JP2000514641A - Methods and compositions for reducing xenograft rejection - Google Patents

Abstract

  (57) [Summary] The present invention relates to methods and compositions for reducing xenograft rejection. In particular, the invention firstly modifies, directly or indirectly, cell surface carbohydrate epitopes such that the carbohydrate epitopes are no longer recognized by natural human antibodies or human cell-mediated immune responses, Transgenic cells, tissues containing transgenic nucleic acid molecules that direct the expression of gene products such as, but not limited to, enzymes that can reduce the response of the human immune system elicited by the presence of such carbohydrate epitopes , Organs and animals. In a preferred embodiment, the transgenic cells, tissues, organs and animals express a nucleic acid molecule that encodes a functional recombinant α-galactosidase A (αGalA) enzyme that alters the carbohydrate epitope Galα (1,3) Gal. In a further preferred embodiment, the transgenic cells, tissues, organs and animals expressing the functional recombinant αGalA are cells, organs, tissues and / or animals of a transgenic pig. Second, the present invention introduces transgenic cells, tissues and / or organs into a human recipient so that the level of severe rejection (HAR) observed in that human recipient is higher than that of non-transgenic cells. Xenograft methods comprising lowering the level of HAR as observed in human recipients receiving tissues and / or organs.

Description

DETAILED DESCRIPTION OF THE INVENTION               Methods and compositions for reducing xenograft rejection 1.Introduction   The present invention relates to methods and compositions for reducing xenograft rejection. In particular, The present invention provides, firstly, direct or indirect modification of cell surface carbohydrate epitopes. In addition, carbohydrate epitopes can be stimulated by natural human antibodies or human cell-mediated immune responses. So that it can no longer be recognized, thereby providing such a carbohydrate epitope Enzymes that can reduce the response of the human immune system elicited by the presence of Transgenic nucleic acids that direct the expression of a gene product (including, but not limited to) It relates to transgenic cells, tissues, organs and animals containing the molecule. preferable In embodiments, the transgenic cells, tissues, organs and animals are carbohydrate A functional recombinant α-galactosidase A that modifies the pitope Galα (1,3) Gal (αGal A) Express the nucleic acid molecule encoding the enzyme. In a more preferred embodiment, the functional Transgenic cells, tissues, organs and animals expressing recombinant αGalA can be Cells, organs, tissues and / or animals of transgenic pigs. Secondly, The present invention provides for the transfer of transgenic cells, tissues and / or organs to human recipients. And as a result, severe rejection (hyperacute reje ction) (HAR) levels in non-transgenic cells, tissues and / or organs. Government is lower than the level of HAR found in donated human recipients And xenotransplantation methods. The present invention is described in the following sections 6 to 11. This will be described with reference to Examples. In the following examples, for example, transgenic cells Expression of functional recombinant αGalA and cell surface Galα (1,3) Gal resulting from the expression A corresponding dramatic reduction in carbohydrates is described (Sections 7 and 10), and Cells in which transgenic cells expressing recombinant αGalA are complement mediated Illustrate a significant reduction in toxicity levels (Section 9); Genetic α-GalA can dramatically reduce Galα (1,3.) Gal levels in vivo. And Is illustrated. 2.Background of the Invention   Due to the considerable shortage of human organs available for transplantation purposes, There is high interest in the use of organ transplants, i.e., "xenografts", in humans. Currently, There are numerous studies on such xenografts. For example, for human xenografts Sandrin et al. (Sandrin, M.S. et al., 1994, Tra nsplant. Rev. 8: 134).   The body's first response to foreign tissue is known as severe rejection (HAR), Painful response, one of the biggest obstacles to the success of xenograft technology. is there. HAR is mostly mediated by antibodies and complement, where Natural reacting with many molecules on xenograft cells, especially endothelial cells, in tubular grafts Human antibodies, mainly the IgG and IgM subclasses (Cooper, D.K.C. et al. , 1994, Immunol. Rev. 141: 31; Sandrin, M.S. and McKenzie, I.F.C., 1994, Imnlunol. Rev. 141: 169). Currently, all or most of the HAR Is due to the presence of a human antibody specific for the chloride epitope Galα (1,3) Gal. It is generally accepted. This is especially Gal⁺Study of absorption by transfected cells And that Galα (1,3) Gal carbohydrates can enhance the reaction in vivo and in vitro. Can be inhibited (Sanadrin, MS et al., 1994, Xenotrans plantation 1:81).   Attempts to eradicate HAR include cobra venom factor or human complement regulatory molecules. (Eg, CD46, CD55 and CD59). In addition, removal or neutralization of complement by various techniques is mentioned. Alternatively, , Logically different removal of antibodies (Oriol, R. et al., 1993, Transplantation 56: 433). And attempts to change the antigen itself. For this latter method, Ga Porcine .ALPHA. (1,3) galactosyltransferase required for l.ALPHA. (1,3) Gal carbohydrate production The gene encoding the enzyme should be used for gene knockout studies by homologous recombination. And to More isolated. Unfortunately, such techniques can not be performed in pigs. I can't. For a review of other methods for preventing the expression of Galα (1,3) Gal, see Sandrin (Sandrin, M.S. et al., 1994, Transplant. Rev. 8: 134). The method comprises: Including the use of a chisense construct, these results are indeterminate and generally Not a thing.   Other methods that have been tried are enzymes that cleave terminal α-linked galactosyl residues. It uses certain α-galactosidase A (Oriol, R. et al., 1993, Transp. lantation 56: 433). Alpha-galactosidase of erythrocytes, lymphocytes and endothelial cells Treatment with A inhibits their reaction with human serum, and Cairns et al. Clarified a similar phenomenon. One meeting record states that bacterial α-gas was added to tissues prior to transplantation. It has been reported that perfusion of the lactosidase A enzyme delays the onset of HAR. (Cairns, T. et al., 1994, Transplant. Proc. 26: 1279). These enzyme treatment methods However, it is difficult. For example, these enzymes are expensive, and Perfusion with elemental is actually a challenge, especially to ensure epitope eradication. It would be difficult.   Thus, in summary, xenografts represent a significant lack of existing human donor organs. Although a potential solution to the foot, the problem of severe rejection is the use of xenografts. Remains a major obstacle to success. 3.Summary of the Invention   The present invention relates to methods and compositions for reducing xenograft rejection.   