell therapies e.g., for delivery to a patient of such proteins as Factor VIII, ⁇ - galactosidase A or ornithine aminotransferase.
  • FIG. 1 illustrates gene targeting of the Ao b gene and germ line transmission of the disrupted allele.
  • A a schematic of the gene targeting construct used for disruption of the Ao b gene is shown. Black boxes represent exons and restriction enzyme sites are as indicated.
  • the neo r gene from pMClneo-poly A was subcloned into the second exon of the A ⁇ b gene and the tk gene from HSV-1 was positioned in the targeting vector as previously described in Johnson et al., (Science 245:1234. 1989).
  • the probes used for Southern blot analysis are also indicated.
  • FIG. 2 illustrates a flow cytometric analysis of MHC class II expression in the periphery of class II deficient animals.
  • a and B single cell suspensions from spleens and lymph nodes of wild type
  • (+/+) mice were analyzed using antibodies specific for Ao b (25-9-17, 34-5-3) arid A ⁇ b (3JP, 1E9) ; control shows background staining obtained with the secondary reagent alone.
  • C and D flow cytometric analysis of lymphocytes from mutant (-/-) animals was performed as described above. None of the class II antigen-specific antibodies revealed specific lymphocyte staining. Flow cytometric analysis was performed in triplicate with one wild type and one mutant animal each time; all trials yielded essentially identical results.
  • FIG. 3 is a tabular representation of the results of a two color flow cytometric analysis of lymphocyte subsets in wild-type and MHC class II deficient animals.
  • the control animal for Experiment 1 was (+/+) and for Experiment 2 was (+/-) .
  • the mutant animals were (-/-) for both Experiment 1 and Experiment 2.
  • FIG. 4 illustrates flow cytometric analysis of MHC class I and class II expression in the periphery of MHC- deficient animals.
  • Single cell suspensions were prepared from spleens of control and MHC-deficient animals and 1 x 10 6 cells were stained with hybridoma supernatants containing class I-specific (A) or class II-specific (B) antibodies, followed by a fluorescein-conjugated Ffab') 2 fragment of goat antibody to mouse IgG.
  • MAbs were 3*-83P (H-2K b ) , 28-14-8S (H-2D b ) , 28-14-8S (H-2D b ) , 25-9-17S (I- A/3 b ) , and 1E9 (I-A ⁇ b ) .
  • Control shows background staining with the secondary reagent alone.
  • FIG. 5 illustrates flow cytometric analysis of T cell subsets in the lymphoid organs of MHC-deficient animals.
  • Single cell suspensions were prepared from thy i, spleens, and lymph nodes of control and MHC- deficient animals and analyzed as described in Fig. 4. Approximately 10,000 events were recorded for each analysis.
  • FIG. 6 illustrates the phenotype and function B of cells in MHC-deficient animals.
  • A Single cell suspensions were prepared from spleens of control and MHC-deficient animals and analyzed as described in Fig. 4. Approximately 10,000 events were recorded for each analysis.
  • FIG. 7 depicts the results of MLR assays using MHC-deficient spleen cells as responder and stimulator.
  • the responder populations were depleted of CD4 + cells where indicated.
  • mice which are devoid of cell surface expression of MHC class II molecules by introducing a loss of function mutation into the Ao b gene (i.e., one of the MHC class II genes) in ES-D3 cells.
  • Ao b gene i.e., one of the MHC class II genes
  • Disruption of the A ⁇ b gene prevented the cell surface expression of I-A molecules on class II-expressing cells.
  • the disruption in the E ⁇ b gene (another of the MHC- class II genes) in the ES-D3 host cells i.e., cells derived from mice of the H- 2 b ha lotype; Doetschman et al., J. Embryol . Exp.
  • Morph . 81_: 27 , 1985 rendered such cells similarly devoid of I-E cell surface expression on class II-expressing cells (Mathis et al. , Proc. Natl . Acad. Sci . USA .80:273, 1983).
