EP0769902A4 - Verfahren zur verringerung der hyperakuten abstossung von xenotransplantaten - Google Patents

Verfahren zur verringerung der hyperakuten abstossung von xenotransplantaten

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Publication number
EP0769902A4
EP0769902A4 EP95923864A EP95923864A EP0769902A4 EP 0769902 A4 EP0769902 A4 EP 0769902A4 EP 95923864 A EP95923864 A EP 95923864A EP 95923864 A EP95923864 A EP 95923864A EP 0769902 A4 EP0769902 A4 EP 0769902A4
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EP
European Patent Office
Prior art keywords
cell
cells
gal
antibodies
genetically altered
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP95923864A
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English (en)
French (fr)
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EP0769902A1 (de
Inventor
Mauro S Sandrin
William L Fodor
Russell P Rother
Stephen P Squinto
Ian F C Mckenzie
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Austin Research Institute
Alexion Pharmaceuticals Inc
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Austin Research Institute
Alexion Pharmaceuticals Inc
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Application filed by Austin Research Institute, Alexion Pharmaceuticals Inc filed Critical Austin Research Institute
Publication of EP0769902A1 publication Critical patent/EP0769902A1/de
Publication of EP0769902A4 publication Critical patent/EP0769902A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • This invention relates to xenotransplantation. More specifically, the invention relates to methods that will prevent or reduce hyperacute rejection of xenogeneic cells, tissues and organs following transplantation into human recipients.
  • the invention provides methods for stably reducing the expression on the surface of a xenogeneic cell of the non-human antigen known as galactose 0.(1,3) galactose. This prevents the phenomenon of antibody-dependent rejection of xenogeneic cells that typically follows exposure to human blood, plasma, or serum (e.g., following xenotransplantation into a human patient) as a result of the binding of preformed natural human antibodies to the surfaces of such cells.
  • Xenotransplantation Surgical problems related to the transplantation of allogeneic organs (i.e., organs from donors of the same species as the transplant recipient) , such as kidney, liver, heart, lung and pancreas, have been largely solved, and immunosuppression has been improved such that these procedures are now routinely performed with a high degree of success (Brent, 1991) .
  • allogeneic organs i.e., organs from donors of the same species as the transplant recipient
  • immunosuppression has been improved such that these procedures are now routinely performed with a high degree of success (Brent, 1991) .
  • a major problem in transplantation medicine today is the provision of sufficient allogeneic donor organs to satisfy the large numbers of patients awaiting a transplant. Given the increasing emphasis on the costs of dialysis and hospitalization incurred by patients awaiting transplantation, there is even greater emphasis on the transplantation of donor organs early in the course of disease.
  • This animal is commonly used commercially, and therefore its use will engender fewer ethical problems than the use of primate donors. Furthermore, the pig is considered a highly suitable donor for anatomical and physiological reasons (Cooper et al., 1991 and Niekrasz, et al., 1992).
  • Immunolocfical Rejection of Xenografts The rejection of transplanted cells, tissues, or organs may involve both an extremely rapid hyperacute rejection (HAR) phase and a slower cellular rejection phase.
  • HAR hyperacute rejection
  • xenogeneic organs, tissues, or cells referred to herein as "xenogeneic" organs, tissues, or cells, or "xenotransplants", or "xenografts”
  • xenogeneic organs, tissues, or cells referred to herein as "xenogeneic" organs, tissues, or cells, or “xenotransplants", or "xenografts”
  • xenogeneic organs, tissues, or cells bind to donor cells, e.g., endothelial cells, and activate attack by the complement arm of the human immune system (Dalmasso, et al., 1992; and Tusso, et al., 1993).
  • HAR is the most significant impediment to the successful xenotransplantation of most cells and tissues, and of all vascularized organs.
  • Methods for the control of the HAR are available. These include interference with the antibody antigen reactions responsible for initiating the HAR response, either by removing the preformed natural antibodies from the circulation or by interference with the binding of the natural antibodies to their specific epitopes (see copending U.S. application Serial No. 08/214,580, entitled “Xenotransplantation Therapies", filed by Mauro S. Sandrin and Ian F.C. McKenzie on March 15, 1994, and PCT publication No.
  • a particularly desirable approach to the prevention of hyperacute rejection is to delete or inhibit the ⁇ .(l,3) galactosyltransferase gene in xenogeneic cells, and to thus eliminate or significantly reduce expression of Gal o-(l,3) Gal epitopes on the surface of such cells (see copending U.S. patent application Serial No. 08/214,580, supra) .
  • This approach eliminates or reduces the binding of preformed natural human antibodies to the xenogeneic cells and, therefore, prevents or reduces the activation of complement and subsequent hyperacute rejection of xenogeneic cells, tissues and organs.
  • Inhibition of complement attack on the xenotransplant may be accomplished by several means, including the use of complement inhibitors such as the 18kDa C5b-9 inhibitory protein and monoclonal antibodies against human C5b-9 proteins as disclosed in U.S. Patent No. 5,135,916, issued August 4, 1992.
