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WO1997016064A1 - Procedes et compositions de reduction du rejet de xenogreffe - Google Patents

Procedes et compositions de reduction du rejet de xenogreffe Download PDF

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Publication number
WO1997016064A1
WO1997016064A1 PCT/US1996/017695 US9617695W WO9716064A1 WO 1997016064 A1 WO1997016064 A1 WO 1997016064A1 US 9617695 W US9617695 W US 9617695W WO 9716064 A1 WO9716064 A1 WO 9716064A1
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WIPO (PCT)
Prior art keywords
transgenic
cells
cell
human
organs
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PCT/US1996/017695
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English (en)
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WO1997016064A9 (fr
Inventor
Yiannis Ioannou
Robert J. Desnick
Mauro S. Sandrin
Ian F. C. Mckenzie
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The Mount Sinai Medical Center
The Austin Research Institute
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Application filed by The Mount Sinai Medical Center, The Austin Research Institute filed Critical The Mount Sinai Medical Center
Priority to AU76686/96A priority Critical patent/AU7668696A/en
Priority to EP96939544A priority patent/EP0877549A4/fr
Priority to JP09517615A priority patent/JP2000514641A/ja
Priority to IL12429396A priority patent/IL124293A0/xx
Publication of WO1997016064A1 publication Critical patent/WO1997016064A1/fr
Publication of WO1997016064A9 publication Critical patent/WO1997016064A9/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • 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
    • 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 present invention relates to methods and compositions for the reduction of xenotransplantation rejection. Specifically, the present invention relates, first, to transgenic cells, tissues, organs and animals containing transgenic nucleic acid molecules that direct the expression of gene products, including, but not limited to enzymes, capable of modifying, either directly or indirectly, cell surface carbohydrate epitopes such that the carbohydrate epitopes are no longer recognized by natural human antibodies or by the human cell-mediated immune response, thereby reducing the human immune system response elicited by the presence of such carbohydrate epitopes.
  • the transgenic cells, tissues, organs and animals express nucleic acid molecules encoding functional recombinant ⁇ -Galactosidase A ( ⁇ GalA) enzyme which modifies the carbohydrate epitope Gala(1,3)Gal.
  • ⁇ GalA ⁇ -Galactosidase A
  • the transgenic cells, tissues, organs and animals expressing the functional recombinant ⁇ GalA are transgenic pig cells, organs, tissues and/or animals.
  • xenotransplants Extensive studies now exist regarding such xenotransplantations. See, e.g.. Sandrin et al. (Sandrin, M.S. et al., 1994, Transplant. Rev. :134), which discusses studies involving the use of pig organs for xenotransplantation to humans.
  • HAR hyperacute rejection
  • the present invention relates to methods and compositions for the reduction of xenotransplantation rejection.
  • the present invention relates, first, to transgenic cells, tissues, organs and animals containing transgenic nucleic acid molecules representing functional carbohydrate epitope-modifying genes which direct the expression of gene products that, either directly or indirectly, bring about modification of cell surface carbohydrate epitopes, including, but not limited to the Gala(1,3)Gal cell surface carbohydrate epitope, in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified Gala(1,3)Gal epitope.
  • Such gene products can include, but are not limited to, carbohydrate epitope-modifying enzymes capable of modifying cell surface carbohydrate epitopes such that the carbohydrate epitopes are no longer recognized by either natural human antibodies or the human cell-mediated immune system, thereby reducing the human immune system response elicited by the presence of such carbohydrate epitopes.
  • the transgenic cells, tissues, organs and animals express transgenic nucleic acid molecules encoding functional recombinant ⁇ -Galactosidase A ( ⁇ GalA) enzyme which modifies the carbohydrate epitope Gala(1,3)Gal by cleaving the terminal ⁇ -linked galactose from the carbohydrate epitope prior to its transfer to the cell surface on different molecules, thus producing cells which are phenotypically Gala(1,3) Gal".
  • the transgenic cells, tissues, organs and animals expressing the functional recombinant ⁇ GalA are transgenic pig cells, organs, tissues and/or animals.
  • the present invention relates to methods for xenotransplantation comprising introducing the transgenic cells, tissues and/or organs into human recipients so that a lower level of hyperacute rejection (HAR) is observed in the human recipients relative to the level of HAR observed in human recipients having received non-transgenic cells, tissues and/or organs, thereby reducing the level of xenotransplantation rejection.