  • a 5.4 kb EcoRI-XhoI fragment of the Ao b gene (encompassing the first four exons) was subcloned into a pBluescript SK(+) vector into which a 3.4 kb BamHI fragment of HSV-1 containing the tk gene was previously blunt cloned into the Nael site (Johnson, et al.. Science 245:1234-1236, 1989).
  • the unique BstEII restriction enzyme site in the second exon of the Ao b gene was changed to a Sail site with linkers, and the 1.1 kb Xhol-Sall fragment of pMClneo-poly A (Stratagene, La Jolla, CA) , containing the neo r gene, was inserted into this site.
  • 2 X 10 7 ES-D3 cells publicly available from the American Type Culture Collection, Rockville, MD; ATCC Accession No.
  • Probe 1 is a 690 bp Bglll-EcoRI fragment which hybridizes to a 6.3 kb Ncol fragment of the endogenous A» b gene and to a 3.8 kb fragment of the disrupted allele resulting from homologous recombination with the depicted targeting construct.
  • Probe 2 is a 705 bp XhoI-EcoRI fragment which hybridizes to a 6.4 kb EcoRI fragment of the endogenous Ao b gene and 5.2 kb fragment of the disrupted allele.
  • the targeting vector incorporates the neomycin resistance (neo r ) gene into the second exon of the Ao b gene, contains 5.4 kb of homologous flanking sequence, and contains the Herpes simplex Virus (HSV-1) thymidine kinase (tk) gene, allowing positive-negative selection of transfectants by the method of Mansour et al. (Nature 136, 348-352, 1988).
  • frozen sections of thymus from wild type (+/+) or mutant (-/-) animals were isolated and examined by immunohistochemistry as follows. Frozen sections (6/x) of thymus from wild type (+/+) and mutant (-/-) mice were hydrated in 0.05 M Tris-HCl (pH 7.5) for 5 min and then blocked with 3% horse serum for 15 min.
  • Sections were then incubated with 50 / vc-l.of purified Ao b - specific monoclonal antibody 25-9-17 (30 ⁇ g/ml diluted in horse serum) for 60 min, followed by incubation with biotinylated horse anti-mouse IgG and avidin-horseradish peroxidase by the method of the manufacturer (Vectastain Elite ABC kit. Vector Laboratories, Burlingame, CA) . Stained sections were developed with 3,3'- diaminobenzidine as chromogen for 4 min.
  • Single suspensions were prepared from spleens and lymph nodes of wild type (+/+) or mutant (-/-) animals, and 1.5 x 10 6 cells were stained for 20 min at 4°C with 50/l of hybridoma supernatant containing class II specific antibodies; antibodies were 25-9-17 and 34-5-3 (A ⁇ ) and 3JP and 1E9 AJ ) .
  • Cells were washed once in FACS media (Hanks Buffered Saline Solution, 3% fetal calf serum, 0.1% NaN 3 ) and then incubated for 20 min at 4°C with fluorescein-conjugated F(ab') 2 fragment of goat anti- mouse IgG ( ⁇ -chain specific) (Cappel, Durham, NC) .
  • thymocyte subsets in class II deficient animals were also examined.
  • Two color flow cytometric analysis of thymocyte subsets in mutant mice was carried out as described above using a FACScan flow cytometer (Becton-Dickinson, Lincoln Park, NJ) and fluorescein-conjugated anti-CD4 (Pharmingen, San Diego, CA) and phycoerythrin-conjugated anti-CD8 (Pharmingen) .
  • FACScan flow cytometer Becton-Dickinson, Lincoln Park, NJ
  • fluorescein-conjugated anti-CD4 Pharmingen, San Diego, CA
  • phycoerythrin-conjugated anti-CD8 phycoerythrin-conjugated anti-CD8
  • CD4 + cells in the periphery of class II deficient mice were markedly reduced in comparison to control littermates (FIG. 3). In contrast to the thymus, however, there were detectable numbers of CD4 + cells in the spleen and lymph nodes (l%-3%) . These cells may represent either class I restricted CD4 + T cells, CD4 + T cell receptor-bearing T cells, or CD4 + natural killer ⁇ like cells. Production and Analysis of MHC Class I and Class II Deficient Mice
  • mice harboring a gene disruption at the /3 2 -microglobulin (j3 2 m) locus were mated to animals carrying a similar mutation in the Aj9 b gene (e.g., those animals described above) .
