WO1994016065A1 - Production de cellules destinees a la transplantation et reduisant le rejet du greffon par l'hote, et cellules ainsi obtenues - Google Patents

Production de cellules destinees a la transplantation et reduisant le rejet du greffon par l'hote, et cellules ainsi obtenues Download PDF

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WO1994016065A1
WO1994016065A1 PCT/US1993/012670 US9312670W WO9416065A1 WO 1994016065 A1 WO1994016065 A1 WO 1994016065A1 US 9312670 W US9312670 W US 9312670W WO 9416065 A1 WO9416065 A1 WO 9416065A1
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cells
εaid
cell
recipient
cellε
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PCT/US1993/012670
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Paul J. Leibowitz
Samuel C. Wadsworth
Chee-Wai Woon
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Exemplar Corporation
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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
    • 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
    • A01K2217/05—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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • This invention relates to a novel method for producing cells in a donor organism for use in transplantation to a recipient organism which reduces transplant rejection and to organisms having such cells.
  • T cells respond to antigens that are present on cell surfaces.
  • T cells function by recognizing foreign antigens in combination with determinants present on their host's own cells. These determinants are cell surface proteins coded for by major histocompatibility complex (MHC) genes, especially MHC class I genes.
  • MHC major histocompatibility complex
  • the MHC proteins are present on the surface of all cell types in an animal. Since MHC class I proteins are highly polymorphic, any two animals are likely to have different MHC proteins. T cells use these differences to distinguish self from non-self.
  • transplanting into an i munologically deficient host e.g., athymic animals
  • transplanting into an immunologically compromised host e.g., an irradiated host or a host treated with an immunosuppressive drug such as cyclosporin
  • transplanting into a syngeneic host e.g., an irradiated host or a host treated with an immunosuppressive drug such as cyclosporin
  • E19 protein a protein, the E19 protein, from adenoviruse ⁇ which affects presentation of MHC class I cell surface antigens on cells infected with the adenovirus. Since T-cell receptors simultaneously recognize both the MHC class I cell surface antigens and the surface viral antigens on adenovirus infected cells, the presence of E19 protein impacts on the fate of adenovirus cell infection.
  • a method of treating a donor organism's cells is provided. These cells are contacted with E19 protein to alter the presentation of MHC class I cell surface antigens on these cells and allow introduction of these cells into a recipient organism while reducing transplant rejection by the recipient organism's immune system.
  • the cells are provided in a form for introduction into the recipient organism to reduce recipient rejection caused by MHC class I cell surface antigens.
  • Variations of the method of this invention include subjecting the cells to E19 protein by the presence in the cells of a vector which carries and expresses the E19 coding sequence or by the presence in the cells of a transgene which expresses the E19 coding sequence.
  • the cells that are removed from the donor organism may constitute a tissue or an organ.
  • the method may also be used for producing a cell in a donor organism for transplantation by introducing DNA into the cell, for example, a vector, that is capable of expressing the E19 protein coding sequence, or by introducing a transgene carrying the E19 coding sequence into a zygote, an early stage embryonic cell, or a stem cell, and allowing this cell to divide and substantially contribute to the development of the donor organism.
  • a vector that is capable of expressing the E19 protein coding sequence
  • transgene carrying the E19 coding sequence into a zygote, an early stage embryonic cell, or a stem cell, and allowing this cell to divide and substantially contribute to the development of the donor organism.
  • the method can be used for gene therapy, or to treat the body for a genetic disorder, a wound, a burn, or a disease in a recipient organism.
  • cells for transplantation which are subject to reduced transplant rejection produced by the method of this invention are provided.
  • Transgenic and chimeric nonhuman organisms produced by the method of this invention are also provided.
  • Fig. 1 depicts recombinant retroviral vectors containing the E19 coding sequence fused to the CMV, ⁇ -actin or DHFR promoter.
  • Fig. 2 depicts three essentially similar fusion constructs with each construct differing substantially only in the promoter region, between the E19 coding sequence and strong general promoters.
  • Fig. 3 depicts three essentially similar fusion constructs with each construct differing substantially only in the promoter region, between the E19 coding sequence and tissue/organ specific promoters.
  • Fig. 4 depicts the retroviral vector pWE-Adeno E19 containing the E19 coding sequence fused to the ⁇ -actin promoter.
  • Fig. 5 depicts a fusion construct between the El9 coding sequence and the rat insulin promoter.
  • the prior art reveals the difficulties that have been encountered in transplanting cells, tissues or organs from one organism to another due to rejection of such transplants by the recipient organism's immune system.
  • the present invention describes a method for reducing or eliminating such transplant rejection by altering certain cell surface antigens on the donor cells with adenovirus E19 protein.
  • a method for treating donor cells to reduce recipient rejection caused by MHC class I cell surface antigens, by contacting the donor cells with E19 protein to alter the presentation of MHC class I cell surface antigens on these cells and allow introduction of these cells into a recipient organism while reducing transplant rejection by the recipient organism's immune system.
  • the cells are provided in a form for introduction into the recipient organism.
  • other conventional i munosuppressant means and methods can be used along with the products and methods of this invention.
  • the term organism is meant to include animals.
  • Animals include mammals, birds, reptiles, amphibians and fish.
  • Preferred animals are mammals and preferred mammals are humans, monkeys, pigs, dogs, cats, sheep, goats, cows, horses and rodents.
  • the most preferred mammal for the recipient organism is a human.
  • Donor cells for transplantation are obtained from an organism.
  • the term donor cell is meant to include cells, a tissue or an organ.
  • Examples of cells include T cells, B cells, islets of La ⁇ gerhans, adrenal medulla cells, osteoblast ⁇ , osteoclasts, epidermal cells, epithelial cells, endothelial cells, neurons, glial cells, ganglion cells, retinal epithelial cells, liver cells, bone marrow cells, myoblast cells, hematopoietic cells, spleen cells, cardiac cell ⁇ , thymus cells, lung cells, blood cells, glandular cells and stem cells.
  • Stem cells include totipotent cells and pluripotent cells.
  • a totipotent cell means a cell that can differentiate into any cell or tissue type (e.g., an embryonic stem cell or a fertilized egg).
  • a pluripotent cell means a cell that can differentiate into many, but not all tissue types.