  • Immunohistological analysis of hyperacutely rejected xenotransplants reveals antibody deposition, complement fixation, and vascular thrombosis as well as neutrophil infiltration (Zehr, et al., 1994; Auchincloss, 1988; Najarian, 1992; Somervile and d'Apice, 1993; and Mejia-Laguna, et al., 1972).
  • HAR and Xenoantigens The targets of natural human antibodies have been the subject of investigations for a number of years, as the identification of these xenoantigens would enable the development of strategies to circumvent hyperacute rejection of xenografts.
  • Galili and colleagues have shown that a large proportion of IgG (1%) in human serum is directed against the Gal or(1,3) Gal epitope expressed as part of a variety of glycosylated molecules found on both cell surfaces and on secreted glycoproteins (Galili et al., 1984; and Thall and Galili, 1990) .
  • This disaccharide epitope is found in all mammals except humans and Old World primates, and naturally occurring preformed anti-Gal 0.(1,3) Gal antibodies are found only in humans and Old World primates, i.e., those species which do not themselves express the epitope (Galili et al., 1987 and Galili et al., 1988) .
  • HAR and Preformed Natural Antibodies The immunoglobulin class of an anti-Gal 0.(1,3) Gal antibody determines the biological role of that antibody in hyperacute rejection.
  • Bach and Platt (Platt et al., 1990; Platt and Bach 1991; Platt et al., 1991; and Geller et al., 1993) consider that IgM is the most important class of immunoglobulin involved in hyperacute xenograft rejection.-
  • Sugars such as melibiose (a disaccharide containing a terminal galactose in an a linkage) coupled to a carrier such as SEPHAROSE can be used to purify anti-Gal o.(l,3) Gal antibodies (Galili et al, 1984 and Galili et al., 1985).
  • human serum was passed over the carrier-sugar matrix in order to prepare serum from which the antibodies reactive with the sugar were removed.
  • the results of testing the cytolytic activity of the sera prepared in these experiments indicate that the majority of the cytotoxic antibodies were removed from the serum by these means (Sandrin et al., 1993A; Sandrin et al., 1993B) .
  • Mammalian cells display a complex variety of carbohydrate antigens on their surfaces.
  • Carbohydrate epitopes are expressed on all mammalian cells by membrane glycoproteins and glycosphingolipids. Profound changes in the structures of these glycoconjugates frequently accompany important biological processes such as differentiation and development. The types and numbers of carbohydrate epitopes present on cells vary in different species and in different tissues within a given species (Yamakawa and Nagai, 1978) .
  • the glycosyltransferases comprise a family of enzymes that transfer sugars from nucleoside diphosphate- sugar conjugates (donor molecules) to acceptor substrate molecules, forming covalent linkages.
  • Acceptor substrates are often olig ⁇ saccharides or oligosaccharide moieties of larger molecules, but may also be specific proteins or lipids.
  • Glycosyltransferases function in a sequential manner, such that the oligosaccharide product of a transferase activity often becomes the acceptor substrate for subsequent transferase activity.
  • the final result generally contains a linear and/or branched polymer of component monosaccharides linked to one another.
  • Glycosyltransferases differ from each other with respect to the nature of the nucleoside diphosphate- carbohydrate donor, the nature of the acceptor substrate, and the glycosidic linkage joining the donor sugar to the acceptor substrate (reviewed by Beyer and Hill, 1982) .
  • glycosyltransferases include the following: galactosyltransferases , fucosyltransferases, sialyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, glucosyltransferases, sulfotransferases, acetylases, and mannosyltransferases.
  • the galactosyltransferases are examples of glycosyltransferases that transfer galactose from a UDP- galactose donor molecule to an acceptor substrate.
  • One such galactosyltransferase UDP-Gal:Gal 0(1,4) Gal NAcGlc c.(l,3) galactosyltransferase (also referred to as 0.(1,3) Gal transferase) , is a Golgi membrane-bound enzyme that catalyzes the following reaction:
  • Gal o.(l,3) Gal transferase and the Gal c.(l,3) Gal 01-R show both species and tissue- specific expression (Galili et al., 1988).
  • the 0.(1,3) Gal transferase is widely expressed in a variety of ⁇ iammalian species, with the notable exception of Old World primates and humans. These mammals do not express the enzyme due to frameshift and nonsense mutations in their genomic sequences encoding this enzyme (Larsen et al., 1990a).
  • Several carbohydrates, including those associated with the H antigen, contain the terminal structure Fucose 0.(1,2) Galactose.
  • the synthesis of the Fucose Q.(1,2) linkage is catalyzed by specific 0.(1,2) fucosyltransferase enzymes.
  • the enzymatic activities of these transferases result in the covalent attachment of L-fucose by an o.(l,2) linkage to a variety of acceptor molecules.
  • the H transferase for example, is a fucosyltransferase that catalyzes a transglycosylation reaction covalently linking a fucose to a specific oligosaccharide acceptor substrate.
  • the fucose is derived from the nucleotide sugar donor molecule GDP-fucose and connected by an o.(l,2) linkage to the Galactose residue of Gal 0(1,3) GlcNAc-R or Gal 0(1,4) GlcNAc-R acceptor substrates (i.e., galactose linked to N-acetylglucosamine in a 0(1,3) or a 0(1,4) linkage, where R represents a glycoprotein, protein, glycolipid, or lipid) .