In particular, the present invention relates, firstly, but not exclusively, to the Galα (1,3) Gal cell surface. Direct or indirect cell surface carbohydrate epitopes, such as carbohydrate epitopes To alter the response of the human immune system caused by the resulting altered epitope Reduced for the response elicited by the unmodified Galα (1,3) Gal epitope A functional carbohydrate epitope-altered gene that directs the expression of the gene product to be altered Transgenic nucleic acid content Transgenic cells, tissues, organs and animals, including offspring. Like that Gene products include, but are not limited to, cell surface carbohydrate epitopes The carbohydrate epitope is no longer a natural human antibody or human cell-mediated Can be prevented from being recognized by the immune system, so that the carbohydrate Carbohydrates reduce the response of the human immune system caused by the presence of tope Pitope-modified enzymes.   In a preferred embodiment of the present invention, the transgenic cells, tissues, organs and The animal is able to transfer terminal α-linked galactose to a different molecule prior to transfer to the cell surface. The carbohydrate epitope Galα (1,3) Gal by cleavage from the carbohydrate epitope Functional, thus producing cells that are phenotypically Galα (1,3) Gal Transgenic encoding recombinant α-galactosidase A (αGalA) enzyme Express the nucleic acid molecule. In a more preferred embodiment, expressing a functional recombinant αGalA Transgenic cells, tissues, organs and animals Cells, organs, tissues and / or animals. Yet another preferred embodiment of the present invention In one embodiment, the αGalA and H transferase genes are Co-expressed in transgenic cells, tissues, organs and animals.   Second, the invention relates to transgenic cells, tissues and / or organs To the recipient and consequently to the severe rejection (HA R) the level provided by the non-transgenic cells, tissues and / or organs; Lower than the level of HAR found in the selected human recipient, Thus, it relates to a xenograft method comprising reducing the level of xenograft rejection.   The present invention will be described by examples shown in the following sections 6 to 11. In the following example For example, expression of functional recombinant αGalA in transgenic cells and its expression Describes a corresponding dramatic decrease in cell surface Galα (1,3) Gal carbohydrates caused by 7 and 10) and functional recombination Transgenic cells expressing αGalA can be complement-mediated cytotoxicity Illustrate a significant reduction in bell (Section 9). Illustrates that α-GalA dramatically reduces Galα (1,3) Gal levels in vivo I do.   The transgenic cells, tissues, organs and animals of the present invention perform various functions. I can. For example, the transgenic cells, tissues and organs of the invention , Can be used as xenografts for introduction into human recipients. Transformer of the present invention Transgenic animals are the source of xenograft material that can be introduced into human recipients. Alternatively, it can be used as a source of production for transgenic cell lines. is there Alternatively, certain transgenic cells of the invention, ie, bone marrow cells, It can also be used to produce the altered red blood cells of the present type. That is, B blood Converts liquid erythrocytes to O-type erythrocytes, which are universal donors can do.   As used herein, a "functional carbohydrate epitope-altered gene" refers to Galα ( 1,3) Cell surface carbohydrates such as, but not limited to, Gal cell surface carbohydrate epitopes Modify the hydrate epitope directly or indirectly to obtain the resulting modified epitope Response of the human immune system to the unmodified Galα (1,3) Gal epitope Encoding a gene product that reduces the response elicited, and its expression Means a nucleic acid sequence indicating   As used herein, “functional carbohydrate epitope-altering enzyme” refers to Galα (1, 3) Cell surface carbohydrates such as, but not limited to, Gal cell surface carbohydrate epitopes Humanized by a modified epitope and resulting modified epitope Response of the immune system to the response caused by the unmodified Galα (1,3) Gal epitope Encoded by a functional carbohydrate epitope-altered gene Means an enzyme.   As used herein, "functional αGalA" or "functional recombinant αGalA" Cell surface carbohydrate epitope Galα (1,3) Gal Response of the human immune system caused by the modified epitopes 3) αGalA enzyme that reduces the response caused by the Gal epitope means. 4.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the agglutination of erythrocytes by α-galactosidase A treatment. Red blood cells In a direct hemagglutination assay showing the effect of pretreatment with α-galactosidase A Yes, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / m l, 15.63 ng / ml, 7.81 ng / ml, 3.91 ng / ml, 1.95 ng / ml, 0.98 ng / ml IB4 lectic Red blood cells treated with untreated red blood cells or normal human serum (NHS) Is red treated with 600 U, 300 U or 150 U of human α-galactosidase A (HG) Blood cells (FIG. 1A) or 50 U, 25 U, 12.5 U or 6.25 U E. coli α-galactosida The cells were incubated with erythrocytes (FIG. 1B) treated with Asease A (EG).   FIG. 2 shows the titration of α-galactosidase A cDNA. COS cells are transformed into α (1 , 3) a fixed amount (2.5 μg) of galactosyltransferase cDNA and α-galactosyltransferase Increasing the amount of tosidase A cDNA (horizontal axis, 0 to 12.5 μg) Transfected. After 48 hours, cells were stained with IB4 lectin. The vertical axis is α (1,3) recognized in cells transfected with galactosyltransferase alone The cell staining intensity is shown assuming that the obtained staining intensity is 100%. Transfection efficiency Was 20-40%.   FIG. 3 shows α-galactosidase in transiently transfected COS cells. 2 shows the activity of Aase. Cell lysates were converted to plasmid α-galactosidase A and α (1,3) galactosyltransferase (the amount is shown in μg) or α-galacto Transfected with sidase A alone or mock transfected P-nitrophenyl-α-D-galactoside was prepared from Used as α-GalA Activity was assayed.   FIG. 4 shows lysis of transfected COS cells with normal human serum. . Pooled normal human serum⁵¹In a Cr-release lysis assay, Tested for lysis of transfected and untransfected COS cells did. (A) Normal human serum is treated with α (1,3) galactosyltransferase. Transfected cells (αGT), transfected with α-galactosidase A Cells (αGdase) and cells transfected with H transferase (HT ), Transfection with α (1,3) galactosyltransferase + Htransferase. Sfected cells (αGT + HT), α (1,3) galactosyltransferase + Cells transfected with α-galactosidase A + H transferase ( αGT + αGdase + HT) and 1 for mock-transfected cells (mock) : 5 dilution was used. (B) mock transfected cells and α (1,3) galact Tosyltransferase transfected cells (αGT), α (1,3) galactos Transferase + α-galactosidase A transfected cells (αGT + αGdase), α (1,3) galactosyltransferase + H transferase Transfected cells (αGT + HT), α (1,3) galactosyltransferase + α-galactosidase A + H transferase transfected cells (αGT + Titration of normal human serum against αGdase + HT). The vertical axis indicates the percentage of dead cells, and the horizontal axis Indicates the dilution of serum.   