  • An example of a pluripotent cell is a hematopoietic stem cell.
  • tissues include dermal tissue, epidermal tissue, adipose tissue, connective tissue, neuronal tissue, lymphoid tissue, glandular tissue, bone tissue and bone marrow tissue.
  • organs include kidney, pancreas, liver, heart, lung, gall bladder, skin, spleen, intestine, colon, stomach, eye, inner ear, esophagus, trachea, veins and arteries.
  • the donor cells can be from an organism of the same species as the recipient organism or from a different species than the recipient organism.
  • the donor cells are contacted with E19 protein.
  • E19 protein By contacted it is meant that the donor cell is exposed to E19 protein. Preferably, this contact is i tracellular. Most preferably, this intracellular contact is the result of intracellular production of E19 protein.
  • the E19 gene encodes a 19 kD glycoprotein from the E3 region of adenoviruses of the subgroups B, C, D and E. (Paabo et al . PNAS USA, 1986, 83: 9665-9669; Wold et al. , J. Biol. Chem., 1985, 260: 2424-2431).
  • E19 protein it is meant the entire 19 kD protein or a portion thereof.
  • E19 coding sequence it is meant the coding sequence for the entire 19 kD protein or a portion thereof.
  • the E19 protein has a dominant mode of action. It is believed that E19 protein binds to and prevents MHC class I molecules from exiting the endoplasmic reticulum of the cells. (Paabo et al. , Adv. Cancer Res., 1989, 52: 151-163; Burgert et al. , PNAS USA, 1987 84: 1356-1360; Cox et al., J. Exp. Med., 1991, 174: 1629-1637).
  • MHC class I cell surface antigens on the cell it is meant that MHC class I molecules are at least partially blocked from becoming cell surface antigens. This alteration in presentation must be sufficient to reduce transplant rejection by a recipient's immune system when the cells are introduced into the recipient organism.
  • Rejection is impairment or destruction of cell structure and/or function to any degree caused by an immune reaction.
  • the rejection alleviated by the methods and products of this invention are those cases of rejection where impairment in function and/or destruction of cell structure is such that the desired effects of using the methods and products of this invention are not obtained for a sustained period of time.
  • recipient rejection refers to rejection of the transplant by the recipient.
  • reduce transplant rejection is meant to include situations where there is no rejection and where there is only partial rejection.
  • the amount of E19 protein that is required for treating the donor cells is that amount which is sufficient to effect such a reduction in transplant rejection.
  • the number of E19 molecules is equal to or greater than the number of MHC class I molecules in a given donor cell.
  • conventional immunosuppressant therapy can be administered simultaneously with transplants in accordance with this invention. For example, cyclosporin, azathioprine, FK-506 and rapamycin can be administered.
  • Intracellular contact of the donor cell to E19 protein can be effected in a variety of ways, including introduction into the donor cell of a vector having the E19 coding region, transfection of DNA containing the E19 coding region and production of transgenic animals containing the E19 coding region.
  • the term vector is meant to include viruses, plasmids, cosmids and YACS.
  • the preferred vector is a virus.
  • Preferred viruses include recombinant retroviral vectors, recombinant adenoviral vectors and recombinant Herpes simplex viral vectors.
  • the genome of conventional recombinant retroviral vectors is comprised of a promoter, a selectable marker and 5' and 3' LTRs (long terminal repeats), as shown in Figure 1.
  • the arrows in the figure show the direction of transcription.
  • the symbol ⁇ refers to a cis-acting sequence on the 5' end of a retroviral genome distal to the 5' LTR that is required to effect packaging of the RNA genome into retroviral particles.
  • the selectable marker on the vector is a neomycin resistance gene (NEO-R) that permits selection with G418.
  • NEO-R neomycin resistance gene
  • the promoter is a strong general or tissue specific internal promoter. Examples of promoters include ⁇ -actin (Lai et al. , PNAS USA, 1989, 86: 10006-10010), CMV or DHFR (Scharfmann et al. , PNAS USA, 1991, 88: 4626-4630).
  • retroviral vector containing the CMV promoter is LNL-SLX CMV
  • an example of a retroviral vector containing the ⁇ -actin promoter is pWE
  • an example of a retroviral vector containing the DHFR promoter is LNL-SLX DHFR.
  • the E19 coding sequence is cloned into the genome immediately downstream from the promoter so that the promoter and coding sequence of the E19 gene are operably linked so as to permit expression of the E19 coding sequence.
  • Selective modifications in the sequence of the LTRs to improve the expression of the E19 coding sequence for a variety of cell and tissue types are utilized, as have been described for other cloned genes. (Hilberg et al . , PNAS USA, 1987, 84: 5232-5236; Holland et al. , PNAS USA, 1987, 84: 8662-8666; Valerio et al. , Gene, 1989, 84: 419-427).
  • Recombinant retroviral vectors capable of transducing and expressing the E19 coding sequence in donor cells are produced by transfecting the recombinant retroviral genome into a suitable (helper virus-free) amphotropic packaging cell line.
  • suitable (helper virus-free) amphotropic packaging cell line examples include PA317 and Psi CRIP (Cornetta et.al.. Human Gene Therapy, 1991, 2: 5-14; Cone & Mulligan, PNAS USA, 1984, 81: 6349-6353).
  • Transfected virus packaging cell lines produce and package the recombinant retroviral vectors, shedding them into the tissue culture media.
  • the retroviral vectors are then harvested and recovered from the culture media by centrifugation as described in Compere et al., MCB, 1989, 9: 6-14.
  • the retroviral vectors are resuspended in 10 mM HEPES.
  • retroviral vectors for introducing the E19 coding sequence into a donor cell are that such vectors, in combination with the choice of an appropriate retroviral packaging cell line, offer wide host range and tissue tropism. Retroviral vectors also permit selective targeting of donor cell and tissue types by appropriate modifications in the LTR.
  • Recombinant adenoviral vectors also have a cell or tissue specific promoter that is operably linked to the E19 coding sequence so as to permit expression of the E19 coding sequence.
  • An example of the construction of such a vector is essentially as described in Freidman et al. , MCB, 1986, 6: 3791-3797; Levrero, et al. , Gene, 1991, 101: 195-202.