  • mice homozygous for both the /3 2 m and class II mutant alleles were mated and subsequently intercrossed, to produce mice homozygous for both the /3 2 m and class II mutant alleles (MHC- deficient mice) .
  • mice homozygous for the disrupted / 3 2 m allele and heterozygous for the disrupted A « b allele were mated to mice heterozygous for the disrupted / 3 2 m allele and homozygous for the disrupted Ao b allele, thereby generating litters which include class I- deficient, class II-deficient, and MHC-deficient, as well as littermate control animals, which are singly mutant homozygotes and multiply mutant heterozygote combinations. All animals were maintained in autoclaved microisolate cages and provided with autoclaved food and water. When housed under these conditions, MHC-deficient animals and their littermates appeared healthy. BALB/cBy mice were purchased from The Jackson Laboratory (Bar).
  • mAbs MHC class I- or class II-specific monoclonal antibodies
  • mAbs were 3-83P (H-2K b ) , 28-14-8S (H-2D b ) , and 25-9-17S (I-Ao: b ) .
  • Cells were then washed once in Hanks balanced salt solution, 3% fetal calf serum, and 0.1% sodium azide and then incubated with a fluorescein-conjugated F(ab') 2 fragment of goat antibody to mouse IgG (r-chain- specific) .
  • mice homozygous for mutations of the ,3 2 m and A « b loci are essentially devoid of both MHC class I and class II molecules.
  • T cells subsets present in the lymphoid organs of MHC-deficient animals was examined.
  • Flow cytometric analysis of thymocytes from MHC-deficient mice using mAbs specific for CD4 and CD8 revealed the presence of normal numbers of double-positive cells but a virtual absence of single positive CD4 + and CD8 + cells (Fig. 5) .
  • these latter two populations were significantly depleted in the spleens and lymph nodes of MCH-deficient animals.
  • spleens from MHC-deficient animals are essentially devoid of mature T cells defined by markers for CD4 and CD8 (Fig. 5) as well as CD3 and A ⁇ / 3 T cell receptor, their cellarity is approximately twice that of littermate controls.
  • markers for CD4 and CD8 Fig. 5
  • CD3 and A ⁇ / 3 T cell receptor markers for CD4 and CD8
  • their cellarity is approximately twice that of littermate controls.
  • spleens from MHC-deficient animals have normal numbers of natural killer cells and ⁇ T cells, but slightly increased numbers of macrophages and granulocytes.
  • mice have normal numbers of IgM + B lymphocytes, but are unable to mount antibody responses to T-dependent antigens due to a lack of CD4 + T helper cells (Grusby et al., Science 253:1417-1420, 1990; Cosgrove et al., Cell .66.:1051-1066, 1991) as shown by the following experiments.
  • Animals were immunized interperitoneally with 100 ⁇ g of trinitrophenol (TNP)- conjugated ficoll in phosphate buffered saline. Animals were bled 3 days prior to immunization, and 8 and 16 days post-immunization. Serum was prepared and stored at 4°C.
  • TNP trinitrophenol
  • TNP-specific antibody responses were measured by coating flat-well microtiter plates overnight at 4°C with 25 ⁇ g/ral of TNP-conjugated bovine serum albumin in Tris buffered saline (TBS) . After washing twice with TBS, wells were blocked with 2% goat serum in TBS, and then washed twice again with TBS. Serial five- old dilutions from 1:100 to 1:12500 were analyzed. Flow cytometric analysis of spleen cells from MHC-deficient animals shows that mature IgM + B cells also develop normally in an environment devoid of MHC class I and class II molecules (Fig. 6A) .