FIG. 5 shows a flow cytometric analysis of anti-Galα (1,3) Gal antibody binding. versus Control, relative of control PIEC and PIEC cells transfected with human αGalA Showing fluorescence levels and relative binding ability of cells to native human anti-GalA (1,3) Gal antibody Is shown.   FIG. 6 shows transgenic and non-transgenic littermates. FIG. 4 shows α-galactosidase enzyme levels in plasma. The bar graph shows human αGalA Of transgenic and non-transgenic littermates expressing Relative amount of plasma αGalA enzyme activity (unit / m l) is shown.   FIG. 7 shows transgenic and non-transgenic littermate mice. Figure 4 shows Galα (1,3) Gal levels in plasma. The bar graph is used for flow cytometry measurement. Thus, transgenic mice expressing human αGalA and non-transgenic mice Relative amount of Galα (1,3) Gal in peripheral blood lymphocytes of transgenic littermates (% I B4 staining). Levels were IB4 in control non-transgenic littermates. Expressed as% of staining. 5.Detailed description of the invention   The present invention provides for directly or indirectly modifying a carbohydrate epitope on a cell surface, Carbohydrate epitopes are no longer generated by natural human antibodies or the human cell-mediated immune system Make it unrecognizable, thereby reducing the presence of such carbohydrate epitopes The more elicited response of the human immune system is triggered by the presence of the unmodified carbohydrate epitope. Enzymes, such as, but not limited to, enzymes that can reduce the response to be evoked Expresses a functional carbohydrate epitope-altered gene that directs the expression of a gene product Including the design, construction and use of transgenic cells, tissues, organs and animals. No.   The next aspect of the invention, namely the carbohydrate epitope-altered gene sequence, and Can be used in combination with the sequence for the construction of a transgene such as a chimeric transgene. Vectors and promoters; methods for producing transgenic cells; Produce transgenic animals and transgenic animals by inbred or crossbreeding. How to establish a product colony; and the xenotransplantation method are described in the following subsections, These are for illustration only and do not limit the invention.   The invention is further illustrated by the examples shown in Sections 6-11 below. In particular, in the embodiment Expression of functional recombinant αGalA in transgenic cells and its expression A corresponding dramatic decrease in cell surface Galα (1,3) Gal carbohydrates caused is described (7th and 7th). And Section 10), and functional recombinant αGalA Cause significant reduction in the level of complement-mediated cytotoxicity (Section 9). In addition, transgenic α-galA can increase the level of Galα (1,3) Gal in vivo. The dramatic reduction is exemplified. 5.1.Carbohydrate epitope-modified gene   The transgenic cells, tissues, organs, and animals of the present invention are functional carbohydrates. One or more functional transgenes that direct the expression of the epitope-altered gene product Carbohydrate epitopes-including modified genes. Such carbohydrate epitopes-modified Mutant genes include, but are not limited to, Galα (1,3) Gal cell surface carbohydrate epitopes. Cell surface carbohydrate epitopes, e.g. Unaltered carbohydrate response of the human immune system caused by altered epitopes Gene products that reduce the response caused by the The nucleic acid sequence to be loaded. Nucleic acids include, but are not limited to, cDNA sequences Or a genomic sequence.   In a preferred embodiment of the present invention, the carbohydrate epitope-altering gene is It is a messenger. In another preferred embodiment of the present invention, the carbohydrate epitope of interest- The modified gene can be used to transfer the transgenic cells, tissues, organs and / or A functional H-transferase whose nucleic acid sequence is well known to those skilled in the art. It is co-expressed with the gene.   Carbohydrate epitopes-modified genes include, but are not limited to, carbohydrate Gene encoding a product epitope-altering enzyme. Preferred embodiment of the present invention In another embodiment, the carbohydrate epitope-altering enzyme is a functional αGalA enzyme. Other of αGalA The enzyme may be, for example, modified by modifying a cell surface carbohydrate epitope. Epitopes cause a reduction in the response of the human immune system to unmodified epitopes , Functional sialidase enzymes and lactose aminidase enzymes.   Further, carbohydrate epitope-modified genes include, for example, those whose expression is Elephant cell surface carbohydrate epitopes, such as Galα (1,3) Gal epi Antisense acts to inhibit transcription of genes required for tope production Nucleic acid sequences encoding oligonucleotide molecules are included. For example, Carbohydrate epitope-modified genes include α (Gal1,3) galactosyltrans Produced by a gene encoding a transferase enzyme such as a ferase enzyme Nucleic acid sequence encoding an antisense oligonucleotide complementary to the transcript to be Column.   Nucleic acid sequences encoding such carbohydrate epitope-altered genes can be obtained by those skilled in the art. Is well known. Carbohydrate epitopes of interest-nucleic acids encoding altered gene products If the sequence is unknown, the nucleic acid sequence may be used as an example, as described in Section 5.1.1 below. The use of αGalA nucleic acid sequences in accordance with standard techniques well known to those skilled in the art. Can be easily obtained.   The nucleic acid sequence encoding the carbohydrate epitope-altered gene product is It can be operably linked to a regulator that directs expression. As used herein Regulators that do so include, but are not limited to, inducible promoters and uninducible promoters. Induced promoters, enhancers, operators and appropriate cells and / or Manipulates and regulates expression of the coding sequence in a subcellular location, other known to those of skill in the art. Factors. As used herein, "suitable location" refers to a cell surface carbohydrate of interest. Alteration of the epitope, resulting in a human being caused by the altered epitope. Diminishes the immune response to the response caused by the unmodified epitope Mean cell and / or subcellular location of expression.   For example, to regulate the sequence encoding a carbohydrate epitope-altered gene Nucleotide regulatory sequences used include carbohydrate epitopes of interest-modified Regulatory sequences that are intrinsic (ie, usually associated) in the gene itself are included. is there Alternatively, a promoter not endogenous to the sequence encoding the carbohydrate epitope-altered gene Functional carbohydrate regulated by a promoter or promoter / enhancer complex -Comprising a sequence encoding nucleotides for the modified gene product A chimeric carbohydrate epitope-altered gene construct can be transformed with the transgenic cell, Genetically engineered as a transgene for use in producing tissues, organs and animals You can also. Align multiple copies of a gene or chimeric gene construct into a vector And multiple copies of the gene or chimeric gene construct can be transfected. It can also be introduced stably into genic cells or founder animals.   