  • acceptor substrates are also the acceptor substrates for the c.(l,3) Gal transferase discussed above, although each transferase utilizes a different nucleotide sugar donor molecule (UDP galactose for 0.(1,3) Gal transferase vs. GDP fucose for H transferase) .
  • UDP galactose for 0.(1,3) Gal transferase vs. GDP fucose for H transferase
  • the o.(l,3) Gal transferase and the H transferase have now been cloned (see copending U.S. patent application Serial No. 08/214,580, supra: Stanley, 1992; and Lowe, 1991) .
  • xenogeneic cells are genetically modified so that they express the glycosyltransferase activity of an exogenous glycosyltransferase (i.e., a glycosyltransferase encoded by a recombinant nucleic acid molecule introduced into - li ⁇
  • the genetically-modified xenogeneic cells of the invention exhibit reduced levels of the xenoantigen Gal 0.(1,3) Gal on their cell surfaces.
  • the genetically- modified xenogeneic cells of the invention are inhibited from binding to preformed naturally occurring human antibodies and are therefore significantly less sensitive to HAR as demonstrated by reduced sensitivity to activation and/or lysis by human complement. In this way, when transplanted into human patients, the rejection of such cells by complement-mediated hyperacute rejection mechanisms is reduced or prevented.
  • the invention provides a method for reducing rejection of a xenogeneic cell following transplantation into a human or an Old World primate comprising:
  • the invention provides an ungulate cell which has been genetically altered by the introduction of an expression vector comprising a nucleic acid sequence encoding a protein having fucosyltransferase activity into a recipient ungulate cell, the introduction of said expression vector causing a substantial reduction in the binding of naturally occurring preformed human -antibodies or naturally occurring preformed Old World primate antibodies to said genetically altered ungulate cell when compared to the binding of said antibodies to the recipient ungulate cell.
  • the invention provides a retroviral packaging or producer cell which has been genetically altered by the introduction of an expression vector comprising a nucleic acid sequence encoding a protein having fucosyltransferase activity into a recipient cell from which the genetically altered retroviral packaging or producer cell is derived, the introduction of said expression vector causing a substantial reduction in the binding of naturally occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies to said genetically altered retroviral packaging or producer cell when compared to the binding of said antibodies to the recipient cell from which the genetically altered * retroviral packaging or producer cell is derived.
  • FIGS. 1 and 4 are photomicrographs of African Green Monkey COS cells which have been fluorescently stained with anti-H antigen mAbs (FIGS. 2 and 3) or with lectins specific for the Gal o.(l,3) Gal epitope (FIGS. 1 and 4) .
  • the bottom panel shows all cells, as seen by phase contrast illumination, and the top panel shows only those cells specifically binding to the mAb or lectin as seen by ultraviolet illumination.
  • the cells in FIG. 1 have been transfected with a vector expressing the Gal o.(l,3) Gal transferase; the cells in FIG. 2 have been transfected with a vector expressing H transferase; and the cells in FIGS. 3 and 4 have been transfected with equal amounts of both vectors.
  • FIG. 5 illustrates the expression of the H epitope and reduced expression of the Gal o.(l,3) Gal epitope in stably transfected porcine kidney cells as analyzed by lectin staining and fluorescence-based flow cytometric analysis of the cells.
  • FIG. 6 demonstrates the loss of human serum IgG and IgM binding to porcine kidney cells associated with the expression of the H epitope and reduced expression of the Gal o.(l,3) Gal epitope as analyzed by fluorescence staining and flow cytometry.
  • FIG. 7 illustrates the enhanced resistance to human serum lysis associated with the expression of the H epitope and consequent reduced expression of the Gal ⁇ (l,3) Gal epitope in stably transfected porcine kidney cells.
  • glycosyltransferases A variety of nucleic acid molecules encoding glycosyltransferases can be used in the practice of the present invention provided that the glycosyltransferase encoded by the nucleic acid molecule is able to reduce the levels of Gal 0.(1,3) Gal epitopes on the surface of a xenogeneic cell in which the exogenous transferase is expressed.
  • this property can be determined by introducing an appropriate expression vector directing the expression of the candidate glycosyltransferase into xenogeneic cells and then testing the cells for cell surface levels of the Gal 0.(1,3) Gal epitope using, for example, human serum, as described below in Example 4.
  • Transferases suitable for use in the methods and cells of the present invention will cause a substantial reduction in the binding of naturally occurring preformed human antibodies to the xenogeneic cells after introduction of the expression vector compared to binding before introduction of the vector.
  • An at least 50% reduction in binding will, in general, comprise a "substantial reduction”.
  • Smaller reductions in binding are also considered “substantial” if they represent a statistically significant reduction, i.e., a reduction that, when analyzed by a standard statistical test, such as the student's T test, will give a probability value, p, less than or equal to 0.05 and, preferably, less than or equal to 0.015. Examples of the construction of such vectors, production of such cells, and the testing of such cells for reduction of preformed natural antibody binding are given below in Examples 1-5.