  • TNP-specific antibody responses of all isotypes can be elicited in MHC-deficient animals following immunization with the T-independent antigen TNP-ficoll (Fig. 6B) .
  • B cells from MHC- deficient animals can be induced to proliferate following stimulation with the mitogen lipopolysaccharide.
  • MHC-deficient cells The capacity of MHC-deficient cells to serve as both responder and stimulator populations in a mixed lymphocyte reaction (MLR) was also examined.
  • Responder spleen cells (2 x 10 5 ) and irradiated (2,000 rad) stimulator spleen cells (4 x 10 5 ) were added to triplicate U-bottom wells in a final volume of 200 ⁇ l of RPMI 1640 supplemented with 20 mM Hepes, 2 mM L- glutamine, 0.1 mM nonessential amino acids, 50 ⁇ ,M-2- mercaptoethanol, 10% fetal calf serum, and 100 ⁇ g/ml gentarnicin.
  • the samples were harvested onto glass fiber filters and [ 3 H]thymidine uptake was measured by ⁇ scintillation counting (30 sec/sample) . Results are expressed as mean counts per min +/- SEM. In some experiments, spleen cells were depleted of CD4 + or CD8 + cells.
  • Spleen cells 50 x 10 6 cells/ml were incubated for 30 min on ice with medium alone, or a 1:4 dilution of ascites containing mAb GK1.5 (anti-CD4) or 2.43 (anti-CD8) .
  • Cells were washed once, resuspended at 40 x 10 6 cells/ml, and then incubated with a 1:5 dilution of rabbit complement (C") (C-Six Diagnostic) for 30 min at 37°C. The cells were then washed twice and counted.
  • C rabbit complement
  • MHC-deficient 129/Sv mice demonstrate only marginal levels of proliferation to the same allogeneic stimulator cells.
  • This proliferation is completely abolished when the MHC-deficient responding population is pretreated with anti-CD4 mAb plus complement, suggesting that the responding population is contained within the small numbers of CD4 + cells found in the periphery of MHC-deficient animals.
  • MHC-deficient cells are used as stimulator cells in MLR (Fig. 7B) , they stimulate very low levels of proliferation of allogeneic responder cells relative to normal 129/Sv stimulator cells.
  • This proliferation is also due to CD4 + cells, as pretreatment of the responder population with anti-CD4 mAb plus complement abrogates the response. This result was surprising since MHC- deficient cells do not express the class II antigens for which allogeneic CD4 + cells might.
  • MHC class II antigen genes make possible the production of both animals and mammalian cells lacking MHC surface antigens; such animals and cells are useful for diagnostic and therapeutic purposes.
  • the animals of the invention may be used as models of human disease characterized by MHC class II surface antigen deficiencies, and thus are useful to test potential therapeutics.
  • the animals may be used to test compounds for those which are useful for the treatment of Bare Lymphocyte Syndrome (an immunodeficiency disease characterized by a lack of all MHC class II antigen expression resulting in an unusual susceptibility to infection and infant or early childhood mortality) .
  • Bare Lymphocyte Syndrome an immunodeficiency disease characterized by a lack of all MHC class II antigen expression resulting in an unusual susceptibility to infection and infant or early childhood mortality
  • an animal of the invention is contacted with a candidate material.
  • Administration is by any known route, but is preferably intravenous and is preferably at a range of concentrations.
  • a sample of lymphoid cells is isolated from the animal (as described herein) and examined for the presence of MHC class II surface antigens (e.g., by the methods described herein) .
  • a useful therapeutic is one which promotes an increase in the number of MHC class II surface antigens on any host cell (e.g., on lymphoid cells) .
  • transgenic sequences encoding particular MHC class II or class I antigens which are either endogenous to the animal or heterologous (e.g., a human sequence in a murine animal) may be introduced into the animal, e.g.. by the methods described herein to create well-defined models for immune disorders which involve, e.g. , the loss of only one murine or one human class of surface antigen.