To produce a gene or chimeric gene construct for use in the present invention, one skilled in the art Recombinant DNA and cloning methods well known in the art can be used (Sambrook Et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Ha rbor Laboratory Press, NY). In this regard, the appropriate carbohydrate epitope -The sequence encoding the modified gene should be located at a convenient position in the sequence at a relevant position. Can be generated from cDNA or genomic clones using restriction sites You. Alternatively or in combination with the above method, for example, a single-stranded circular DNA molecule A vector such as M13 or phagemid capable of producing Used in combination with nucleotides and specific strains of E. coli (Kunkel, T.A. et al., 1 987, Meth. Enzymol. 154: 367-382), synthetic oligonucleotides and PCR Limerase chain reaction) (Ho et al., 1989, Gene 77: 51-59; Kamman, M. et al., 1). 989, Nucl. Acids Res. 17: 5404) using site-directed mutagenesis techniques To generate a sequence encoding the required carbohydrate epitope-modified nucleotides. It can also be done. Carbohydrate epitopes-modified nucleotide regulatory sequences It can be obtained from a genomic clone using techniques. Then isolate the appropriate sequence Cloned and used directly for the production of transgenic cells or animals be able to. The sequence may also be used to create a carbohydrate epitope-altered gene. Chimeric gene constructs that use regulatory sequences other than those endogenous are also described herein. Genetic engineering can also be performed using the methods described in. These chimeric genes Using the offspring construct for the production of transgenic cells or animals You can also.   The description given in 5.1.1 below is specific Epitope-modified gene αGalA, but the present invention is not limited to this. It is not something to be done. As will be appreciated, the general disclosure regarding this gene is Carbohydrate epitopes-equally applicable to modified genes as is . 5.1.1.αGalA gene   Any nucleic acid molecule that directs the expression of a functional αGalA gene product will Used as transgenes in the production of transgenic cells, tissues, organs and animals. Can be used. As described in Section 3 above, "functions" Specific αGalA ”or“ functional recombinant αGalA ”is a cell surface carbohydrate epitope Gal Human immunity caused by altered epitopes resulting from alteration of α (1,3) Gal The response of the system to the response caused by the unmodified Galα (1,3) Gal epitope ΑGa1A enzyme.   Such αGa1A genes include, but are not limited to, functional αGa1A Encoding prokaryotic species such as E. coli, and eukaryotic species, plants such as coffee And αGa1A gene sequences derived from human and non-human animal sequences Can be For example, the human αGalA amino acid sequence is well known. For example, U.S. Pat. See 356,804, which is incorporated herein by reference in its entirety.   It is known that homologs of the human αGalA gene sequence are present in other species. Array If not known, undue experimentation may be performed by molecular biology techniques well known in the art. Can be identified and isolated without performing For example, the isolated αGalA gene The child sequence is known to be labeled to express αGalA from the organism of interest. Constructed from mRNA obtained from a cell type expected to be expressed or expressed It can be used for screening a cDNA library. Hybridi In general, the conditions for the incubation are as follows. Low stringency when derived from organisms other than There will be. Alternatively, labeled fragments may also be prepared using appropriate stringency conditions. For screening genomic libraries derived from the organism of interest. Can be. Such low stringency conditions are well known to those skilled in the art. Expected that libraries and tag sequences will vary depending on the particular organism from which they are derived Is done. For guidance on 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 Pu blishing Associates and Wiley Interscience, N.Y.).   Furthermore, the previously unknown αGalA gene sequence is based on the known αGalA amino acid sequence. PC using two degenerate oligonucleotide primer pools designed It can be isolated by performing R. Reaction template expresses αGalA gene MRs generated from cell lines or tissues known or expected to It may be cDNA obtained by reverse transcription of NA. The PCR product is Sequence and determine the amplified sequence is the desired αGalA sequence That can be guaranteed. The PCR fragment is then subjected to various methods by various methods. It can be used to isolate DNA clones. For example, the amplified fragment Can be used for screening of Teriophage cDNA library . Alternatively, labeled fragments can be used to screen genomic libraries. it can.   PCR techniques can also be used to isolate full-length cDNA sequences. For example, R NA can be prepared according to standard methods using a suitable cell or tissue source, ie, Isolate from those known or predicted to express functional αGalA Can be Amplify on the RNA for priming of the first strand synthesis Using oligonucleotide primers specific for the most 5 'end of the fragment To perform a reverse transcription reaction. The resulting RNA / DNA hybrid is Then, using a standard terminal transferase reaction, ring The hybrid can be digested with RNaseH. And then second strand synthesis can be initiated with a poly-C primer . Thus, the cDNA sequence upstream of the amplified fragment can be easily isolated. . For a review of cloning methods that can be used, see, for example, Sambrook et al., 1989, Mo. lecularCloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y .; And Ausubel et al., 1989, Current Protocols in Molecular Biology, (Green Publ. ishing Associates and Wiley Interscience, N.Y.).   Note that, due to the degeneracy of the nucleotide coding sequence, In addition to those isolated by the technique, other αGalA DNA sequences are also functional αGalA genes. It should be understood that the product can be encoded. In particular, the functional αGalA gene May include a nucleic acid sequence encoding the amino acid sequence of a functional αGalA gene product. it can. For example, αGalA nucleic acid sequences can be used under conditions of high stringency (eg, 0. 5M NaHPO_FourIn 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, Hybridization to filter-bound DNA at 65 ° C, and 68 ° C, 0.1 x Washing with SSC / 0.1% SDS (Ausubel F.M. et al., eds., 1989, Current Protocols in Mole cular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley  & Sons, Inc., New York, p. 2.10.3)), for example, US Pat. No. 5,356,804 Known αGalA gene coding sequence, such as the human αGalA gene sequence disclosed in Nucleic acid sequence that hybridizes to the complement and encodes a functional αGalA gene product And / or under less stringent conditions, eg, moderate stringency Under the conditions of a gently (for example, washing at 42 ° C., 0.2 × SSC / 0.1% SDS (Ausubel Et al., 1989, supra)), but still produce a functional αGalA gene. A nucleic acid sequence encoding the product. 