  • HAR hyperacute rejection
  • the invention is demonstrated by way of the Examples presented in Sections 6-11, below, which describe the expression of functional recombinant ⁇ GalA in transgenic cells and the corresponding dramatic reduction of cell surface Gal (1,3)Gal carbohydrate such expression causes (Sections 7 and 10) , further demonstrate that transgenic cells expressing functional recombinant ⁇ GalA elicit a significantly reduced level of complement-mediated cytoxicity (Section 9) , and still further demonstrate that transgenic ⁇ - galA dramatically reduces the level of Gala(1,3)Gal in vivo.
  • the transgenic cells, tissues, organs and animals of the invention can serve a variety of functions.
  • the transgenic cells, tissues and organs of the invention can be used as xenotransplants for introduction into human recipients.
  • the transgenic animals of the invention can be used as sources for xenotransplant material to be introduced into human recipients or, alternatively, as sources for the production of transgenic cell lines.
  • specific transgenic cells of the invention namely bone marrow cells, may be used to produce red blood cells exhibiting an altered ABO phenotype, that is, can convert blood group B erythrocytes into erythrocytes of universal donor group 0.
  • the term "functional carbohydrate epitope-modifying gene”, as used to herein, refers to a nucleic acid sequence which encodes and directs the expression of a gene product that, either directly or indirectly, brings about modification of a cell surface carbohydrate epitope, including, but not limited to, the Gala(1,3)Gal cell surface carbohydrate epitope, in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified Gala(1,3)Gal epitope.
  • functional carbohydrate epitope-modifying enzyme refers to an enzyme, encoded by a functional carbohydrate epitope-modifying gene, which modifies a cell surface carbohydrate epitope, including, but not limited to the Gal (1,3)Gal cell surface carbohydrate epitope, in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified Gala(1,3)Gal epitope.
  • ⁇ GalA or “functional recombinant ⁇ GalA”, as used to herein, refers to an ⁇ GalA enzyme which modifies the cell surface carbohydrate epitope Gala(1,3)Gal in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified Gala(1,3)Gal epitope.
  • COS cells transiently co-transfected with a constant amount of ⁇ (l,3)galactosyltransferase cDNA (2.5 ⁇ g) and increasing amounts of ⁇ -Galactosidase A cDNA (horizontal axis, 0-12.5 ⁇ g) .
  • ⁇ (l,3)galactosyltransferase cDNA 2.5 ⁇ g
  • ⁇ -Galactosidase A cDNA horizontal axis, 0-12.5 ⁇ g
  • Cell lysates were prepared from COS cells transfected with plasmids ⁇ -Galactosidase A and ⁇ (l,3)galactosyltransferase (amounts in ⁇ g as indicated) or ⁇ -Galactosidase A alone or mock-transfected and assayed for ⁇ -Gal A activity using p-nitophenyl- ⁇ -D-galactoside as substrate.
  • aGT + aGdase + HT H transferase-transfected cells
  • mock mock-transfected cells
  • B Titer of normal human serum on mock-transfected cells and on ⁇ (1,3)galactosyltransferase-transfected cells (aGT)- transfected cells, ⁇ (l,3)galactosyltransferase + ⁇ -
  • H transferase-transfected cells (aGT + aGdase + HT) .
  • the vertical axis shows the percentage of dead cells and the horizontal axis dilutions of serum.
  • Figure 5. flow cytometric analysis of anti-Gala(1,3)Gal antibody binding.
  • Bar graphs are shown depicting the relative amounts (in units/ml) of plasma ⁇ GalA enzymatic activity in transgenic mice expressing human ⁇ GalA and non-transgenic littermates.
  • FIG. 7 Gala(1,3)Gal levels in the plasma of transgenic mice and non-transgenic littermates. Bar graphs are shown depicting the relative amounts (in % IB4 staining) of Gala(1,3)Gal in peripheral blood lymphocytes of transgenic mice expressing human ⁇ GalA and non-transgenic littermates, as obtained by flow cytometry measurements. Levels are expressed as a percentage of the control non-transgenic littermate IB4 staining.
  • the present invention involves the design, construction and use of transgenic cells, tissues, organs and animals which express functional carbohydrate epitope-modifying genes which direct the expression of gene products, including but not limited to, enzymes, capable of modifying, either directly or indirectly, cell surface carbohydrate epitopes such that the carbohydrate epitopes are no longer recognized by either natural human antibodies or the human cell-mediated immune system, thereby reducing the human immune system response elicited by the presence of such carbohydrate epitopes, relative to the response elicited by the presence of the unmodified carbohydrate epitopes.