  • These replication deficient recombinant adenoviral vectors are based on an Ade-5 plasmid.
  • the viral El region is deleted from the plasmid pMLP6 (Logan & Shenk, PNAS USA, 1984, 81: 3655-3659) by digesting with the restriction endonucleases Bglll and Rsal .
  • the remainder of the plasmid retains the left-most 194 bp of the Ade-5 genome.
  • the entire promoter-E19 fusion gene unit from any of the essentially similar plasmid constructs shown in Figure 2 and Figure 3 is excised with appropriate restriction endonucleases, cut and recloned into the Ade-5 plasmid.
  • the recombinant genomes are generated by mixing the linearized plasmid above with the subgenome fragment of adenovirus DNA representing the 3.85 to 100 map units prepared by digesting the In340 viral genome with Clal or Xbal.
  • the DNAs are transfected into 293 cells (Graham et al. , J. Gen. Virol, 1977, 36: 59-72) as described in Berkner & Sharp, Nuc. Acid. Res., 1983, 11: 6003-6020).
  • Intracellular recombination between the 3.85 to 100 map unit segment and each of the cellular promoter-E19 constructs results in the generation of recombinant adenoviral vectors .
  • adenoviral vectors infect a wide variety of cells and tissues in susceptible hosts.
  • the host range includes cotton rats, hamsters, dogs, chimpanzees and humans (Hsu et al., J. Infectious Diseases, 1992, 166: 769-775).
  • Such vectors have the distinct advantage of not requiring mitotically active cells for infection.
  • Viral vectors are used to introduce the E19 coding sequence into donor cells by in vitro or in vivo infection.
  • in vitro infection donor cells from target tissues of an organism are obtained, grown in culture and infected with a recombinant viral vector containing the E19 coding sequence.
  • in vivo infection a recombinant viral vector is administered to the organism so as to result in general systemic infection or tissue specific or organ specific infection.
  • aerosol administration of recombinant adenoviral vectors results in infection of respiratory epithelial cells (Hsu et al. , J. Infectious Diseases, 1992, 166: 769-775; Rosenfeld et al.
  • plasmids can be used to expose donor cells to E19 protein.
  • the term plasmid is meant to describe a DNA molecule that can replicate autonomously or integrate in a host such as a bacterium or yeast.
  • the DNA molecule can contain, in addition, DNA sequences such as those encoding the E19 protein and a promoter controlling its expression in a mammalian cell.
  • the term plasmid also refers to the same DNA molecule above after the DNA molecule is purified away from the host cell such as occurs when using the plasmid DNA for introduction into mammalian cells, when using portions of the DNA molecule for additional ' DNA cloning experiments, and when a portion of the DNA molecule is used in a substantially purified form.
  • Such plasmids may contain fusion constructs between the El coding sequence and a strong general promoter or between the E19 coding sequence and a tissue or organ specific promoter.
  • strong general promoters are the mouse H-2K promoter (Schuh et al. , Nature, 1990, 346: 757-760, the human ⁇ -actin promoter (Leavitt et al., MCB, 1984, 1961-1969) and the human CMV promoter (Boshart et al. , Cell, 1985, 41: 521-530).
  • Figure 2 depicts such fusion constructs.
  • tissue or organ specific promoters examples include the liver specific albumin promoter (Heckel et al., Cell, 1991, 62: 447-456), the cardiac specific alpha myosin heavy chain promoter (Subramaniam et al., J. Biol. Chem., 1991, 266: 24613-24620) and lung alveolar epithelium specific promoter SP-C promoter (Wikenheiser et al. , Cancer Res., 1992, 52: 5342-5352).
  • Figure 3 depicts such fusion constructs.
  • DNA from plasmid constructs can be used to introduce the E19 coding sequence into donor cells grown n vitro by DNA-mediated cell transfection methods.
  • the DNA used for transfection is preferably prepared by the alkali-lysis method and preferably purified on cesium chloride equilibrium gradients as described in Sambrook et al. , Molecular Cloning: A Laboratory Manual, CSHL, 1989.
  • the DNA is introduced into the donor cells by one of the following methods: calcium phosphate precipitation (Ausubel et al. , Current Protocols in Molecular Biology, 1987, Wiley-Interscience.), DEAE-dextran method (Ausubel et al .
  • the DNA from vectors carrying the E19 coding sequence can also be u ⁇ ed to directly target ⁇ pecific ti ⁇ ues or organs of a donor organism in vivo.
  • Examples include (i) receptor mediated endocytosi ⁇ of intravenously administered DNA for delivery to hepatocytes in the liver (Wu et al. , J. Biol. Chem., 1991, 266:14338-14342; Wil ⁇ on et al. , J. Biol. Chem., 267:963-967); (ii) lipo ⁇ ome ⁇ that encapsulate DNA for delivery (Nicolau et al.
  • Intracellular contact of the donor cell to E19 protein can al ⁇ o be achieved by production of tran ⁇ genic or chimeric organi ⁇ m ⁇ containing the E19 coding region.
  • tran ⁇ genic organism it is meant an organism that gain ⁇ new genetic information from the introduction of exogenous DNA into its own, or into an ancestor' ⁇ germ line.
  • exogenous DNA it is meant DNA that is not normally found in- the genome of the non-tran ⁇ genic organism.
  • chimeric organism it is meant an organism in which some of its cell ⁇ have gained new genetic information from the introduction of exogenous DNA into the organi ⁇ m, or an ancestor of the organism, preferably at an embryonic stage.
  • transgene it is meant an exogenou ⁇ gene that ha ⁇ been added to the germ line of an organi ⁇ m.
  • Tran ⁇ genic or chimeric organisms containing the E19 coding sequence are obtained by introducing into a cell a DNA construct containing the E19 coding ⁇ equence.
  • the DNA con ⁇ truct ha ⁇ the E19 coding ⁇ equence under the control of a promoter. If ⁇ pecific promoter ⁇ are u ⁇ ed, specific populations of cell ⁇ , tissue ⁇ or organ ⁇ of the tran ⁇ genic or chimeric organi ⁇ m may be targeted.