5.2.Production of transgenic animals   Without limitation, mice, rats, rabbits, guinea pigs, pigs, miku Micro-pig and non-human primates (eg, baboons, squirrel monkeys and chicks) Any kind of animal (such as a pancreas) can be used to produce the transgenic animals of the present invention. Pigs and micropigs are preferred.   The transgenic animal has at least one transgene (t A non-human animal containing a foreign gene called ransgene). In this example, Is a carbohydrate epitope-altered gene. Preferably, the introduction remains The gene is included in the animal's reproductive system and is therefore transmitted to the animal's offspring. Is available. In such cases, the animal may be found in a `` founder '' Things ".   The transgenic animal has the transgene in all or not all of its cells. Can be contained in a few cells (ie, a transgenic animal can It may be genetically mosaic. ). For example, Jacobovits, 1994, Curr. See Biol., 4: 761-763. But for xenotransplantation Means that the cells, tissues or organs to be introduced into the human recipient Product epitope-must contain and express the modified gene.   The transgene can be a single transgene or a concatamer (eg, head-to-tail) Tandem or head-to-head tandem). The transgene also For example, Lasko et al. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6. 236), it can also be selectively introduced into specific cell types and activated. . The regulatory sequences required for such cell type-specific activation are specific to the particular control being considered. It depends on the cell type and will be apparent to those skilled in the art.   Any method known in the art can be used to introduce a transgene into an animal and It can be used to create a founder line of nick animals. With such a method Include, but are not limited to, pronuclear microinjections (Hoppe, PC and Wagner, T .; E., 1989, U.S. Patent No. 4,873,191); raw Retrovirus-mediated gene targeting to inbreeding lines (Van der Putten et al., 198 5, Proc. Natl. Acad. Sci., USA82: 6148-6152); Gene targeting in embryonic stem cells (T hompson et al., 1989, Cell.56: 313-321; Wheeler, M.B., 1994, WO 94/26884, Are incorporated herein by reference in their entirety); electroporation of embryos ( Lo, 1983, Mol Cell. Biol.Three.: 1803-1814); cells gkun; transfection Transduction; retrovirus infection; adenovirus infection; adenovirus-associated Infection; liposome-mediated gene transfer; naked DNA transfer: and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell57: 717-723) and the like. Of such a way For a review, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.11 Five : 171-229, which is incorporated herein by reference in its entirety.   Once founders are created, they can be used to colonize certain animals. For example, it can be crossed, homologous, out-bred or cross-bred. That's it Examples of such mating methods include, but are not limited to, the establishment of various strains. Outcrossing of founders with more than one integration site; additional expression of each transgene Combined transgenics expressing transgenes at higher levels for current effects Homologous breeding of various strains to produce E. coli; increased expression and DNA analysis To eliminate the need for animal screening by Crossover of heterozygous transgenic animals to produce homozygous animals; Crossing of various heterozygous lines to produce complex heterozygous or homozygous lines Difference; can examine the effect of modifying the allele on transgene expression Crossbreeding of animals into various inbred genetic backgrounds to include You.   Among the preferred transgenic animals are transgenic ungulates, Examples include, but are not limited to, transgenic pigs. Such a tiger Methods for constructing transgenic animals are well known to those skilled in the art. For example, International Application No. WO 94/26884 and WO 95/047 44 (incorporated herein by reference in their entirety). 5.3.Transgenic cells, tissues and organs   Transgenic cells include, but are not limited to, transgenic cells. Bone marrow cells, peripheral blood stem cells, hepatocytes, kidney cells, blood island cells, etc. Should be considered within range. In addition, tissues or organs are But not liver, kidney, muscle, lung, pancreas, skin thyroid, parathyroid, adrenal cortex, adrenal Any of the renal medulla, thymus, cartilage, bone, etc. should be considered within the scope of the present invention. You.   The transgenic cells, tissues and organs of the present invention can be any of a variety of It can be produced by a method. For example, the transgenic cells of the invention , Tissues and organs can be obtained from the transgenic animals described in Section 5.2 above. Can be.   With respect to transgenic cells, they are derived from the transgenic animals of the present invention. Primary cultures of the cells to be derived can be used, or preferably, Progenitor cell lines can be obtained. Such continuous cell lines are known in the art. Methods, for example, Small et al., 1985, Mol. Cell. Biol.Five: Use the method described in 642-648 Can be obtained.   In addition to obtaining cells from the transgenic animals of the invention, the cells of interest Type of cell that expresses a functional carbohydrate epitope-modified gene product in the cell Transfected with a carbohydrate epitope-altering sequence that can Light transgenic cells can also be obtained. Transgenic nuclei of cells Transfection with an acid sequence is performed by standard methods, for example, as described in Section 5.2 above. It can be performed using the method described above. Further, for example, Ausubel, 1989 , Current Protocols in Molecular Biology, (Green Publishing Associates a nd Wiley Interscience, N.Y.). Transfected cells are Presence of sgenic nucleic acid sequence, transgenic carbohydrate epitope-modified The expression and accumulation of the gene product should be evaluated. In addition, the transgenic cells can be modified cell surface carbohydrates The ability to show pitopes should be evaluated. 5.4.Selection and characterization of transgenic cells, tissues, organs and animals   Transgenic cells produced according to the methods detailed in sections 5.2 and 5.3, Use tissues, organs and animals as a suitable xenograft or xenograft source Screening and evaluation to select cells, tissues, organs and animals that can be Value should be done.   