  • availability of animals and mammalian cells lacking MHC surface antigens provides an abundant source of universal donor tissue for transplant. In one particular example, such tissue includes epithelial cells useful for the promotion of wound healing.
  • the epithelial cells may be derived from a MHC surface antigen-deficient transgenic animal or may be derived from cultured epithelial cells (e.g., keratinocytes) which harbor inactivated MHC antigen genes (produced, e.g., by the general methods described herein; in Zijlstra et al., Nature 342:435. 1989; or in WO 91/01140, hereby incorporated by reference) .
  • the cells may be engineered (by standard techniques) to produce and secrete a growth factor which further speeds the healing of a lesion.
  • whole organs e.g., hearts isolated from MHC antigen-deficient transgenic animals may be transplanted into human, recipients.
  • Donor organs are preferably isolated from larger transgenic animals such as pigs (also created by the general methods described herein) .
  • Donor tissue is administered to the patient using standard transplantation techniques.
  • the animal donors preferably include a null mutation in at least one MHC class II antigen gene (e.g., the murine genes: Att, A / 3, E ⁇ , or E / 9; the human genes: HLA-DP, HLA-DQ, or HLA- DR; or the porcine genes: SLA class II) and a null mutation in at least one MHC class I antigen gene (e.g., the / 3 2 -microglobulin gene from (B 2 m) mice, humans, or pigs; see, e.g., Zijlstra et al., Nature 342:435, 1989 or WO 91/01140, hereby incorporated by reference),
  • Such genes may be sequentially inactivated using a homologous replacement system (e.g., that described herein).
  • animals lacking both MHC class I and MHC class II antigens may be produced by breeding animals deficient in one class of antigens with animals deficient in the other class.
  • the MHC class II antigen-deficient mice described herein may be crossed with /?2-microglobulin-deficient, and thus MHC class I antigen-deficient, mice (e.g., those described in Zijlstra et al. , Nature 342:435. 1989) to produce offspring which are deficient in all MHC surface antigens as demonstrated above.
  • MHC antigen-deficient tissue may also be used as a general vehicle for somatic cell therapies.
  • Any gene of interest e.g. , the human Factor VIII gene
  • Any gene of interest may be introduced into a transgenic cell of the invention such that the protein product is expressed by the cell. This is accomplished, e.g., by positioning the gene (e.g., the Factor VIII gene) downstream of a viral enhancer.
  • General methods for insertion of genes and somatic cell expression are described in Mansour et al. (Nature 336:348. 1988) and U.S. Pat. 4,868,116, hereby incorporated by reference.
  • the transgenic cells lack MHC antigens, the problems normally associated with immune rejection are circumvented, and useful cells may therefore be derived from any organism.
  • the gene targeting methods described herein may be used to specifically inactivate both alleles of the MHC class II genes (e.g. , using methods described herein combined with those described in Riele et al.. Nature 348:649 r
  • the MHC class I proteins e.g., by inactivating the ,92-microglobulin gene
  • the patient's own cells e.g., the patient's isolated lymphocytes.
  • the engineered cells can then be used as host cells for the production of a therapeutic protein (as described above) .
  • the MHC class II antigen-deficient animals of this invention can be used to examine the role of the class II antigens in any of several complicated immunological processes characterized by interaction between antigen presenting cells and T cells.
  • the animals may also be used generally to examine the absence of class II expression .in vivo. This is not possible to do in humans, e.g., in BLS patients, since they receive bone marrow transplants at an early age.

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Abstract

Mammifères transgéniques non humains, dont les cellules présentent une surface dépourvue de tous les antigènes de la classe MHC II et d'un ou de plusieurs antigènes de la classe MHC I. On décrit également l'utilisation de ces mammifères comme modèles pour l'étude des immunodéficiences provoquées par l'absence des antigènes MHC et comme sources de tissus de donneur universel en vue d'une greffe ou d'une thérapie des cellules somatiques.