  • a 2.3 kb mou ⁇ e albumin promoter ha ⁇ been reported to direct the expre ⁇ ion of genes in the liver of transgenic mice (Heckel et al., Cell., 1991, 62: 447-456), a 3.7 kb human SP-C (pulmonary Surfactant-C) promoter will direct the expres ⁇ ion of tran ⁇ gene ⁇ to the bronchial and alveolar epithelium of adult lungs (Wikenheiser et al. , Cancer Re ⁇ ., 1992, 52: 5342-5352), a 5.8 kb alpha myo ⁇ in heavy chain promoter will direct the expression of transgene ⁇ in both the atrial and ventricular cells of the adult heart (Subramaniam et al.
  • tissue specific promoter ⁇ to produce transgenic animal ⁇ can also be used, as de ⁇ cribed in Jaenisch, Science, 1988, 240; 1468-1474; Hanahan, Science, 1989, 240: 1265-1274.
  • the expression of the adeno-E19 gene may also be placed under the transcriptional control of a ⁇ trong general promoter that would direct the expression in a wide range of cells and tissue ⁇ of the donor.
  • a ⁇ trong general promoter that would direct the expression in a wide range of cells and tissue ⁇ of the donor.
  • Example ⁇ are the human B-actin promoter (Leavitt et al. , 1984, 4: 1961-1969) and the 1.9 kb mou ⁇ e H-2K promoter (Schuh et al . , Nature, 1990, 346: 757-760) .
  • Introduction of the DNA into a cell include ⁇ introduction into a ⁇ ingle cell derived from a ⁇ omatic or germ cell, into an egg, into a ⁇ per , into a zygote, into an embryo, into a po ⁇ t-natal human or nonhuman organi ⁇ m, into cell ⁇ derived from an embryo or post-natal human or nonhuman organism, or into a cell from a cell culture, e.g. , an embryonic stem cell. If embryonic stem cells are used, after introduction of the DNA into such cells, the cells may be transferred to blastocy ⁇ t ⁇ to produce line ⁇ of tran ⁇ genic nonhuman organi ⁇ m ⁇ .
  • the cell containing the DNA with the E19 coding ⁇ equence is allowed to divide and substantially contribute to the development of the donor organism.
  • the resulting transgenic or chimeric organism ⁇ have their MHC cla ⁇ I molecule ⁇ downregulated a ⁇ a re ⁇ ult of production of E19 protein in the cell ⁇ containing the E19 coding sequence.
  • Such organisms are a source of cells, ti ⁇ ue ⁇ and organ ⁇ for tran ⁇ plantation.
  • the cells are provided in a form for introduction into a recipient organism.
  • Thi ⁇ form can be no change from the form and physical structure of the cells during the contacting ⁇ tep.
  • Such a form require ⁇ that the cell ⁇ be in an appropriate condition for introduction with regard to factor ⁇ including phy ⁇ iological ⁇ tate, chemical environment and phy ⁇ ical condition.
  • the donor cell ⁇ are removed after treatment with E19 by ⁇ tandard procedure ⁇ 'known to those ⁇ killed in the art. For example, surgical removal of cells, tis ⁇ ue ⁇ or organ ⁇ from whole organi ⁇ m ⁇ is effected. If cell cultures are the source of donor cells, cells are removed from the culture ⁇ . The removed cell ⁇ are then transplanted into the recipient. The removed cells may be directly tran ⁇ planted, or they may be stored prior to transplantation. Examples of methods of transplantation include standard surgical grafting procedure ⁇ , topical application, or injection into the blood stream, a tissue or an organ of the recipient organism.
  • a method for treating a genetic di ⁇ order, a wound, a burn, a disease, or effecting gene therapy whereby cells are transplanted into the recipient organism which can substantially alleviate the abnormal condition, the transplanted cells having been treated with E19 protein to alter the presentation of MHC clas ⁇ I cell ⁇ urface antigen ⁇ so as to allow the transplantation to the recipient organism while reducing tran ⁇ plant rejection by the recipient organi ⁇ m' ⁇ immune system.
  • substantially alleviate it is meant a complete or partial cure or relief of the abnormal condition.
  • keratinocytes may be introduced to replace skin in the ca ⁇ e of burn ⁇ or to replace ⁇ kin removed from the recipient organi ⁇ m for u ⁇ e at another ⁇ ite; i ⁇ let ⁇ of Langerhan ⁇ may be introduced for production of in ⁇ ulin for recipient's suffering from diabetes; retinal epithelial cell ⁇ may be introduced to treat vi ⁇ ual disorders such as macular degeneration; immune cells may be used to treat immune deficiency; and myoblasts may be introduced to treat mu ⁇ cle wa ⁇ ting, ⁇ uch a ⁇ Duchenne' ⁇ muscular dystrophy. The number of cells introduced will depend upon the particular abnormal condition and the manner in which the cells are introduced. In other embodiment ⁇ , tissue ⁇ or entire organs are introduced to replace disea ⁇ ed or otherwise abnormal tis ⁇ ue or organ ⁇ .
  • the method ⁇ of thi ⁇ invention are for treating an organi ⁇ m to achieve a de ⁇ ired result over a su ⁇ tained time period.
  • Such a time period may be short and yet be useful, since immunosuppre ⁇ ant therapy when used in conjunction with the method of this invention, can provide su ⁇ tained time periods over hours, days, months and years in some ca ⁇ e ⁇ .
  • the reduction in immune rejection i ⁇ such that the transplanted cell ⁇ which may be in the form of ti ⁇ ue ⁇ , organ ⁇ or parts thereof, will resist transplant rejection over the life of the organism into which the treated cell ⁇ are tran ⁇ planted, although a ⁇ horter time period of weeks or le ⁇ , of re ⁇ i ⁇ tance to ⁇ evere damage to the tran ⁇ planted cell ⁇ by recipient rejection can be u ⁇ eful.
  • the transplant is used in veterinarian uses for treating animals who in turn are producing useful substance ⁇ , any prolonging of life and attendant prolonging production of ⁇ uch ⁇ ub ⁇ tance ⁇ can be important and u ⁇ eful.
  • longer time period ⁇ of at lea ⁇ t a month or more are desirable, with periods of at least six months to a year or more being most desirable.