Initial screening is based on the fact that transgene integration has occurred. It can be performed by means of Zan blot analysis or PCR. Transgenic Carbohydrate epitopes in cells, tissues, organs and animals-modified gene mRN The level of A expression is also, but not limited to, Northern blot analysis of the sample. , In situ hybridization analysis and reverse transcriptase-PCR (rt-PC R) and the like.   mRNA or protein (detected immunocytochemically using appropriate antibodies) Carbohydrate epitope-modified transgene expressing at easily detectable levels Genetic cells, tissues, organs and / or animals are then treated with the modified cell surface Further evaluation should be performed to identify animals displaying carbohydrate epitopes. For example, the histopathological evaluation of transgenic material is based on the cell surface Use antibodies specific for pitopes and combine them with standard techniques well known to those skilled in the art. Let's do it. 5.5.Use of transgenic cells, tissues, organs and animals   The transgenic cells, tissues, organs and animals of the present invention can be used in vitro and It can serve a number of functions, both in vivo. For example, this transgenic Material can be xenograft material or xenograft It can serve as a source of material. Xenograft of the transgenic material of the present invention Use as a measure of the level of severe rejection (HAR) observed in human recipients A human recipient receiving a non-transgenic cell, tissue and / or organ. Lower than the level of HAR observed in the recipient, Helps reduce the level of rejection.   Alternatively, certain transgenic cells of the invention, ie, bone marrow cells, It can be used for the production of red blood cells with altered BO phenotype. That is, red blood cells of blood group B Can be converted into type O red blood cells, which are a group of donors of the whole blood type.   For xenografts using the transgenic material of the invention, the materials provided Any technique for transplanting into recipients is available. Such techniques are known to those skilled in the art. It is well known. The transfer method includes, for example, the cells described in section 5.3 above (these cells). (Including, but not limited to, blood cells and bone marrow cells) Tissues and organs as listed in section 5.3 (heart, liver, lung and kidney tissues and And / or organs). 6. Example:Hemagglutination is reduced by α-galactosidase A treatment   Examples shown here demonstrate that αGalA results in reduced hemagglutination I do. 6.1.Materials and methods Hemagglutination assay: Washing rabbit erythrocytes (Galα (1,3) Gal +) in PBS And resuspended in a 2% (v / v) suspension. Cells can be left untreated or at 37 ° C Two hours, various concentrations (see legend in FIG. 1) of human α-galactosidase A or Treatment with α-galactosidase A from E. coli (Boehringer Mannheim, Germany) Was. Wash cells and allow red blood cells to clot Collection assay was performed using the IB4 lectin (Griffonia simplicifolia(Sigma, St. . Louis, MO; Hayes, C.E. And Goldstein, I.J., 1974, J.M. Biol. Chem.249: 1904) was diluted in a 50 μl aliquot with a microtiter plate. Α-galactosidase A-treated erythrocytes and untreated erythrocytes The end-point titer of hemagglutination was measured after 2 hours. Was. 6.2.result   A terminal galactose residue is cleaved from Galα (1,3) Gal of α-galactosidase A The ability to cleave was examined by using rabbit erythrocytes as targets. Ceramide pentahexoside on the surface of erythrocytes is Galα (1,3) Gal (Galil, U. et al., 1988, J. Biol. Chem.263: 17755). Galα (1,3) Gal-specific lectinGriffonia simplicifoliaIB4 (Hayes, C.E. and Goldstein, I.J., 1974, J.E. Biol. Chem.249: 1904) with α- It was used as an indicator of antigen density before and after galactosidase A treatment. Untreated red blood cells Aggregated with the use of 0.98 ng / ml lectin (FIG. 1A). Red blood cells After treatment with E. coli α-galactosidase A, significantly more After lectin is required and erythrocytes are treated with 150 U of human α-galactosidase A Requires 7.81 ng / ml lectin and 15.63 ng / ml after treatment with 300 U. , And 125 ng / ml after treatment with 600 U (FIG. 1a). Similar results, It is also obtained after treating erythrocytes with bacterial α-galactosidase A and treated with 6.25 U. After processing, 62.5 ng / ml is required, and after 25 U processing, 125 ng / ml, and 50 U processing. After the treatment, it was 250 ng / ml (FIG. 1b). That is, erythrocytes are converted to α-galactosidase. Treatment with ZeA reduces the level of Galα (1,3) Gal on the cell surface up to 255-fold, This shows that the method is suitable for reducing the amount of antigen on erythrocytes. 7. Example:Expression of α-galactosidase A cDNA reduces Galα (1,3) Gal Bring   In the examples shown here, cells transfected with αGalA cDNA were Untransfected levels of the surface Galα (1,3) Gal carbohydrate epitope Demonstrates reduction compared to cells. 7.1.Materials and methods cDNA, transfection and serology: The plastic used in these studies Sumid was expressed in a mammalian expression vector p91023 (B) using human α-galactosidase AcD. P91-AGA encoding NA (Ioannou, Y.A., et al., 1992, J. Cell Biol.119: 1137 ); Against the human α-galactosidase A cDNA with the mammalian expression vector pCDNA1 phAGA (Invitrogen) encoding cDNA; porcine α (1,3) galactosyl tra PPGT-3 (referred to as ppGT), which encodes a transferase cDNA (Sandrin, M.S. And McKenzie, IFCC, 1994, Immunol. Rev.141: 169) and human CD48 Encoding pHuLy-m3.7 (referred to as pCD48) (Vaughan, H.A. et al., 1991, Immunogenetic s33: 113), which are based on standard methods (Ausubel, FM et al., 1994, Current Pr. otocols in Molecular Biology, Wiley-Interscience, New York). Made. COS-7 cells were obtained from Dulbecco's Modified Eagle's Medium (DMEM) (Trace  Biosciences Pty. Ltd., Castle Hill, NSW, Australia) AE-dextran method (Vaughan, H.A. et al., 1991, Immunogenetics)33: 113) And supplemented with 10% Nu-serum (Collaborative Research Inc., Bedford, Mass.) Transfect using DMEM (1-20 μg DNA / 10 cm dish) These cells were examined 48 hours later for cell surface expression. Cell surface carbohydrate epitotes Direct Fluorescence of the Group Galα (1,3) Gal Using FITC-Binding IB4 Lectin went. Cell surface staining of CD48 in control transfections showed CD48 (ASH1360, Austin Research Institute) and FITC-conjugated goat anti-mouse IgG A specific moclonal antibody (mAb) was used. Human α-galactosida Expression ofase A was determined by apoptosis of cells permeated with Triton X-100 after formaldehyde fixation. Initiatively purified rabbit anti-α-galactosidase A antibody (Ioannou, Y.A. et al. 1992, J.A. Cell Biol.1191137), then FITC-conjugated goat anti-rabbit IgG Was evaluated by internal staining. Fluorescence was detected by microscopy.   α-Galactosidase A and protein assays: Wash cells twice with PBS And 1% Triton X-100 / sodium phosphate (pH 7.0) / 150 mM NaCl on ice Dissolved in 1 mM EDTA buffer containing / protease inhibitor for 20 minutes. Meltdown The product was centrifuged at 13,000 g for 15 minutes at 4 ° C., and the supernatant was collected, and α-galactosidase A activity Use p-nitrophenyl-α-D-galactoside as substrate (Kint, J.A., 1970, Science270: 1268). Protein concentration The assay was performed using the bovine serum albumin as a standard, using the Bradford assay. (Bradford, M.M., 1976, Anal. Biochem. 72:248). 7.2.result   Transfection of cells with human α-galactosidase A cDNA To test if Galα (1,3) Gal levels can be reduced, α-galactosidase A is replaced with C-expressing α-galactosyltransferase. Co-expressed in OS cells. 