  • carbohydrate epitope-modifying gene sequences and vectors and promoters which can be used in conjunction with such sequences for the construction of transgenes, including chimeric transgenes; methods for producing transgenic cells; methods for producing transgenic animals and establishing transgenic animal colonies by inbreeding or crossbreeding; and methods for xenotransplantation.
  • the transgenic cells, tissues, organs and animals of the invention contain one or more functional transgenic carbohydrate epitope-modifying genes which direct the expression of functional carbohydrate epitope-modifying gene products.
  • a carbohydrate epitope-modifying gene comprises a nucleic acid sequence which encodes a gene product that, either directly or indirectly, brings about modification of a cell surface carbohydrate epitope, including, but not limited to the Gala(1,3)Gal cell surface carbohydrate epitope, in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified carbohydrate epitope.
  • the nucleic acid can include, but is not limited to, a cDNA sequence or a genomic sequence.
  • the carbohydrate epitope-modifying gene is an ⁇ GalA gene.
  • the carbohydrate epitope-modifying gene of interest is coexpressed in the transgenic cells, tissues, organs and/or animals of the invention with a functional H transferase gene, the nucleic acid sequence of which is well known to those of skill in the art.
  • Carbohydrate epitope-modifying genes can include, but are not limited to genes which encode carbohydrate epitope- modifying enzymes.
  • the carbohydrate epitope-modifying enzyme is a functional ⁇ GalA enzyme.
  • such enzymes can include, for example, functional sialidase enzymes and lactosaminidase enzymes which modify cell surface carbohydrate epitopes such that the modified epitopes elicit a reduced human immune system response relative to the unmodified epitopes.
  • carbohydrate epitope-modifying genes can include, for example, nucleic acid sequences which encode antisense oligonucleotide molecules which act to inhibit the transcription of genes whose expression is necessary for the production of the cell surface carbohydrate epitope of interest, e.g.. the Gala(1,3)Gal epitope.
  • carbohydrate epitope-modifying genes can include nucleic acid sequences which encode antisense oligonucleotides complementary to transcripts produced by genes which encode transferase enzymes such as ⁇ (Gall,3)galactosyltransferase enzymes.
  • nucleic acid sequences encoding such carbohydrate epitope-modifying genes are well known to those of skill in the art. If there exists an instance in which the nucleic acid sequence encoding the carbohydrate epitope-modifying gene product of interest is not known, such a nucleic acid sequence can readily be obtained utilizing standard techniques well known to those of skill in the art, as discussed, below, in Section 5.1.1., using ⁇ GalA nucleic acid sequences as an example.
  • the nucleic acid sequences encoding the carbohydrate epitope-modifying gene products can be operatively associated with regulatory elements that direct the expression of the coding sequences.
  • regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression of the coding sequences within the appropriate cellular and/or subcellular location.
  • Appropriate location in this context, refers to a cellular and/or subcellular location of expression that results in a modification of the cell surface carbohydrate epitope of interest which results in a reduction in the human immune response elicited by the modified epitope relative to that response elicited by the unmodified epitope.
  • Carbohydrate epitope-modifying nucleotide regulatory sequences can be obtained from genomic clones utilizing the same techniques. Appropriate sequences may then be isolated, cloned, and used directly to produce transgenic cell or animals. The sequences may also be used to engineer the chimeric gene constructs that utilize regulatory sequences other than those endogenous to the carbohydrate epitope-modifying gene, again using the techniques described here. These chimeric gene constructs would then also be used in the production of transgenic cells or animals.
  • the term “functional ⁇ GalA” or “functional recombinant ⁇ GalA”, as used to herein, refers to an ⁇ GalA enzyme which modifies the cell surface carbohydrate epitope Gal (1,3)Gal in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that elicited by the unmodified Gala(1,3)Gal epitope.
  • Such ⁇ GalA genes include, but are not limited to, ⁇ GalA gene sequences from prokaryotic species, such as E. coli. and eukaryotic species, plant, such as coffee, as well as human and non-human animal sequences, which encode functional ⁇ GalA.
  • the human ⁇ GalA amino acid sequence is, for example, well known. See, e.g.. U.S. Patent No. 5,356,804, which is incorporated herein by reference in its entirety.
  • the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
  • Such low stringency conditions will be well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, (Green Publishing Associates and Wiley Interscience, N.Y.).