  • Thi ⁇ invention also includes transgenic and chimeric nonhuman organisms which have cells for transplantation which are subject to reduced transplant rejection by a recipient's immune ⁇ y ⁇ tem a ⁇ a re ⁇ ult of the cell ⁇ producing E19 protein ⁇ o a ⁇ to alter the presentation of MHC class I cell surface antigens.
  • This invention further includes a cell from such a tran ⁇ genic organism, a ⁇ well a ⁇ a cell which ha ⁇ DNA which carrie ⁇ and expresses the E19 coding sequence.
  • Example 1 Generation of Alloqeneic or Xenoqeneic Epidermal Tis ⁇ ue ⁇ for Tran ⁇ plantation
  • a recombinant retroviral vector carrying the Ade-2 E19 gene is prepared as follows.
  • the Ade-2 E19 gene i ⁇ i ⁇ olated on a 540 bp BamHI fragment from the plasmid pCMVgpl9Kneo by BamHI restriction endonuclease dige ⁇ tion.
  • the BamHI E19 fragment i ⁇ then ⁇ ubcloned in the BamHI cloning site of the retroviral vector pWE (Choudary et al. , Cold Spring Harbor Symp. Quant. Biol., 1986, 51: 1047-1032) by conventional recombinant DNA techniques (Sambrook et al.
  • the E19 gene is inserted downstream from the strong internal 275 bp chicken ⁇ -actin promoter.
  • the arrow indicates the direction of transcription.
  • the cis-acting ⁇ sequence is shown.
  • the retroviral genome also contains a neo ycin resi ⁇ tance gene (NEO-R) that permit ⁇ ⁇ election with G418, and 5' and 3' LTRs.
  • the recombinant retroviral construct is propagated as a plasmid in E.coli strain HB101 in the presence of ampicillin.
  • recombinant retroviruse ⁇ 10 ug of the recombinant retroviral DNA is transfected into the amphotropic retroviral packaging cell line PA317 (Miller & Buttimore. MCB, 1986, 6: 2895-2902) by the calcium pho ⁇ phate precipitation method (Ausubel et al. ; Current Protocols in Molecular Biology, 1987, Wiley-Interscience). Tran ⁇ fected retroviral packaging cell ⁇ are ⁇ elected in DMEM (Gibco/BRL, Grand Island, NY) supplemented with 10% FBS containing 500 ug/ml G418 (Gibco/BRL, Grand Island, NY). Clonal viral producing cell lines are establi ⁇ hed in culture.
  • Viral production is carried out by plating the producer lines at 80% confluence in DMEM supplemented with 10% FBS (Gibco/BRL, Grand Island, NY). Media containing shed viru ⁇ i ⁇ harvested 24 hours after plating.
  • Viral titers are determined by infecting NIH 3T3 cell ⁇ in culture in the pre ⁇ ence of Polybrene (8 ug/ml) (Sigma, St. Louis, MO). Viral stock ⁇ are prepared by centrifugation at 10,000 g at 40°C for 12 hours. Viral pellets are re ⁇ uspended in lOmM HEPES containing 0.5% dimethyl sulfoxide and stored at -70°C or in liquid nitrogen before use.
  • DMEM Dulbecco' s modified Eagle medium
  • FBS fetal bovine ⁇ erum
  • keratinocyte medium a 7:3 mixture of DMEM with L-glutamine and Ham's F12 (Gibco) medium supplemented with 0.18 M adenine (Boehringer Mannheim, Indianapolis, IN), 10% FBS, insulin (5 ug) (Sigma), transferrin (5 ug/ml) (Gibco), hydrocorti ⁇ one (0.4 ug/ml) (Gibco), 2 nM 3,3' ,5-triiodo-L-thyronine (Sigma)), and cholera toxin 0.1 nM (Calbiochem-Behring, San Diego, CA) (Stock ⁇ chlaeder et al.
  • DMEM Dulbecco' s modified Eagle medium
  • FBS fetal bovine ⁇ erum
  • FBS fetal bovine ⁇ erum
  • 1-2 x 10 epidermal cell ⁇ are ⁇ eeded in ti ⁇ ue culture fla ⁇ k ⁇ (Corning, Corning, NY) on feeder layer of irradiated (20 Gy) 3T3 fibroblasts (Green et al. , PNAS USA; 1979, 76: 5665-5668). Cultures are maintained at 37°C, 5% C0 2 in a humidified incubator. Epidermal growth factor (Chiron, Emeryville, CA) was added to the culture at 10.ng/ml and confluent cultures passaged by trypsinization and replating on irradiated murine feeder layer ⁇ at a cell density of 1 x 10 cell/ml.
  • Epidermal growth factor Choiron, Emeryville, CA
  • Keratinocyte ⁇ are infected either by co-cultivation with irradiated (20 Gy) PA317 cell ⁇ producing the recombinant E19 retroviru ⁇ or by conventional infection with recombinant retroviral ⁇ tock ⁇ .
  • Cell ⁇ are infected at 30% confluency with viru ⁇ (1 x 10 colony-forming units (cfu)/ml) in Polybrene (8 ug/ml) at 37°C for 4 hours. The cells are returned to fresh media overnight ⁇ upplemented with G418 (500 UG/ml) and fed every 2-3 days until confluency.
  • the transduced keratinocytes are examined for the expre ⁇ ion of E19 by immunoblot analy ⁇ i ⁇ of total cell ly ⁇ ate ⁇ using anti-EI9 antisera.
  • the lo ⁇ of MHC class I molecules on the cell surface resulting from E19 expression i ⁇ analyzed by immunofluorescence staining with specific MHC monoclonal antibodies (Pharmingen, San Diego, CA; Harlan, Indianapolis, IN) coupled to fluorescent dyes like Rhodamine or Fluorescein (Sigma, St. Loui ⁇ , MI).
  • Tran ⁇ duced keratinocyte ⁇ are prepared for grafting by seeding in 2 cm x 4 2 cm pieces of a biodegradable mesh composed of polyglycolic acid (Dexon; Davi ⁇ and Geek Inc., Danbury, Conn.) (Hansbrough, et al, Surgery, 1992, 111: 438-446).