PCT/US1992/007992 1991-09-19 1992-09-21 Mammiferes transgeniques depourvus des antigenes des classes mhc i et ii WO1993005817A1 (fr)

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US76282391A 1991-09-19 1991-09-19
US762,823 1991-09-19

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Cited By (18)

* Cited by examiner, † Cited by third party
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EP0596887A1 (fr) * 1991-01-10 1994-05-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Souris obtenue par genie genetique presentant des modifications des genes de la classe ii du complexe majeur d'histocompatibilite
WO1994028122A1 (fr) * 1993-05-26 1994-12-08 Ontario Cancer Institute Mammiferes transgeniques chez qui manque l'expression d'isoformes cd45 particuliers
WO1994028123A1 (fr) * 1993-05-26 1994-12-08 Ontario Cancer Institute Mammiferes ne possedant pas l'expression du transgene cd28
WO1995003331A1 (fr) * 1993-07-23 1995-02-02 Zeneca Limited Double transgene de la classe ii du complexe majeur d'histocompatibilite humain et ses utilisations
WO1995006744A2 (fr) * 1993-09-03 1995-03-09 Chiron Viagene, Inc. Procedes de suppression de la reponse immunitaire par therapie genique
WO1997045550A2 (fr) * 1996-05-31 1997-12-04 Baxter International Inc. Vecteur mini-adenoviral
WO1998054345A1 (fr) * 1997-05-30 1998-12-03 Baxter International Inc. Vecteur mini-adenoviral
US6403370B1 (en) 1997-02-10 2002-06-11 Genstar Therapeutics Corporation Oncolytic/immunogenic complementary-adenoviral vector system
FR2827302A1 (fr) * 2001-07-13 2003-01-17 Genoway Cellule et animal transgenique modelisant la presentation antigenique humaine et leurs utilisations
US8847005B2 (en) 2011-10-28 2014-09-30 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US9043996B2 (en) 2011-10-28 2015-06-02 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex animals
US9591835B2 (en) 2011-10-28 2017-03-14 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex animals
US9615550B2 (en) 2011-10-28 2017-04-11 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US9848587B2 (en) 2013-02-20 2017-12-26 Regeneron Pharmaceuticals, Inc. Humanized T cell co-receptor mice
US10154658B2 (en) 2013-02-22 2018-12-18 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US10314296B2 (en) 2013-02-22 2019-06-11 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US11259510B2 (en) 2015-04-06 2022-03-01 Regeneron Pharmaceuticals, Inc. Humanized T cell mediated immune responses in non-human animals
US11813318B2 (en) 2011-04-20 2023-11-14 University Of Washington Beta-2 microglobulin-deficient cells

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* Cited by examiner, † Cited by third party
Title
CELL, Volume 66, issued 06 September 1991, D. COSGROVE et al., "Mice Lacking MHC Class II Molecules", pages 1051-1066. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 260, No. 26, issued 15 November 1985, D. LARHAMMAR et al., "Sequence of Gene and cDNA Encoding Murine Major Histocompatibility Complex Class II Gene A Beta 2", pages 14111-14119. *
NATURE, Volume 312, issued 22 November 1984, W.I. WOOD et al., "Expression of Active Human Factor VIII from Recombinant DNA Clones", pages 330-336. *
NATURE, Volume 342, issued 23 November 1989, M. ZIJLSTRA et al., "Germ-Line Transmission of a Disrupted Beta-2-Microglobulin Gene Produced by Homologous Recombination in Embryonic Stem Cells", pages 435-438. *
NUCLEIC ACIDS RESEARCH, Volume 11, No. 15, issued 1983, J.J. HYLDIG-NIELSEN et al., "The Complete Nucleotide Sequence of the I-E Alpha D Immune Response Gene", pages 5055-5071. *

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0596887A4 (en) * 1991-01-10 1997-02-26 Inst Nat Sante Rech Med Genetically engineered mice containing alterations in the mhc class ii genes.