  • a previously unknown ⁇ GalA gene sequence may be isolated by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of known ⁇ GalA amino acid sequences.
  • the template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from cell lines or tissue known or suspected to express an ⁇ GalA gene.
  • the PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the desired ⁇ GalA sequences.
  • the PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment may be used to screen a bacteriophage cDNA library.
  • the labeled fragment may be used to screen a genomic library.
  • PCR technology may also be utilized to isolate full length cDNA sequences.
  • RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source, i.e. , one known to or suspected of expressing functional ⁇ GalA.
  • a reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
  • the resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer.
  • cDNA sequences upstream of the amplified fragment may easily be isolated.
  • cloning strategies which may be used, see e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, (Green Publishing Associates and Wiley Interscience, N.Y.). It is to be understood that, due to the degeneracy of the nucleotide coding sequence, other ⁇ GalA DNA sequences, in addition to those either described above or isolated via the techniques described above, can also encode a functional ⁇ GalA gene product.
  • a functional ⁇ GalA gene can comprise any nucleic acid sequence which encodes the amino acid sequence of a functional ⁇ GalA gene product.
  • an ⁇ GalA nucleic acid sequence can include a nucleic acid sequence that hybridizes to the complement of the coding sequence of a known ⁇ GalA gene such as, for example, the sequence of the human ⁇ GalA gene disclosed in U.S. Patent No. 5,356,804, under highly stringent conditions, e.g..
  • mice of any species, including but not limited to mice, rats, rabbits, guinea pigs, pigs, micro-pigs, and non ⁇ human primates, e.g. , baboons, squirrel monkeys and chimpanzees may be used to generate the transgenic animals of the invention, with pigs and micro-pigs being preferred.
  • a transgenic animal is a non-human animal containing at least one foreign gene, called a transgene, in its genetic material.
  • this transgene represents a carbohydrate epitope-modifying gene.
  • the transgene is contained in the animal's germ line such that it can be transmitted to the animal's offspring. In such an instance, the animal is referred to as a "founder animal”.
  • Transgenic animals may carry the transgene is all their cells or in some, but not all their cells (i.e.. the transgenic animals may be genetically mosaic) . See, for example, techniques described by Jacobovits, 1994, Curr. Biol., 4.:761-763.
  • the cells, tissues or organs which are to be introduced into human recipients should contain and express the carbohydrate epitope-modifying gene of interest.
  • the transgene may be integrated as a single transgene or in concatamers, e.g.. head-to-tail tandems or head-to-head tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al.
  • transgenic cells, tissues, organs and animals that are produced in accordance with the procedures detailed in Sections 5.2 and 5.3 should be screened and evaluated to select those cells, tissues, organs and animals which may be used as suitable xenotransplant material or xenotransplant material sources..
  • Initial screening may be accomplished by Southern blot analysis or PCR techniques to that integration of the transgene has taken place.
  • the level of carbohydrate epitope-modifying gene mRNA expression in the transgenic cells, tissues, organs and animals may also be assessed using techniques which include but are not limited to Northern blot analysis of samples, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR) .
  • the carbohydrate epitope-modifying transgenic cells, tissues, organs and/or animals that express mRNA or protein (detected immunocytochemically, using appropriate antibodies) at easily detectable levels should then be further evaluated to identify those animals which display modified cell surface carbohydrate epitopes.
  • histopathological evaluation of transgenic material can be carried out using antibodies directed against the cell surface epitope of interest, coupled with standard techniques well known to those of skill in the art.
  • Transfer methods include, for example, methods of introducing cells such as those listed, above, in Section 5.3, including but not limited to blood cells and bone marrow cells, and methods for introducing tissues and organs such as those listed, above, in Section 5.3, including heart, liver, lung and kidney tissues and/or organs.
  • ⁇ -Galactosidase A and Protein Assays Cells were washed twice with PBS and lysed in 1% TritonX-100/Sodium phosphate pH 7.0/150 mM NaCl/l mM EDTA buffer containing protease inhibitors on ice for 20 min. Lysates were centrifuged for 15 min at 13000g at 4°C, supematants collected and assayed for ⁇ -Galactosidase A activity using p- nitophenyl- ⁇ -D-galactoside as substrate (Kint, J.A. , 1970, Science 270:12681. Protein concentrations were determined by Bradford assay using bovine serum albumin as standard (Bradford, M.M. , 1976, Anal. Biochem. 72:248) .