  • reduction in the binding of naturally occurring preformed antibodies can be determined by staining and counting stained cells as described below in Example 2, or by FACS analysis as described in Example 4 below in which case a quantitative readout can be obtained by measuring the areas under the various FACS curves and the shifts in the positions of those curves, or by measurement of changes in complement resistance as described in Example 5 below.
  • glycosyltransferases which are able to reduce the levels of Gal o-(l,3) Gal epitopes on the surface of a xenogeneic cell in which the transferase is expressed, effect this reduction by competition for a shared acceptor substrate.
  • transferases suitable for use in the methods and cells of the invention transfer donor sugars to 0(1,3) Glc NAc-R or 0(1,4) Glc NAc-R acceptor substrates.
  • preferred transferases include those that transfer donor sugars to 0(1,3) Glc NAc-R or 0(1,4) Glc NAc-R acceptor substrates and create covalent linkages other than the Gal c.(l,3) Gal linkage upon such transfer.
  • Preferred transferases to be used in the practice of the invention include fucosyltransferases. With regard to these transferases, it is believed that the addition of a terminal fucose residue to the 0(1,3) Glc NAc-R or 0(1,4) Glc NAc-R acceptor substrate of the 0.(1,3) galactosyltransferase prevents the addition of a Gal o-(l,3) Gal epitope to the acceptor substrate.
  • fucosyltransferases that can be tested for use in the process of the present invention include the o.(l,2) fucosyltransferase (H transferase; Larsen et al., 1990A) and the 0.(1,3/1,4) fucosyltransferase (Weston et al., 1992) .
  • H transferase is preferred and the human H transferase is particularly preferred.
  • This transferase is responsible for synthesis of the H antigen which is the universal donor 0-blood group antigen and utilizes the same acceptor substrates as the 0.(1,3) Gal transferase.
  • sialyltransferases e.g., the 0.(2,6) sialyltransferase (see Lowe, 1991) , may be used in the practice of the invention.
  • nucleic acid molecules encoding glycosyltransferases the cells and methods of the invention more generally comprise nucleic acid molecules encoding any and all proteins that have glycosyltransferase activity, including, in particular, fucosyltransferase activity.
  • proteins may be in the form of intact glycosyltranferases, but may also be in the form of proteins comprising active mutant glycosyltranferases such as those comprising active fragments of glycosyltranferases. See, for example, Kukowska, et al., 1991.
  • Vectors for expression of recombinant glycosyltransferases In addition to the foregoing, the present invention provides vectors for the expression of recombinant glycosyltransferases in xenogeneic cells at levels effective to reduce the expression of Gal 0.(1,3) Gal epitopes by the xenogeneic cells into which the vectors have been introduced.
  • Recombinant polynucleotides encoding glycosyltransferases that are appropriate for use in such vectors include those encoding the transferases discussed above.
  • a particularly preferred polynucleotide is that encoding human H transferase, SEQ ID NO: 3.
  • the nucleic acid encoding the desired exogenous glycosyltransferase may be inserted into an appropriate parent expression vector, i.e., an expression vector that contains a site for inserting protein-encoding nucleic acid molecules, and also contains (in the appropriate orientation for expression) the necessary elements for the transcription and translation of an inserted protein- encoding sequence.
  • an appropriate parent expression vector i.e., an expression vector that contains a site for inserting protein-encoding nucleic acid molecules, and also contains (in the appropriate orientation for expression) the necessary elements for the transcription and translation of an inserted protein- encoding sequence.
  • Particularly preferred transcriptional and translational signals allow for expression of the desired glycosyltransferase in a wide variety of xenogeneic cell types.
  • a candidate parent expression vector can be tested for suitability for use in the practice of the present invention by the insertion of a nucleic acid fragment encoding the human H transferase into a site appropriate for expression in the parent expression vector, as described below in Example 1 for the APEX-1 vector, and testing cells containing the resulting expression vector for susceptibility to human complement-mediated damage as described below in Example 5.
  • the transcriptional and translational control sequences in mammalian expression vector systems to be used in genetically altering vertebrate cells may be provided by various sources, including viral sources.
  • viral sources including viral sources.
  • commonly used promoters and enhancers known to be generally operable in many mammalian cell types are derived from Polyoma virus, Adenovirus, Simian Virus 40
  • SV40 SV40
  • CMV human cytomegalovirus immediate-early gene 1 promoter and enhancer
  • Cell-type specific promoters may also be used to express glycosyltransferases in particular cell types if desired.
  • a particularly preferred eukaryotic vector for the expression of glycosyltransferases in the methods and cells of the invention is pAPEX-1, SEQ ID NO:4 (see also copending U.S. patent application Serial No. 08/252,493, filed June 1, 1994, entitled "Porcine E-Selectin”) .
  • pAPEX-1 is a derivative of the vector pcDNAI/Amp
  • transferase-encoding polynucleotide fragments will be subcloned into the parent vector, typically following digestion with appropriate restriction endonucleases. Fragments for such subcloning can be obtained by PCR amplification, restriction endonuclease digestion, and the like. These fragments and the parent vectors are assembled into a transferase expression vector using standard methods such as PCR fusion or enzymatic ligation (Sambrook, et al., 1989; Ausubel et al., 1992).