  • Epidermal grafts are performed on allogeneic and xenogeneic recipients. In animal experimentation, the procedure is performed with approval of the Animal Research Committee and in accordance with the guidelines of the National Institute of Health. In an example of a small animal recipient, the shaved dorsolateral surface of the torso is washed with povidone-iodine and 70% isopropanol. A 2 cm x 2 cm skin section is exci ⁇ ed under ane ⁇ the ⁇ ia and me ⁇ h-keratinocyte graft of equivalent ⁇ ize i ⁇ placed over the wound. The graft i ⁇ sutured in place and dressed.
  • a 2 mm square piece of Gelfoam (Upjohn, Kalimazoo, MI) containing 2 ug of basic fibroblast growth factor may be applied to the connective ti ⁇ sue with the graft (Louis & Verma, PNAS USA, 1988, 85: 3150-3154) .
  • Example 2 Generation of a Transgenic Pancreatic ⁇ -islet Expre ⁇ sing E19 for Transplantation
  • the Ade-2 E19 gene i ⁇ placed down ⁇ tream from the rat in ⁇ ulin promoter (Hanahan, Nature, 1985, 315: 115-122).
  • the con ⁇ truct is assembled as follows.
  • Plasmid pCMVgpl9neo is constructed by inserting the 540 bp BamHI Ade-2 genome containing the E19 gene into the unique BamHI site of the plasmid vector pcDNAlNEO (Invitrogen, San Diego, CA) ⁇ o that the E19 gene i ⁇ under the tran ⁇ criptional control fo the CMV promoter in the pla ⁇ mid.
  • the Ade-2 E19 gene containing the complete coding ⁇ equence i ⁇ recovered a ⁇ a 550 bp Hindlll-Xbal fragment from the plasmid pCMVgpl9Kneo by cleaving with the restriction endonuclease ⁇ Hindlll and Xbal .
  • the E19 and rat in ⁇ ulin promoter gene fragments are purified on agarose gels and ligated to produce the rat insulin promoter-E19 fusion gene construct, by conventional recombinant DNA techniques (Sambrook, et al., Molecular Cloning: A Laboratory Manual, CSHL, 1989), a ⁇ shown in Figure 5.
  • the arrow indicate ⁇ the direction of tran ⁇ cription.
  • the preferred method for producing a tran ⁇ genic animal is by microinjection of DNA containing the E19 coding sequence into the pronucleus of a zygote a ⁇ de ⁇ cribed in Hogan et al. , Manipulating the Mou ⁇ e Embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1986); Hammer, et al. , Nature, 1985, 315: 680-683.
  • Tran ⁇ genic donors include pigs, mice, rats, rabbits, sheep, monkeys, cats, dogs, goats, cows and horses.
  • Transgenic animals are identified by performing PCR (Innis et al. , PCR Protocol ⁇ : A guide to method ⁇ and applications, Academic Pres ⁇ , 1990) and Southern Blot analy ⁇ i ⁇ (Southern, J. Mol. Biol., 1975, 98: 503) of genomic DNA prepared from tail ⁇ ample ⁇ . Tran ⁇ genic animal line ⁇ are established. Expres ⁇ ion of the E19 tran ⁇ gene in the ⁇ -cell ⁇ of the pancrea ⁇ are examined by Northern Blot analysis (Sambrook et al. , Molecular Cloning: A laboratory Manual, CSHL, 1989) of total RNA prepared from the islet ⁇ and by immunoblot analysis with E19-specific antisera (Wold, et al. , J.
  • Pancrea ⁇ are harvested from transgenic animals es ⁇ entially a ⁇ de ⁇ cribed in Nakazato et al., 1992, Surgery, 111: 37-47.
  • the pancreatic duct ⁇ are cannulated and the excised organ i ⁇ immer ⁇ ed in ice cold Hank ⁇ ' balanced ⁇ alt ⁇ olution (HBSS) .
  • the organ i ⁇ then perfu ⁇ ed with Type X collagenase (Sigma) in HBSS (1 mg/ml), 10 ml/min at 37°C essentially as described in Alderson et al., Transplantation, 1987, 43: 579-581).
  • the capsule around the glands is removed. The perfusion is terminated when the glands turn mucoid.
  • the tissues are then removed from the ductal system, disaggregated in ice-cold HBSS and filtered through a 500 um ⁇ tainle ⁇ ⁇ teel ⁇ ieve.
  • the filtrate is concentrated at 800 g for 4 minutes at 4°C.
  • the tis ⁇ ues are 'Stored re ⁇ u ⁇ pended in HBSS at 4°C.
  • the ti ⁇ ue ⁇ are ⁇ ubjected to a final centrifugation at 800 G for 4 minute ⁇ and re ⁇ uspended in a fluid/tissue volume of 2:1 in HBSS.
  • the islet ⁇ will ⁇ ettle between the top two concentration ⁇ and may be recovered, wa ⁇ hed in cold HBSS and u ⁇ ed directly for implantation.
  • Diabetes is induced within a week after a single injection of 45 mg/kg of body weight of streptozotocin dis ⁇ olved in lOmM citrate buffer, pH 4.5 into the tail vein. Animals with serum glucose level in exces ⁇ of 20 mM/liter are con ⁇ idered diabetic. Animal ⁇ are u ⁇ ed 6-8 week ⁇ after treatment. Following recovery from the surgical implantation of donor transgenic islet ⁇ (400 i ⁇ let ⁇ ) grafts are examined for viability and immune rejection by conventional pathological methods. Short term graft function is monitored by as ⁇ e ⁇ ment of blood gluco ⁇ e, in ⁇ ulin and glucagon levels as described in Alderson, et al, Br. J. Surgery, 1984, 71: 756. Long term autograft, allograft and xenograft function are a ⁇ e ⁇ ed by analyzing mortality rate ⁇ following withdrawal from in ⁇ ulin therapy.
  • the mou ⁇ e ⁇ -myo ⁇ in heavy chain 5'-flanking region ha ⁇ been cloned (Gulick, et al. , 1991, 266: 9180-9185) and characterized (Subramaniam et al. , 1991, 266: 24613-24620).
  • the 5.8 kb 5'-flanking region included the first two exon ⁇ and part of the third exon wa ⁇ able to direct the expre ⁇ ion of tran ⁇ gene in the atrial and ventricular ti ⁇ ue ⁇ of the adult heart (Subramaniam et al. , 1991, 266: 24613-24620).