  • ⁇ -Galactosidase A expressed in ⁇ -Galactosidase A-transfected cells
  • lysates were assayed using p-nitophenyl- ⁇ -D-galactoside as a substrate for ⁇ - Galactosidase A.
  • ⁇ -Galactosidase A activity in lysates from mock-transfected cells was 15 nmol/h/mg protein (Fig. 3) .
  • Lysates from cells transfected with ⁇ -Galactosidase A cDNA alone gave enzyme activity at 48 nmol/h/mg protein, as did lysates from cells co-transfected with ⁇ (l,3)galactosyltransferase and ⁇ -Galactosidase A (38, 35 and 56 nmol/h/mg protein for 2.5, 5 and 12.5 mg ⁇ -Galactosidase A cDNA respectively) (Fig. 3) .
  • ⁇ -Galactosidase A cDNA Lysates from cells transfected with ⁇ -Galactosidase A cDNA alone gave enzyme activity at 48 nmol/h/mg protein, as did lysates from cells co-transfected with ⁇ (l,3)galactosyltransferase and ⁇ -Galactosidase A (38, 35 and 56 nmol/h/mg protein for 2.5, 5 and 12.5
  • COS cells were transiently co-transfected with (i) ⁇ (1,3)galactosyltransferase + H transferase cDNAs; (ii) ⁇ (1,3)galactosyltransferase + ⁇ -Galactosidase A cDNAs; or (iii) ⁇ (1,3)galactosyltransferase + H transferase + ⁇ - Galactosidase A cDNAs, and were stained on the cell surface with IB4 or UEA1 and permeabilized cells were stained for ⁇ - Galactosidase A.
  • Dye released from the cells was measured using a Millipore Cytofluor 2350 fluorescence plate reader (490nm excitation, 530nm emission) and total cell associated dye was determined from a 1% SDS cell lysate and specific dye release calculated as a percent of total. Other techniques. Other techniques were as described, above, in Section 7.1.
  • the Example presented in this Section demonstrates that the successful use of transgenic ⁇ GalA expression in pig cells to reduce the cell surface level of Gal (1,3)Gal.
  • Stable cell lines were generated which express human ⁇ - galactosidase under a cytomegalovirus promoter.
  • the cell lines were produced using standard calcium phosphate transfection and neomycin selection. Cells were tested for their ability to bind natural human anti-Gala(1,3)Gal
  • transgenic mouse lines expressing human ⁇ - galactosidase under an H2-K b promoter were generated using standard techniques. Results from C57BL/6 mice heterozygous for the human ⁇ -galactosidase gene demonstrated that the transgene was incorporated into the genome and was transmitted between generations, ⁇ -galactosidase enzyme levels in the plasma of transgenic mice were measured as at least four-fold higher than the level measured in non- transgenic littermates (Fig. 6) . Peripheral blood lymphocytes from each transgenic line were tested for the level of Gala (1,3) Gal by staining the cell surface with IB4 (a lectin specific for Gala(1,3)Gal) and measuring by standard flow cytometry. Results are depicted in Fig. 7, with levels being expressed as a percentage of the control non-transgenic littermate IB4 staining.
  • Transgenic mice showed between 34% and 50% reduction in their level of Gala(1,3)gal depending on the line tested, thus demonstrating the successful in vivo reduction of the epitope via the use of transgenic ⁇ GalA.

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Abstract

La présente invention concerne des procédés et des compositions de réduction du rejet de xénogreffes. De façon spécifique, l'invention concerne principalement des cellules, des tissus, des organes et des animaux transgéniques contenant des molécules d'acides nucléiques transgéniques dirigeant l'expression de produits géniques incluant, entre autres, des enzymes capables de modifier directement ou indirectement les épitopes des glucides de la surface des cellules, de façon que ces épitopes de glucides ne soient plus reconnus par les anticorps humains naturels ou par la réponse immunitaire à médiation cellulaire de l'homme. On arrive ainsi à réduire la réponse du système immunitaire humain mise en évidence par la présence de tels épitopes de glucides. Selon une réalisation préférée, les cellules, tissus, organes et animaux transgéniques expriment des molécules d'acides nucléiques codant l'enzyme fonctionnelle α-Galactosidase A (α-GalA) recombiné qui modifie l'épitope de glucide Galα(1,3)Gal. Selon un mode de réalisation plus préférentiel, les cellules, tissus, organes et animaux transgéniques exprimant l'α-GalA fonctionnelle recombiné sont des cellules, des organes, des tissus et/ou des animaux transgéniques d'origine ou de nature porcine. L'invention concerne également des techniques de xénogreffe consistant à introduire, chez un receveur humain, des cellules, tissus et/ou organes transgéniques de façon à constater chez de tels receveurs humains un niveau de rejet hyperaigu inférieur au niveau de rejet hyperaigu observé chez les receveurs humains ayant reçu des cellules, tissus et/ou organes non transgéniques.