  • nucleic acid molecules encoding the glycosyltransferases used in the methods and cells of the invention can be synthesized by chemical means (Talib, et al., 1991) .
  • Expression vectors preferably also contain selectable markers, such as a beta lactamase antibiotic resistance gene for plasmid selection and propagation in microbial cells in the presence of an antibiotic such as ampicillin, and the neomycin gene for selection and propagation of stable mammalian transfectants, e.g., in the presence of the cytotoxic aminoglycoside G418.
  • Introduction of nucleic acid molecules into cells via transfection or transduction As known in the art, introduction of nucleic acid molecules into cells can be accomplished by numerous methods, typically by transfection or transduction.
  • Transfection methods include the addition of chemical carriers such as DEAE/dextran, calcium phosphate, or amphipathic lipids (in which case the procedure is generally referred to in the art as lipofection) to the nucleic acid molecules before or during the addition of those molecules to the cells to be transfected.
  • Transfection methods also include mechanical means, such as electroporation, electric field mediated transfer (also referred to as Baekonization, see, for example, U.S. Pat. No. 4,849,355, entitled "Method Of Transferring Genes Into Cells" and U.S. Pat. No.
  • transduction is a preferred method of nucleic acid molecule introduction into xenogeneic endothelial cells.
  • the first step needed to use transduction methods in the practice of the present invention is incorporating the genetic sequence of the glycosyltransferase into a viral vector, e.g., a retroviral vector. Thereafter, the retroviral vectors are incorporated into retroviral vector particles using packaging cells.
  • retroviral nucleic acids to construct retroviral vectors and packaging cells is accomplished using techniques known in the art. See, for example, Ausubel, et al., 1992, Volume 1, Section III (units 9.10.1 - 9.14.3); Sambrook, et al., 1989; Miller, et al., 1989; Eglitis, et al., 1988; U.S. Patents Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263; as well as PCT Patent Publications Nos.
  • retroviral vectors for use in the practice of the invention can be prepared and used as follows.
  • a retroviral vector comprising a nucleic acid sequence encoding a glycosyltransferase is constructed from a parent retroviral vector. Examples of such parent retroviral vectors are found in, for example, Korman, et al., 1987; Morgenstern, et al., 1990; U.S. Patents Nos.
  • a preferred parent retroviral vector is the Moloney murine leukemia virus-derived expression vector pLXSN (Miller, et al., 1989).
  • the parent retroviral vector used in the practice of the present invention will be modified to include a glycosyltransferase encoding sequence and will be packaged into non-infectious (replication incompetent) transducing retroviral particles (virions) using an amphotropic packaging system, preferably one suitable for use in gene therapy applications.
  • a preferred packaging cell is the PA317 packaging cell line (ATCC CRL 9078) .
  • the generation of "producer cells” is accomplished by introducing retroviral vectors into the packaging cells.
  • the producer cells generated by the foregoing procedures are used to produce the retroviral vector particles. This is accomplished by culturing of the cells in a suitable growth medium.
  • the virions are harvested from the culture and administered to the target cells which are to be transduced.
  • target cells include isolated xenogeneic cells, cells of a xenogeneic organ or tissue, and other cells to be protected from antibody binding and complement attack, as well as xenogeneic progenitor cells, including stem cells such as embryonic or hematopoietic stem cells, which can be used to generate transgenic cells, tissues, or organs.
  • virions are added to the target xenogeneic cells to be transduced by co-culture of the target cells with the producer cells.
  • Suitable buffers and conditions for stable storage and subsequent use of the virions can be found in, for example, Ausubel, et al., 1992.
  • Cells, tissues, and organs In general, any xenogeneic cell, tissue or organ may be utilized in the practice of the present invention. Preferred cells are of ungulate origin, and particularly preferred cells are of pig origin.
  • the glycosyltransferase nucleic acid constructs of the invention can be used to engineer cultured cells of various types for subsequent use in transplantation. Examples of useful cell types include endothelial cells, fibroblastic and other skin cells, hepatic cells, neuronal and glial cells, pancreatic islet cells, hematopoietic cells, blood cells, lens cells, corneal cells, and stem cells.
  • glycosyltransferase nucleic acid constructs of the invention can be used to alter retroviral packaging cells or retroviral producer cells so that such cells exhibit a substantial reduction in the binding of naturally occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies when compared to the binding of said antibodies to packaging or producer cells which have not been so altered.
  • the expression "altered retroviral packaging/producer cells" is used to describe either or both of said altered packaging or producer cells.
  • Such altered retroviral packaging/producer cells may be from any species that expresses Gal c.(l,3) Gal epitopes, including cells of rodent or canine origin.
  • altered retroviral packaging/producer cells may be used to provide gene therapy treatment in a patient in need of such treatment, e.g., for therapeutic control of neoplastic tumors.
  • altered retroviral producer cells producing a retroviral vector particle providing a therapeutic benefit are implanted into the patient.