  • the Ade-2 E19 gene is placed downstream from the 5.8 kb -myosin heavy chain promoter.
  • Thi ⁇ i ⁇ accomplished as follows.
  • the 540 bp Ade-2 E19 gene is recovered as a BamnHI-fragme t by digesting the plasmid pCMVgpl9Kneo with the re ⁇ triction endonuclea ⁇ e BamHI. The ends of the fragment are filled in with Klenow to produce blunt ends that are compatible for ligation with the promoter fragment.
  • the ⁇ -myosin heavy chain promoter i ⁇ i ⁇ olated as a 1.2 kb Maelll proximal fragment and 4.65 kb BamHI-Ndel distal fragment from the plasmid a-5.5 (Subramaniam et al., 1991, 266: 24613-24620).
  • the ends of the proximal 1.2 KB fragment are filled in with Klenow and ligated to the 540 bp E19 gene into the Smal site of the plasmid vector pUC18 (N.E. Biolab ⁇ , Beverly, MA) ⁇ o that the E19 gene i ⁇ operably linked to the ⁇ hort promoter.
  • the fu ⁇ ion con ⁇ truct exci ⁇ ed from thi ⁇ pla ⁇ mid is 1.7 kb Ndel-BamHI.
  • the Ndel site is located close to the 5'-end of the promoter fragment.
  • the remaining 5'-flanking region of the ⁇ -myosin heavy chain promoter is added on by ligating the distal 4.65 kb BamHI-Nde ⁇ to the fu ⁇ ion con ⁇ truct above at the Ndel site into the BamHI cloning ⁇ ite of pUCl ⁇ .
  • the antigenicity of the transgenic donor heart tis ⁇ ue a ⁇ a reflection of the ability of the expressed E19 to interfere with MHC class I presentation i ⁇ asse ⁇ ed by implanting biopsy samples of the donor tis ⁇ ue ⁇ into allogeneic and xenogeneic host ⁇ a ⁇ de ⁇ cribed (Davi ⁇ et al. , Tran ⁇ plantation, 1980, 29: 189-192).
  • Tran ⁇ genically modified heart ti ⁇ ues (atrial and ventricular) are prepared from neonatal donors along with reference tissue (e.g., skin) samples from the same, as well a ⁇ from non-tran ⁇ genie donor ⁇ implanted ⁇ ubcutaneou ⁇ ly behind the ear ⁇ .
  • Graft survival is a ⁇ e ⁇ ed by electrocardiography (Davi ⁇ et al. , Tran ⁇ plantation, 1980, 29: 189-192).
  • Skin graft ⁇ are a ⁇ sessed by routine pathology.
  • a transgenic organ is produced that can be transplanted into a recipient to replace a loss of function in the donor.
  • the experimental animals are of matching size to accommodate for cardiac demand of the recipient on the grafted organ.
  • Transplantation i ⁇ carried out between allogeneic ⁇ train ⁇ of rat ⁇ or between xenogeneic rat ⁇ and ham ⁇ ter ⁇ .
  • Technique ⁇ for heart tran ⁇ plant ⁇ for rodent ⁇ are well de ⁇ cribed in the literature with modification ⁇ employed for the choice of ve ⁇ el ⁇ and sites for grafting (Marni & Ferrero, Tran ⁇ plantation, 1987, 43: 575-577; Lee et al. , Tran ⁇ plantation, 1982, 33: 438-442; Miller et al. , Tran ⁇ plantation, 1985, 39: 555-558).
  • the ⁇ e include the renal vessel ⁇ , the carotid artery and the jugular, the iliac ve ⁇ sels and the abdominal aorta.
  • Example 3 i ⁇ repeated with the u ⁇ e of conventional rapamycin immunosuppres ⁇ ant therapy application to the recipient and advantageou ⁇ results are obtained.
  • mice suitable for transgenic experiments are obtained from ⁇ tandard commercial ⁇ ource ⁇ ⁇ uch as Charle ⁇ River (Wilmington, MA), Taconic (Germantown, NY) and Harlan Sprague Dawley (Indianapoli ⁇ , IN). Swi ⁇ Web ⁇ ter and female mice are u ⁇ ed for embryo retrieval and tran ⁇ fer. B6D2F males are used for mating and vasectomized Swiss Webster stud ⁇ are u ⁇ ed' to stimulate pseudopregnancy. Va ⁇ ectomized mice and rat ⁇ are obtained from the ⁇ upplier.
  • DNA clone ⁇ are cleaved with appropriate enzy e ⁇ and the DNA fragment ⁇ are electrophore ⁇ ed on 1% agarose gels in TBE buffer.
  • the DNA bands are visualized by staining with ethidium bromide, excised, and placed in dialysi ⁇ bag ⁇ containing 0.3 M sodium acetate, pH 7.0.
  • DNA is electroeluted into the dialysi ⁇ bag ⁇ , extracted with phenol-chloroform (1:1), and precipitated by two volume ⁇ of ethanol.
  • the DNA is redis ⁇ olved in 1 ml of low salt buffer (0.2 M NaCl, 20 M tri ⁇ , pH 7.4, and 1 mM EDTA) and purified on an Elutip-D column.
  • the column i ⁇ fir ⁇ t primed with 3 ml of high salt buffer (1 M NaCl, 20 mM tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer.
  • the DNA ⁇ olutions are pas ⁇ ed through the column three time ⁇ to bind DNA to the column matrix.
  • DNA concentration ⁇ are measured by absorption at 260 ran in a UV spectrophotomer.
  • the DNA concentration is adju ⁇ ted to 3 ⁇ g/ml in 5 mM tri ⁇ , pH 7.4 and 0.1 mM EDTA. Additional method ⁇ for DNA preparation are found in Hogan et al. , Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1986).
  • a preferred method for producing transgenic animals for this invention is through the use of embryonic ⁇ tem cells. This method is preferred becau ⁇ e the proces ⁇ of homologou ⁇ recombination i ⁇ inherently inefficient and procedures have been devised to identify and i ⁇ olate ⁇ tem cell ⁇ with the desired insertion event.
  • This method is preferred becau ⁇ e the proces ⁇ of homologou ⁇ recombination i ⁇ inherently inefficient and procedures have been devised to identify and i ⁇ olate ⁇ tem cell ⁇ with the desired insertion event.