2.5 μg α (1,3) galactosyl transferer IB4 (specific for Galα (1,3) Gal) COS cells stained with a typical lectin) show approximately 60% of the cell surface development of Galα (1,3) Gal. (FIG. 2), while only α-galactosidase A cDNA (2.5 μg) Expressing cells did not show surface staining with IB4 (FIG. 2). α (1,3) Gala Ctosyltransferase cDNA + α-galactosidase A cDNA (2. 5 μg of cDNA) As a result, IB4 staining was significantly reduced by 50% (FIG. 2), and co-expressed α-galactosida Increasing the amount ofase A cDNA to 12.5 μg further reduced it by 25%, or 75%. The bottom was confirmed. α (1,3) galactosyltransferase and control cDN IB4 stained cells co-transfected with A (human CD48) Similar to cells expressing only lactosyltransferase (ie, 60 %), Which is the low level observed in IB4 staining with α-galactosidase A. The bottom shows the change in the cell surface level of Galα (1,3) Gal by α-galactosidase A. Accurately, not just the result of the co-transfection method Is shown.   α-Gala that is expressed in cells transfected with α-galactosidase A To determine the level of custosidase A, the lysate was treated with p-nitrophenyl- Use of α-D-galactoside as a substrate for α-galactosidase A And assayed by In lysates from mock transfected cells α-Galactosidase A activity was 15 nmol / h / mg protein (FIG. 3). α Lysates from cells transfected with galactosidase A cDNA alone Shows enzymatic activity at 48 nmol / h / mg protein, which corresponds to α (1,3) galactosyl Co-transfected with transferase and α-galactosidase A Similar to cell-derived lysate (2.5, 5 and 12.5 mg of α-galactosidase 38, 35 and 56 nmol / h / mg protein, respectively, for zeA cDNA) (FIG. 3) . Thus, in all cases of α-galactosidase A transfection, Α-galactosidase A activity is less than background levels. Is three times higher, and α-galacto in transiently transfected COS cells. It was confirmed that Sidase A was expressed and active. These findings indicate that c The expression of α-galactosidase A after DNA transfection was , 3) indicate that Gal levels can be significantly reduced. 8. Example:Co-expression of α-galactosidase A and H transferase is Causes a cumulative decrease in Galα (1,3) Gal   In the example shown here, co-expression of αGalA and H transferase was It has been demonstrated that the expression of the cell surface carbohydrate epitope Galα (1,3) Gal is cumulatively reduced. Testify. 8.1.Materials and methods   The method used here is as described in section 7.1 above. 8.2.result   Expresses human H transferase both in vitro and in vivo Stable down-regulation of the Galα (1,3) Gal epitope in cells has been previously reported. Was. The effect observed by α-galactosidase A has an effect on H transferase. To determine whether it is independent of the resulting reduction in expression, A series of co-transfection experiments was performed. COS cells were transformed into (i) α (1,3) Galactosyltransferase + H transferase cDNA; (ii) α (1 , 3) galactosyltransferase + α-galactosidase A cDNA; Is (iii) α (1,3) galactosyltransferase + H transferase + α -Transient co-transfection with galactosidase A cDNA on the cell surface Was stained with IB4 or UEA1, and the permeated cells were reacted with α-galactosidase A. And stained.   α (1,3) galactosyltransferase cDNA + α-galactosidase A In cells expressing cDNA, α (1,3) galactosyltransferase cDN A significant decrease in IB4 staining was confirmed compared to cells expressing only A . The decrease in IB4 staining was due to α (1,3) galactosyltransferase c Reduction seen in cells co-transfected with DNA + H transferase Was smaller than. α (1,3) galactosyltransferase + H transfer Rase + α Cells co-transfected with galactosidase A cDNA are essentially No B4 staining was shown. That is, the staining level was mock transfected. It was almost the same as the cells. α-galactosidase A or H transferase Control transfection with cDNA alone was performed using anti-α-galactosidase. Strong staining with antibody A or UEA1, but no cross-reactivity with IB4 Was. These results indicate that H-transferase and α-galactosidase A Has an additive effect on the ability to reduce the expression of Galα (1,3) Gal on the cell surface It clearly states that   Similar effects can be seen in cells that constitutively express Galα (1,3) Gal To address the question of whether human α-galactosidase A is A constant transfection product is identified as the porcine endothelial cell line P, which is Galα (1,3) Gal +. Generated using IEC. Galα (1,3) Gal levels in these cells are reduced I was This indicates that overexpression of α-galactosidase A reduces this epitope. It is an effective way to make it work. 9. Example:α-Galactosidase A transgenic expression is complement mediated cytotoxicity Reduce gender   In the example shown here, the expression of αGalA is reduced as a result of the expression, The problem of severe rejection in xenograft methods in that a dramatic decrease in bell is observed Demonstrate an effective solution to 9.1.Materials and methods Complement lysis assay: Α-galactosidase A, α (1,3) galactosyltrans COS-7 cells transfected with ferase and H-transferase Complement-mediated lysis was described previously (Vaughan, H.A. et al., 1994, Transplantation58: 8 79), and grow the cells in a 96-well plate. ence), wash and calcein Exposure to AM (Molecular Probes Inc.) (final 10 μM) for 30 minutes, then complement source For 30 minutes at 37 ° C. in the presence of whole human serum. Released from cells The dye to be used is coupled to a Millipore Cytofluor 2350 fluorescent plate reader (excitation 490 nm). 1% SDS cell lysate, measured using 530 nm emission and total dye bound to cells And specific dye release was calculated as a percentage of the total.   Other method: Other methods were as described in section 7.1 above. 9.2.result   Porcine α (1,3) galactosyltransferase cDNA clone of COS cells Transfection as detected by IB4 lectin binding. This results in the expression of Galα (1,3) Gal on the cell surface of some cells (（60%), These cells also became strongly reactive with natural antibodies in human serum. α (1,3 COS cells transfected with galactosyltransferase cDNA Susceptibility to lysis by human serum⁵¹Using Cr release assay Examined. As a result, after treatment with human serum, Galα (1,3) Gal⁺COS cells Specific lysis at 62% compared to 5% background lysis of these cells Was found to have occurred (FIG. 4). Transfected COS cells bind IB4 Was also tested and found to be 65% positive. That is, the dissolution level is It was proportional to the number of cells expressing the Galα (1,3) Gal epitope. α (1,3) galactosyl CDNA clone encoding transferase + α-galactosidase A In transfected COS cells, 17% lysis was observed and α (1,3) In the case of tosyltransferase + H transferase, 9% lysis was observed. Was done. When transfecting COS cells with all three cDNA clones, A background lysis of 4% was observed. In contrast, human serum is trans Untransfected COS cells or α-galactosidase A only, Spherase only or mock transfected C It did not cause significant lysis of OS cells above background. Human blood Galα (1,3) Gal in the presence of Kiyoshi's dilution up to> 1/256⁺Specific lysis of COS cells Only, α (1,3) galactosyltransferase up to a dilution of 1/64 COS cells expressing + α-galactosidase A, α (1,3) G up to 1/8 dilution Lysis of cells expressing lactosyltransferase + H transferase Admitted. 