  • the implantation is preferably made into or adjacent to the tumor.
  • such altered producer cells are protected from HAR upon transplantation (implantation) into a human or Old World primate patient.
  • packaging vectors are introduced into suitable host cells such as those found in, for example, Miller and Buttimore, Mol. Cell Biol.. 6:2895-2902, 1986; Markowitz, et al., J. Virol.. 62:1120-1124, 1988; Cosset, et al., J ⁇ Virol.. 64:1070-1078, 1990; U.S. Patents Nos.
  • the retroviral vector includes a psi site and one or more exogenous nucleic acid sequences selected to perform a desired function, e.g., an experimental, diagnostic, or therapeutic function. These exogenous nucleic acid sequences are flanked by LTR sequences which function to direct high efficiency integration of the sequences into the genome of the ultimate target cell. (See also the discussion of transduction set forth above.)
  • RWPs retroviral vector particles
  • genes and DNA fragments can be incorporated into RWPs for use in gene therapy. These DNA fragments and genes may encode RNA and/or protein molecules which render them useful as therapeutic agents. Protein encoding genes of use in gene therapy include those encoding various hormones, growth factors, enzymes, lymphokines, cytokines, receptors, and the like.
  • genes which can be transferred are those encoding polypeptides that are absent, are produced in diminished quantities, or are produced in mutant form in individuals suffering from a genetic disease.
  • Other genes of interest are those that encode proteins that, when expressed by a cell, can adapt the cell to grow under conditions where the unmodified cell would be unable to survive, or would become infected by a pathogen.
  • Genes encoding proteins that have been engineered to circumvent a metabolic defect are also suitable for transfer into the cells of a patient.
  • Such genes include the transmembrane form of CD59 discussed in copending U.S. patent application No. 08/205,720, filed March 3, 1994, entitled “Terminal Complement Inhibitor Fusion Genes and Proteins" and copending U.S. patent application No. 08/206,189, filed March 3, 1994, entitled “Method for the Treatment of Paroxysmal Nocturnal Hemoglobinuria".
  • RWPs can be used to introduce nucleic acid sequences encoding medically useful RNA molecules into cells.
  • RNA molecules include anti-sense molecules and catalytic molecules, such as ribozymes.
  • RWPs that can transduce non-dividing cells may be preferred.
  • RWPs are disclosed in copending U.S. patent applications Serial Nos. 08/181,335 and 08/182,612, both entitled “Retroviral Vector Particles for Transducing Non-Proliferating Cells” and both filed January 14, 1994. These patent applications also discuss specific procedures suitable for producing packaging vectors and retroviral vectors as well as the use of such vectors to produce packaging cells and producer cells, respectively.
  • Transgenie animals provide a preferred source of the cells, tissues, and organs of the invention.
  • the nucleic acid molecules of the invention are used to generate engineered transgenic animals, preferably ungulates (i.e., hooved animals such as pigs, cows, goats, sheep, and the like) , that express the carbohydrate products of glycosyltransferases on the surfaces of their cells (e.g., endothelial cells) using techniques known in the art.
  • These techniques include, but are not limited to, microinjection (e.g., of pronuclei) , electroporation of ova or zygotes, electric field mediated transfer (i.e., Baekonization, supra; see also Zhao and Wong, 1991) , nuclear transplantation, and/or the stable transfection or transduction of embryonic stem cells derived from the animal of choice.
  • Electric field mediated transfer i.e., Baekonization, is a preferred method of producing the transgenic animals of the invention.
  • a common element of these techniques involves the preparation of a transgene transcription unit.
  • a transgene transcription unit comprises a DNA molecule which generally includes: 1) a promoter, 2) the nucleic acid sequence of interest, i.e., the sequence encoding a glycosyltransferase, and 3) a polyadenylation signal sequence. Other sequences, such as enhancer and intron sequences, can be included if desired.
  • the unit can be conveniently prepared by isolating a restriction fragment of a plasmid vector which expresses the glycosyltransferase protein in, for example, mammalian cells.
  • the restriction fragment is free of sequences which direct replication in bacterial host cells since such sequences are known to have deleterious effects on embryo viability.
  • transgenic animals The most well known method for making transgenic animals is that used to produce transgenic mice by superovulation of a donor female, surgical removal of the egg, injection of the transgene transcription unit into the pro-nuclei of the embryo, and introduction of the transgenic embryo into the reproductive tract of a pseudopregnant host mother, usually of the same species. See Wagner, U.S. Patent No. 4,873,191, Brinster, et al., 1985, Hogan, et al., 1986, Robertson 1987, Pedersen, et al., 1990.
  • transgenic swine are routinely produced by the microinjection of a transgene transcription unit into pig embryos. See, for example, PCT Publication No. W092/11757.
  • this procedure may, for example, be performed as follows. First, the transgene transcription unit is gel isolated and extensively purified through, for example, an ELUTIP column (Schleicher & Schuell, Keene, NH) , dialyzed against pyrogen free injection buffer (lOmM Tris, pH7.4 + O.lmM EDTA in pyrogen free water) and used for embryo injection.