  • transgenic mice by pronuclear injection are described in detail in: Hogan et al., 1986, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Procedures for producing transgenic rats are similar to those for mice (Hammer et al . , 1990, Cell 63, 1099-1112).
  • Tail samples (1-2 cm) are removed from three week old animals. DNA is prepared and analyzed by both Southern blot and PCR to detect transgenic founder (F_) animals and the progeny (F ⁇ and F 2 ).

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Abstract

Procédé de traitement de cellules au moyen de la protéine E19, afin de modifier la présentation d'antigènes de surface cellulaire de classe I du complexe d'histocompatibilité majeure (MHC) sur ces cellules, et de permettre ainsi l'introduction de ces dernières dans un organisme récepteur tout en réduisant le rejet du greffon par le système immunitaire de l'organisme récepteur. Les cellules peuvent être mises en contact avec une protéine E19 par l'introduction, dans ces cellules, d'un vecteur qui porte et exprime la séquence codante de la protéine E19, ou par l'introduction d'un transgène qui exprime ladite séquence. Ce procédé peut être utilisé pour traiter un trouble génétique, une plaie, une brûlure ou une maladie, ou pour soumettre un organisme recépteur à la thérapie génique. Des organismes chimères ou transgéniques, et lesdites cellules sont également décrites.
PCT/US1993/012670 1992-12-31 1993-12-30 Production de cellules destinees a la transplantation et reduisant le rejet du greffon par l'hote, et cellules ainsi obtenues WO1994016065A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006744A2 (fr) * 1993-09-03 1995-03-09 Chiron Viagene, Inc. Procedes de suppression de la reponse immunitaire par therapie genique
WO1995006717A2 (fr) * 1993-09-03 1995-03-09 Viagene, Inc. Procedes de suppression des rejets de greffes
WO1995026740A1 (fr) * 1994-03-31 1995-10-12 Diacrin, Inc. Procedes ameliores de transplantation a l'aide de cellules modifiees et d'agents inhibiteurs de lymphocytes t
WO1996014874A1 (fr) * 1994-11-10 1996-05-23 University Of Manitoba Procede de therapie genique mettant en ×uvre une suppression de reponse immunitaire
FR2730411A1 (fr) * 1995-02-14 1996-08-14 Centre Nat Rech Scient Association medicamenteuse utile pour la transfection et l'expression in vivo d'exogenes
WO1996031241A1 (fr) * 1995-04-04 1996-10-10 Cell Genesys, Inc. Transplantation de cellules genetiquement modifiees presentant de faibles taux de proteines de classe i du complexe majeur d'histocompatibilite sur leur surface
US6156306A (en) * 1994-08-17 2000-12-05 Albert Einstein College Of Medicine Of Yeshiva University Pancreatic β-cells for allogeneic transplantation without immunosuppression
US6451571B1 (en) 1994-05-02 2002-09-17 University Of Washington Thymidine kinase mutants
US7485293B1 (en) 1999-02-18 2009-02-03 Faustman Denise L Method for inhibiting transplant rejection

Citations (1)

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WO1992010573A1 (fr) * 1990-12-04 1992-06-25 Board Of Regents, The University Of Texas System PROCEDES ET COMPOSITIONS DESTINES A LA SUPPRESSION D'UNE TRANSFORMATION A MEDIATION DE $i(NEU)

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WO1992010573A1 (fr) * 1990-12-04 1992-06-25 Board Of Regents, The University Of Texas System PROCEDES ET COMPOSITIONS DESTINES A LA SUPPRESSION D'UNE TRANSFORMATION A MEDIATION DE $i(NEU)

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M. ANDERSSON ET AL.: "Reduced allorecognition of adenovirus-2 infected cells.", THE JOURNAL OF IMMUNOLOGY, vol. 138, no. 11, 1 June 1987 (1987-06-01), BALTIMORE, MD, USA, pages 3960 - 3966, XP002018521 *
S. PÄÄBO ET AL.: "Adenovirus proteins and MHC expression.", ADVANCES IN CANCER RESEARCH, vol. 52, 1989, NEW YORK, NY, USA, pages 151 - 163, XP002018520, DOI: doi:10.1016/S0065-230X(08)60212-2 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006744A2 (fr) * 1993-09-03 1995-03-09 Chiron Viagene, Inc. Procedes de suppression de la reponse immunitaire par therapie genique
WO1995006717A2 (fr) * 1993-09-03 1995-03-09 Viagene, Inc. Procedes de suppression des rejets de greffes
WO1995006744A3 (fr) * 1993-09-03 1995-04-06 Viagene Inc Procedes de suppression de la reponse immunitaire par therapie genique
WO1995006717A3 (fr) * 1993-09-03 1995-04-06 Viagene Inc Procedes de suppression des rejets de greffes
WO1995026740A1 (fr) * 1994-03-31 1995-10-12 Diacrin, Inc. Procedes ameliores de transplantation a l'aide de cellules modifiees et d'agents inhibiteurs de lymphocytes t
US6451571B1 (en) 1994-05-02 2002-09-17 University Of Washington Thymidine kinase mutants
US6156306A (en) * 1994-08-17 2000-12-05 Albert Einstein College Of Medicine Of Yeshiva University Pancreatic β-cells for allogeneic transplantation without immunosuppression
WO1996014874A1 (fr) * 1994-11-10 1996-05-23 University Of Manitoba Procede de therapie genique mettant en ×uvre une suppression de reponse immunitaire
FR2730411A1 (fr) * 1995-02-14 1996-08-14 Centre Nat Rech Scient Association medicamenteuse utile pour la transfection et l'expression in vivo d'exogenes
WO1996025177A1 (fr) * 1995-02-14 1996-08-22 Rhone-Poulenc Rorer S.A. Association medicamenteuse utile pour la transfection et l'expression in vivo d'exogenes
WO1996031241A1 (fr) * 1995-04-04 1996-10-10 Cell Genesys, Inc. Transplantation de cellules genetiquement modifiees presentant de faibles taux de proteines de classe i du complexe majeur d'histocompatibilite sur leur surface
US7485293B1 (en) 1999-02-18 2009-02-03 Faustman Denise L Method for inhibiting transplant rejection

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