WO1997016064A9 - Procedes et compositions de reduction du rejet de xenogreffe - Google Patents

Procedes et compositions de reduction du rejet de xenogreffe

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WO1997016064A9
WO1997016064A9 PCT/US1996/017695 US9617695W WO9716064A9 WO 1997016064 A9 WO1997016064 A9 WO 1997016064A9 US 9617695 W US9617695 W US 9617695W WO 9716064 A9 WO9716064 A9 WO 9716064A9
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transgenic
cells
cell
human
organs
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PCT/US1996/017695
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English (en)
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WO1997016064A1 (fr
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Priority to JP09517615A priority Critical patent/JP2000514641A/ja
Priority to AU76686/96A priority patent/AU7668696A/en
Priority to IL12429396A priority patent/IL124293A0/xx
Priority to EP96939544A priority patent/EP0877549A4/fr
Publication of WO1997016064A1 publication Critical patent/WO1997016064A1/fr
Publication of WO1997016064A9 publication Critical patent/WO1997016064A9/fr

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  • 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.
  • 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.
  • HAR hyperacute rejection
  • xenotransplants Extensive studies now exist regarding such xenotransplantations. See, e.g.. Sandrin et al. (Sandrin, M.S. et al., 1994, Transplant. Rev. 8:134), 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 Gala(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 O.
  • 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 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 Gal (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.
  • Figure 1 Hemagglutination of red blood cells following ⁇ -Galactosidase A treatment.
  • 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.
  • 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.
  • nudeotide regulatory sequences used to regulate the carbohydrate epitope-modifying gene coding sequences can include the regulatory sequences endogenous to (i.e.. normally associated with) the carbohydrate epitope- modifying gene of interest itself.
  • chimeric carbohydrate epitope-modifying gene constructs containing the nudeotide coding sequence for a functional carbohydrate epitope-modifying gene product, regulated by a promoter or promoter/enhancer complex not endogenous to the carbohydrate epitope-modifying gene coding sequence may be engineered as the transgene to be used in the production of the transgenic cells, tissues, organs and animals of the invention.
  • Multiple copies of the gene or chimeric gene construct may be arranged in the vector, and multiple copies of the gene or chimeric gene construct may be stably introduced into the transgenic cells or founder animals.
  • recombinant DNA and cloning methods which are well known to those skilled in the art may be utilized (see Sambrook et al.. 1989, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY) .
  • appropriate carbohydrate epitope-modifying gene coding sequences may be generated from cDNA or genomic clones using restriction enzyme sites that are conveniently located at the relevant positions within the sequence.
  • site directed mutagenesis techniques involving, for example, either the use of vectors such as M13 or phagemids, which are capable of producing single stranded circular DNA molecules, in conjunction with synthetic oligonucleotides and specific strains of Escherichia coli (E. coli) (Kunkel, T.A. et al.. 1987, Meth. Enzymol. 154:367-382) or the use of synthetic oligonucleotides and PCR (polymerase chain reaction) (Ho et al. , 1989, Gene 22:51-59; Kamman, M. et al.. 1989, Nucl. Acids Res.
  • vectors such as M13 or phagemids
  • Carbohydrate epitope-modifying nudeotide 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.
  • ⁇ GalA GENES Any nucleic acid molecule which directs the expression of a functional ⁇ GalA gene product can be used as a transgene in the production of the transgenic cells, tissues, organs and animals of the present invention. As discussed in
  • the term “functional ⁇ 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 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.
  • ⁇ GalA gene sequences are known to exist in other species. In those instances whereby sequences are not well known, they may be identified and isolated, without undue experimentation, by molecular biological techniques well known in the art. For example, an isolated ⁇ GalA gene sequence may be labeled and used to screen a cDNA library constructed from mRNA obtained from a cell type known to or suspected of expressing ⁇ GalA derived from the organism of interest. Hybridization conditions will generally be of a lower stringency when the cDNA library was derived from an organism different from the type of organism from which the labeled sequence was derived.
  • 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 nudeotide 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. Bi ., 4 ⁇ :761-763. For xenotransplantation purposes, however, 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. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89.:6232-6236) .
  • the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • Any technique known in the art may be used to introduce the transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No.
  • founder animals may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
  • breeding strategies include but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the transgene.
  • transgenic animals are transgenic ungulates, including but not limited to transgenic pigs. Methods for constructing such transgenic animals are well known to those of skill in the art. See, e.g.. international application numbers WO 94/26884 and WO 95/04744, which are hereby incorporated by reference in their entirety.
  • transgenic cell including but not limited to transgenic bone marrow cells, peripheral blood stem cells, liver cells, kidney cells, islet cells, etc.