  • Embryos are recovered from the oviduct of a hormonally synchronized, ovuiation induced sow, preferably at the pronuclear stage. They are placed into a 1.5 ml icrofuge tube containing approximately 0.5 ml of embryo transfer media (phosphate buffered saline with 10% fetal calf serum) . These are centrifuged for 12 minutes at 16,000 x g in a microcentrifuge. Embryos are removed from the microfuge tube with a drawn and polished Pasteur pipette and placed into a 35 mm petri dish for examination. If the cytoplasm is still opaque with lipid such that the pronuclei are not clearly visible, the embryos are centrifuged again for an additional 15 minutes.
  • embryo transfer media phosphate buffered saline with 10% fetal calf serum
  • Embryos to be microinjected are placed into a drop of media (approximately 100 ⁇ l) in the center of the lid of a 100 mm petri dish. Silicone oil is used to cover this drop and to fill the lid to prevent the medium from evaporating.
  • the petri dish lid containing the embryos is set onto an inverted microscope equipped with both a heated stage (37.5-38°C) and Hoffman modulation contrast optics (200X final magnification) .
  • a finely drawn and polished micropipette is used to stabilize the embryos while about 1-2 picoliters of injection buffer containing approximately 200-500 copies of the purified transgene transcription unit is delivered into the nucleus, preferably the male pronucleus, with another finely drawn and polished micropipette.
  • Embryos surviving the microinjection process as judged by morphological observation are loaded into a polypropylene tube (2 mm ID) for transfer into the recipient pseudopregnant sow. Offspring are tested for the presence of the transgene by isolating genomic DNA from tissue removed from the tail of each piglet and subjecting this genomic DNA to nucleic acid hybridization analysis with transgene SD ecific probes or PCR analysis with transgene specific primers.
  • ES cells embryonic stem cells
  • Patent Publication No. WO 93/02188 and Robertson, 1987 Patent Publication No. WO 93/02188 and Robertson, 1987.
  • ES cells are grown as described in, for example, Robertson, 1987, and in U.S. Patent No. 5,166,065 to Williams et al., 1988.
  • Genetic material is introduced into the embryonic stem cells by, for example, electroporation according, for example, to the method of McMahon, et al., 1990, or by transduction with a retroviral vector according, for example, to the method of Robertson, et al., 1986, or by any of the various techniques described by Lovell-Badge, 1987.
  • Chimeric animals are generated as described, for example, in Bradley, 1987. Briefly, genetically modified ES cells are introduced into blastocysts and the modified blastocysts are then implanted in pseudo-pregnant female animals. Chimeras are selected from the-offspring, for example by the observation of mosaic coat coloration resulting from differences in the strain used to prepare the ES cells and the strain used to prepare the blastocysts, and are bred to produce non-chimeric transgenic animals.
  • transgenic animals prepared in accordance with the invention are useful as model systems for testing the xenotransplantation of their engineered cells, tissues, and organs and as sources of engineered cells, tissues, and organs for xenotransplantation.
  • the expression of functional glycosyltransferases by endothelial cells and/or other cell types in the tissues and organs of the transgenic animals of the present invention will provide reduced susceptibility to hyperacute complement-mediated rejection following exposure of those cells, tissues, and organs to complement in human blood, plasma, serum, lymph, or the like, e.g., following xenotransplantation into humans or Old World primates.
  • reduced susceptibility to HAR is provided because naturally occurring preformed human or Old World primate antibodies have fewer binding sites on the transgenic cells of the invention.
  • the human H transferase gene was cloned from cDNA prepared from Human Epidermoid Carcinoma cells (HEC cells, ATCC CRL 1555 #A-431) utilizing the Polymerase Chain Reaction (PCR) .
  • Cytoplasmic RNA was prepared from approximately 5X10 6 cells, and first strand cDNA was synthesized .from 5 ⁇ g of RNA in a final volume of lOO ⁇ l using the following reaction conditions: lOmM Tris-HCl PH8.3; 50mM KC1; 1.5m MgCl 2 ; 500ng oligo(dT) 15 (Promega Corporation, Madison, Wisconsin); lOmM DTT; 0.25mMdNTPs (dG, dC, dA, dT) ; and 20U Avian Myeloblastosis Virus reverse transcriptase (Seikagaku of America, Inc., Rockville, Maryland) at 42°C for one hour.
  • PCR was performed following cDNA synthesis using 4 ⁇ l of first strand cDNA reaction mixture as template and the following primers: a 34 base 5' primer homologous to the ⁇ ' untranslated region of the H transferase cDNA (SEQ ID N0:1; 5' -GGCCACGAAA AGCGGACTGT GGATCCGCCA CCTG-3'), where the underlined sequence represents a unique BamHI site; and a 38 base 3' primer homologous to the 3' UTR cf the H transferase cDNA (SEQ ID N0:2; 5' -CAGGAACACC A CAAGCTTC TCGAGAAGATGC CAGGCC-3'), in which the underlined sequence represents a unique Xhol site.