PCT/US1996/017695 1995-11-03 1996-11-01 Procedes et compositions de reduction du rejet de xenogreffe WO1997016064A1 (fr)

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AU76686/96A AU7668696A (en) 1995-11-03 1996-11-01 Methods and compositions for the reduction of xenotransplantation rejection
EP96939544A EP0877549A4 (fr) 1995-11-03 1996-11-01 Procedes et compositions de reduction du rejet de xenogreffe
JP09517615A JP2000514641A (ja) 1995-11-03 1996-11-01 異種移植拒絶を低減するための方法および組成物
IL12429396A IL124293A0 (en) 1995-11-03 1996-11-01 Method and compositions for the reduction of xenotransplantation rejection

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US60/006,200 1995-11-03

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999021415A1 (fr) * 1997-10-28 1999-05-06 Stem Cell Sciences Pty. Ltd. Transfert nucleaire pour la production d'un embryon d'animal transgenique
US6399578B1 (en) 1998-12-09 2002-06-04 La Jolla Pharmaceutical Company Conjugates comprising galactose α1,3 galactosyl epitopes and methods of using same
US7547522B2 (en) 2002-08-14 2009-06-16 Immerge Biotherapeutics, Inc. Method to enrich for α(1,3)-galactosyltransferase null pig cells
US7560538B2 (en) 2003-11-05 2009-07-14 University Of Pittsburgh Porcine isogloboside 3 synthase protein, cDNA, genomic organization, and regulatory region
US7795493B2 (en) 2002-08-21 2010-09-14 Revivicor, Inc. Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase
WO2011012297A1 (fr) 2009-07-30 2011-02-03 F. Hoffmann-La Roche Ag Traitement enzymatique d'anticorps
US8106251B2 (en) 2002-08-21 2012-01-31 Revivicor, Inc. Tissue products derived from porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase
WO2013050335A1 (fr) 2011-10-05 2013-04-11 F. Hoffmann-La Roche Ag Procédé de production d'anticorps de glycoforme g1
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US6399578B1 (en) 1998-12-09 2002-06-04 La Jolla Pharmaceutical Company Conjugates comprising galactose α1,3 galactosyl epitopes and methods of using same
US7547522B2 (en) 2002-08-14 2009-06-16 Immerge Biotherapeutics, Inc. Method to enrich for α(1,3)-galactosyltransferase null pig cells
US10130737B2 (en) 2002-08-21 2018-11-20 Revivicor, Inc. Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US7795493B2 (en) 2002-08-21 2010-09-14 Revivicor, Inc. Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US11172658B2 (en) 2002-08-21 2021-11-16 Revivicor, Inc. Porcine animals lacking expression of functional alpha 1, 3 galactosyltransferase
US8106251B2 (en) 2002-08-21 2012-01-31 Revivicor, Inc. Tissue products derived from porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase
US10912863B2 (en) 2002-08-21 2021-02-09 Revivicor, Inc. Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US7560538B2 (en) 2003-11-05 2009-07-14 University Of Pittsburgh Porcine isogloboside 3 synthase protein, cDNA, genomic organization, and regulatory region
US8524470B2 (en) 2009-07-30 2013-09-03 Hoffman-La Roche, Inc. Enzymatic antibody processing
WO2011012297A1 (fr) 2009-07-30 2011-02-03 F. Hoffmann-La Roche Ag Traitement enzymatique d'anticorps
US9420770B2 (en) 2009-12-01 2016-08-23 Indiana University Research & Technology Corporation Methods of modulating thrombocytopenia and modified transgenic pigs
US9187564B2 (en) 2010-10-05 2015-11-17 Hoffmann-La Roche Inc. Antibodies against human TWEAK and uses thereof
WO2013050335A1 (fr) 2011-10-05 2013-04-11 F. Hoffmann-La Roche Ag Procédé de production d'anticorps de glycoforme g1

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IL124293A0 (en) 1998-12-06
EP0877549A4 (fr) 2002-01-02
AU7668696A (en) 1997-05-22
EP0877549A1 (fr) 1998-11-18

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