  • transgenic bone marrow cells including but not limited to transgenic bone marrow cells, peripheral blood stem cells, liver cells, kidney cells, islet cells, etc.
  • tissue or organ including but not limited to, liver, kidney, muscle, heart, lung, pancreas, skin thyroid, parathyroid, adrenal cortex, adrenal medulla, thymus, cartilage, bone, etc. are to be considered within the scope of the present invention.
  • transgenic cells, tissues and organs of the invention may be produced by a variety of methods which are well known to those of skill in the art.
  • the transgenic cells, tissues and organs of the invention may be obtained from the transgenic animals described, above, in Section 5.2.
  • primary cultures of cells derived from the transgenic animals of the invention may be utilized, or, preferably, continuous cell lines can be generated.
  • continuous cell lines can be obtained utilizing techniques well known to those of skill in the art, such as, for example, techniques described by Small et al., 1985, Mol. Cell. Biol. 5:642-648.
  • cells of a cell type of interest may be transfected with carbohydrate epitope-modifying sequences capable of expressing a functional carbohydrate epitope modifying gene product within the cell, thus yielding transgenic cells of the invention.
  • Transfection of cells with transgenic nucleic acid sequences can be accomplished by utilizing standard techniques such as, for example, those techniques described, above, in Section 5.2. Additionally, see, for example, Ausubel, 1989, Current Protocols in
  • Transfected cells should be evaluated for the presence of the transgenic nucleic acid sequences, for expression and accumulation of the transgenic carbohydrate epitope-modifying gene product. Further, the transgenic cells should be evaluated for an ability to exhibit modified cell surface carbohydrate epitopes of interest.
  • 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 5 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 10 those of skill in the art.
  • the transgenic cells, tissues, organs and animals of the invention can serve a number of functions, both in vitro and in vivo.
  • the transgenic material can serve as xenotransplantation material or as the source for xenotransplantation material.
  • the use of the transgenic material of the invention as xenotransplantation material serves to lower level of hyperacute rejection (HAR) observed in 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
  • specific transgenic cells of the invention 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 O.
  • any technique for transplanting donor material into recipients can be utilized. Such techniques are well known to those of skill in the art. Transfer methods include, for example, methods of introducing
  • 3 _ 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.
  • red cells were agglutinated using lectin at 0.98 ng/ml (Fig. la) .
  • substantially more lectin was required to agglutinate the red cells: 7.81 ng/ml of lectin after treatment of red blood cells with 150 U of human ⁇ -Galactosidase A, 15.63 ng/ml after 300 U and 125 ng/ml after 600 U (Fig. la) .
  • Example presented herein demonstrates that cells transfected with an ⁇ GalA cDNA brings about a decrease in the level of cell surface Gal ⁇ (l,3)Gal carbohydrate epitope, relative to non-transfected cells.
  • phAGA which encodes cDNA for human ⁇ -Galactosidase A cDNA in mammalian expression vector pCDNAl (Invitrogen)
  • pPGT-3 which encodes porcine ⁇ (l,3)galactosyltransferase cDNA
  • pCD48 pHuLy-m3.7
  • COS-7 cells were maintained in Dulbecco's modified Eagles Medium (DMEM) (Trace Biosciences Pty. Ltd., Castle Hill, NSW, Australia) and were transfected (1-20 ⁇ g DNA/10 cm dish) using the DEAE-Dextran method (Vaughan, H.A. et al., 1991, Immunogenetics 3_3_:113) using DMEM supplemented with 10% Nu-Serum (Collaborative Research Inc. , Bedford, MA) ; 48h later cells were examined for cell surface expression.
  • DMEM Dulbecco's modified Eagles Medium
  • Direct fluorescence of the cell surface carbohydrate epitope Gal ⁇ (l,3)Gal was performed with FITC-conjuga ed IB4 lectin.
  • a monoclonal antibody (mAb) specific for CD48 (ASH1360, Austin Research Institute) and FITC-conjugated goat anti- mouse IgG were used for cell surface staining of CD48 in control transfections.
  • the expression of human ⁇ - Galactosidase A was assessed by internal staining of formaldehyde-fixed and TritonX-lOO-permeabilized cells with affinity purified rabbit anti- ⁇ -Galactosidase A antibodies (Ioannou, Y.A. et al., 1992, J. Cell Biol. 119:1137) followed by FITC-conjugated goat anti-rabbit IgG. Fluorescence was detected by microscopy.
  • ⁇ -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/1 mM EDTA buffer containing protease inhibitors on ice for 20 min. Lysates were centrifuged for 15 min at 13000g at 4°C, supernatants collected and assayed for ⁇ -Galactosidase A activity using p- nitophenyl- ⁇ -D-galactoside as substrate (Kint, J.A. , 1970, Science 270:1268) . Protein concentrations were determined by Bradford assay using bovine serum albumin as standard (Bradford, M.M. , 1976, Anal. Biochem. 72:248) .
  • IB4 staining cells co- transfected with ⁇ (l,3)galactosyltransferase and control cDNA was similar to cells expressing ⁇ (l,3)galactosyltransferase alone (i.e. 60%) indicating that the observed reductions in IB4 staining with ⁇ -Galactosidase A are an accurate reflection of ⁇ -Galactosidase A altering cell surface levels of Gal (1,3)Gal and not merely a result of the co-transfection procedure.
  • ⁇ -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) ⁇ (l,3)galactosyltransferase + H transferase cDNAs; (ii) ⁇ (l,3)galactosyltransferase + ⁇ -Galactosidase A cDNAs; or (iii) ⁇ (l,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.
  • 35 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.
  • 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. 1 , 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.