10. Example:Transgenic αGalA expression in pig cells To Gal Decrease in α (1,3) Gal level   The examples presented in this section use transgenic αGalA expression in porcine cells. The following shows an example in which the cell surface level of Galα (1,3) Gal was successfully reduced using the method described above. 10.1.Materials and methods   10.2 below. The techniques used in the studies described in Sections are based on the methods and techniques described in Sections 6-9 above. And standard protocols were followed. 10.2.result   The results described here show that α-galactosidase in the transgenic method Further exemplifies the efficacy on the removal of Galα (1,3) Gal by use of Ze.   In particular, research has shown that Galα (1,3) Gal⁺Α-Galact in the porcine endothelial cell line PIEC To determine whether expression of the sidase cDNA alters the cell surface expression of Galα (1,3) Gal. I went to see.   Expresses human α-galactosidase under the cytomegalovirus promoter A stable cell line was generated. The cell line is a standard calcium phosphate transfer And neomycin selection. Natural human anti-Galα (1, 3) Determine the binding ability of cells to Gal antibody When tested by flow cytometry analysis, as shown in FIG. Antibody binding was shown to be 10-fold smaller than EC cells, indicating that the cell surface Galα (1,3) Gal Shows a significant decrease in   The results show that COS cells expressing human α-galactosidase were transformed into Galα (1,3) Ga l One of the results shown in Section 7 above, which showed a decrease in cell surface expression levels Match. 11. Example:Galα (1,3) G in vivo by α-galactosidase al Decline   In the examples provided in this section, the use of transgenic human α-galactosidase is described. The following shows an example in which the in vivo level of Galα (1,3) Gal was successfully reduced using the method described above. 11.1.Materials and methods   11.2 below. The techniques used in the studies described in Sections are based on the methods described in Sections 6-9 above. And standard protocols were followed. 11.2.result   H2-K^bSome express human α-galactosidase under the promoter A transgenic mouse line was generated using standard techniques. Human α-gala Results from C57BL / 6 mice heterozygous for the custosidase gene Indicated that the transgene was inserted into the genome and transmitted between generations. Tran Alpha-galactosidase enzyme levels in plasma of sgenic mice are Values at least four times higher than those measured in transgenic littermates were measured. (FIG. 6).   The level of Galα (1,3) Gal in peripheral blood lymphocytes from each transgenic line, Stain the cell surface with IB4 (lectin specific for Galα (1,3) Gal) Tested by measurement by typical flow cytometry. Fig. 7 shows the results. As indicated, the level was the control non-transgenic It is expressed as a percentage (%) of the littermate mouse to IB4 staining.   The transgenic mice are dependent on their Galα (1,3) Gal level, depending on the system tested. Showed a 34% to 50% reduction in This means that transgenic αGalA Demonstrate the successful in vivo reduction of epitopes by the use of   As will be apparent, many modifications and variations to the invention described herein are It is possible without departing from the spirit and scope of the invention. Implement specific embodiments Although shown by way of example only, the present invention is limited only by the accompanying claims. It is what is done.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12P 21/02 A61K 45/00 // A61K 38/46 C12N 5/00 B 45/00 A61K 37/54 ( 72) Inventor Ionnu, Janis United States 10128 New York, NY 96 East 96th Street 306 (72) Inventor Desnick, Robert. Jay United States 10012 New York, New York, West 4 T. Street 329 (72) Inventor Sandrin, Morrow, S. Australia 3056 Victoria, Brunswick, Berkeley Street, 211 (72) Inventor Mackenzie, Ian, F , Sea. Australia 3056 Brunswick, Victoria, Brunswick Road, 359

Claims (1)

[Claims] 1. Functional carbohydrate epitopes that alter cell surface carbohydrate epitopes-modified variants Transgenic carbohydrate epitope-modified nucleic acid sequence directing expression of a gene product Transgenic cells containing rows. 2. The transgenic carbohydrate epitope-modified nucleic acid sequence has a functional αGa 2. A transgenic αGalA nucleic acid sequence that directs expression of the lA enzyme. A transgenic cell as described. 3. In addition, an H-transcript that directs the expression of a functional H-transferase gene product 2. The transgenic cell of claim 1, comprising a spherase nucleic acid sequence. 4. 2. The cell surface carbohydrate epitope of claim 1, wherein the cell surface carbohydrate epitope is Galα (1,3) Gal. Transgenic cells. 5. A trans comprising the transgenic cell according to claim 1, 2, 3, or 4. Genic organization. 6. A trans comprising the transgenic cell according to claim 1, 2, 3, or 4. Genic organ. 7. A trans comprising the transgenic cell according to claim 1, 2, 3, or 4. Genic animals. 8. 4. The transgenic cell is a porcine cell. 5. The transgenic cell according to 4. 9. Wherein the transgenic tissue is a transgenic pig tissue. Item 6. The transgenic tissue according to Item 5. Ten. The transgenic organ is a transgenic pig organ. Item 7. The transgenic organ according to Item 6. 11． The method according to claim 7, wherein the transgenic animal is a transgenic pig. The transgenic animal as described. 12． The transgenic cell according to claim 1, 2, 3 or 4, is used as a human recipient. And consequently, the level of severe rejection observed in the human recipient is reduced by the Cells without transgenic nucleic acid sequences Lower than the level of severe rejection observed in human recipients of A xenotransplantation method comprising: 13. Introducing the transgenic tissue of claim 5 into a human recipient, The severe rejection level observed in the human recipient is the transgenic Severe rejection observed in human recipients receiving cells that do not contain nucleic acid sequences A xenograft method comprising lowering compared to a level. 14. Introducing the transgenic organ according to claim 6 into a human recipient, and consequently The severe rejection level observed in the human recipient is the transgenic Severe rejection observed in human recipients receiving cells that do not contain nucleic acid sequences A xenograft method comprising lowering compared to a level. 15． The transgenic animal of claim 7 is introduced into a human recipient, and the result is The severe rejection level observed in the human recipient is the transgenic Severe rejection observed in human recipients receiving cells that do not contain nucleic acid sequences A xenograft method comprising lowering compared to a level.