EP0596887A1 (fr) * 1991-01-10 1994-05-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Souris obtenue par genie genetique presentant des modifications des genes de la classe ii du complexe majeur d'histocompatibilite
US6664107B1 (en) 1993-05-26 2003-12-16 Ontario Cancer Institute, University Health Network CD45 disrupted nucleic acid
WO1994028122A1 (fr) * 1993-05-26 1994-12-08 Ontario Cancer Institute Mammiferes transgeniques chez qui manque l'expression d'isoformes cd45 particuliers
WO1994028123A1 (fr) * 1993-05-26 1994-12-08 Ontario Cancer Institute Mammiferes ne possedant pas l'expression du transgene cd28
US5616491A (en) * 1993-05-26 1997-04-01 Ontario Cancer Institute Knockout mice
WO1995003331A1 (fr) * 1993-07-23 1995-02-02 Zeneca Limited Double transgene de la classe ii du complexe majeur d'histocompatibilite humain et ses utilisations
WO1995006744A2 (fr) * 1993-09-03 1995-03-09 Chiron Viagene, Inc. Procedes de suppression de la reponse immunitaire par therapie genique
WO1995006744A3 (fr) * 1993-09-03 1995-04-06 Viagene Inc Procedes de suppression de la reponse immunitaire par therapie genique
WO1997045550A2 (fr) * 1996-05-31 1997-12-04 Baxter International Inc. Vecteur mini-adenoviral
WO1997045550A3 (fr) * 1996-05-31 1998-04-09 Baxter Int Vecteur mini-adenoviral
US6403370B1 (en) 1997-02-10 2002-06-11 Genstar Therapeutics Corporation Oncolytic/immunogenic complementary-adenoviral vector system
WO1998054345A1 (fr) * 1997-05-30 1998-12-03 Baxter International Inc. Vecteur mini-adenoviral
FR2827302A1 (fr) * 2001-07-13 2003-01-17 Genoway Cellule et animal transgenique modelisant la presentation antigenique humaine et leurs utilisations
WO2003006639A1 (fr) * 2001-07-13 2003-01-23 Genoway Cellule et animal transgenique modelisant la presentation antigenique humaine et leurs utilisations
US11813318B2 (en) 2011-04-20 2023-11-14 University Of Washington Beta-2 microglobulin-deficient cells
US9043996B2 (en) 2011-10-28 2015-06-02 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex animals
US10869466B2 (en) 2011-10-28 2020-12-22 Regeneran Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US9591835B2 (en) 2011-10-28 2017-03-14 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex animals
US9615550B2 (en) 2011-10-28 2017-04-11 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US9700025B2 (en) 2011-10-28 2017-07-11 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex animals
US9585373B2 (en) 2011-10-28 2017-03-07 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US10045516B2 (en) 2011-10-28 2018-08-14 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex animals
US8847005B2 (en) 2011-10-28 2014-09-30 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US10219493B2 (en) 2011-10-28 2019-03-05 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US11219195B2 (en) 2011-10-28 2022-01-11 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US10779520B2 (en) 2011-10-28 2020-09-22 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex animals
US10986822B2 (en) 2011-10-28 2021-04-27 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US9848587B2 (en) 2013-02-20 2017-12-26 Regeneron Pharmaceuticals, Inc. Humanized T cell co-receptor mice
US10820581B2 (en) 2013-02-20 2020-11-03 Regeneron Pharmaceuticals, Inc. Humanized T cell co-receptor mice
US10314296B2 (en) 2013-02-22 2019-06-11 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US11224208B2 (en) 2013-02-22 2022-01-18 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US10154658B2 (en) 2013-02-22 2018-12-18 Regeneron Pharmaceuticals, Inc. Genetically modified major histocompatibility complex mice
US11259510B2 (en) 2015-04-06 2022-03-01 Regeneron Pharmaceuticals, Inc. Humanized T cell mediated immune responses in non-human animals

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