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)

Priority Applications (4)

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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
AU76686/96A AU7668696A (en) 1995-11-03 1996-11-01 Methods and compositions for the reduction of xenotransplantation rejection
IL12429396A IL124293A0 (en) 1995-11-03 1996-11-01 Method 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

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US620095P 1995-11-03 1995-11-03
US60/006,200 1995-11-03

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WO1997016064A1 WO1997016064A1 (fr) 1997-05-09
WO1997016064A9 true WO1997016064A9 (fr) 1997-08-28

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JP (1) JP2000514641A (fr)
AU (1) AU7668696A (fr)
IL (1) IL124293A0 (fr)
WO (1) WO1997016064A1 (fr)

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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
ES2338111T3 (es) 2002-08-21 2010-05-04 Revivicor, Inc. Animales porcinos que carecen de cualquier expresion de alfa 1,3 galactosiltransferasa funcional.
WO2005047469A2 (fr) 2003-11-05 2005-05-26 University Of Pittsburgh Proteine isogloboside 3 synthase porcine, adnc, organisation genomique, et region regulatrice
JP2007529278A (ja) 2004-03-17 2007-10-25 レビビコア， インコーポレイテッド 機能的α１，３ガラクトシルトランスフェラーゼを欠く動物に由来する組織生成物
AU2010278290A1 (en) 2009-07-30 2012-02-09 F. Hoffmann-La Roche Ag Enzymatic antibody processing
US9420770B2 (en) 2009-12-01 2016-08-23 Indiana University Research & Technology Corporation Methods of modulating thrombocytopenia and modified transgenic pigs
JP5941049B2 (ja) 2010-10-05 2016-06-29 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ ヒトｔｗｅａｋに対する抗体およびその使用
SG2014012470A (en) 2011-10-05 2014-06-27 Hoffmann La Roche Process for antibody g1 glycoform production

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US5356804A (en) * 1990-10-24 1994-10-18 Mount Sinai School Of Medicine Of The City Of New York Cloning and expression of biologically active human α-galactosidase A
EP0755402B1 (fr) * 1994-04-13 2008-05-14 Biotransplant, Inc Porc negatif pour l'alpha(1,3) galactosyltranferase
JPH10504707A (ja) * 1994-06-15 1998-05-12 アレクション・ファーマシューティカル・インク 異種移植片の超急性拒否反応を減少させる方法

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