  • a 34 base 5' primer homologous to the ⁇ ' untranslated region of the H transferase cDNA SEQ ID N0:1; 5' -GGCCACGAAA AGCGGACTGT GGATCCGCCA CCTG-3
  • PCR reactions consisted of 35 cycles of 95°C - 1 minute, 52°C - 1 minute, and 72°C - 1.5 minutes. These 35 cycles were followed by a single ten minute extension at 72° C. An approximately 1300 bp band representing the PCR product was seen following agarose gel electrophoresis of an aliquot of the PCR reaction.
  • This PCR product was cloned into a plasmid vector using the T/A cloning kit (Invitrogen, San Diego, CA) .
  • the pCRII plasmid vector included in this kit served as the recipient, and the resulting plasmid construct was amplified in ⁇ . coli and purified.
  • Positive clones were identified by restriction endonuclease digestion and the insert was subsequently s ⁇ quenced to confirm that the plasmid construct contained the human H transferase cDNA sequence shown in SEQ ID NO:3.
  • Positive clones were identified by restriction mapping with BamHI-XhoI and Stul.
  • Plasmid pAPEXl-HT referred to hereinafter as pHT, was the result of these cloning and subcloning steps.
  • pAPEX-1 (SEQ. ID No:4) is a derivative of the vector pcDNAI/Amp (Invitrogen, San Diego CA) which was modified as follows to increase protein expression in mammalian cells. First, since the intron derived from the gene encoding the SV40 small-t antigen has been shown to decrease expression of upstream coding regions (Evans and Scarpulla, 1989) , this intron was removed from pcDNAI/Amp by digestion with Xbal-Hpal, followed by treatment with the Klenow fragment of DNA polymerase and all four dNTPs.
  • the resulting blunt ended 4.2 kb fragment was gel purified and self ligated to yield a closed circular plasmid.
  • a 5' -untranslated region adenovirus/immuno- globulin hybrid intron was introduced into the plasmid by replacing a 0.5 kb Ndel-NotI fragment with the corresponding 0.7 kb Ndel-NotI fragment from the vector pRc/CMV7SB (obtained from Dr. Joseph Goldstein, University of Texas Southwest Medical Center, Dallas, TX) .
  • the resulting CMV promoter expression cassette was shuttled as an Ndel-Sfil fragment into the vector pGEM-4Z (Promega, Madison WI) by ligation to an Ndel-Sfil fragment (containing pGEM-4Z) obtained from a pGEM based expression vector containing a CMV-promoter and an SV40 origin of replication (Davis et al., 1991).
  • pGEM-4Z Promega, Madison WI
  • Transferase COS cells (ATCC # CRL 1650) were transiently transfected with CMV-based expression vectors. These vectors were pGT, containing an insert comprising a sequence (SEQ ID NO:5) encoding the pig 0.(1,3) Gal transferase oriented for CMV promoter-driven expression in the parent vector pCDNAI (Invitrogen, San Diego, CA) , and pHT (described above) , encoding the human H transferase. Clones containing pig 0.(1,3) Gal transferase cDNAs have been deposited with the Australian Government Analytical Laboratories, 1 Suakin Street, Pymble, N.S.W.
  • COS cells maintained in DMEM with 10% FBS were seeded into 6-well tissue culture plates and were subsequently transfected with pHT and/or pGT. Transfected cells were examined for the expression of the Gal 0.(1,3) Gal epitope or the H epitope 48 hours after transfection.
  • FIGS. 1-4 show the results of phase contrast (P) and fluorescence (F) microscopy of the cells obtained in these experiments.
  • FIGS. 1-4 are photomicrographs of African Green Monkey COS cells which have been fluorescently stained with ASH-1952 (FIGS. 2 and 3) or with IB4 (FIGS. 1 and 4) .
  • the bottom panel shows all cells, as seen by phase contrast illumination, and the top panel shows only those cells specifically binding to the mAb or lectin as seen by ultraviolet illumination.
  • the cells in FIG. 1 have been transfected with pGT; the cells in FIG. 2 have been transfected with pHT; and the cells in FIGS. 3 and 4 have been transfected with equal amounts of both vectors.
  • cotransfections were also done using expression vectors derived from a parent CMV-based expression vector (pCDM8; Seed and Aruffo, 1987) encoding either Ly-9 (Sandrin et al., 1992) or CD48 (Vaughan et al., 1991). Staining for the Ly-9 epitope was carried out using monoclonal antibody anti-Ly-9.2 (Sandrin et al., 1992). Staining for the CD48 epitope was carried out using an anti-CD48 monoclonal antibody (HuLy-m3; Vaughan et al., 1991) .
  • a porcine kidney cell line (LLC-PK ⁇ ATCC# CRL 1392) was transfected with plasmid pHT (directing the expression of H transferase) and plasmid pSV2neo
  • Transfection was carried out by the calcium phosphate co-precipitation method and transfected cells were cultured in DMEM + 10% fetal bovine serum + G418
  • the cell surface expression of the H epitope was analyzed on G418 resistant colonies by indirect immunofluorescence performed with ASH-1952 (identified as "anti-H mAB” in FIG. 5) or with the H epitope specific lectin UEAI (EY Laboratories, Inc., San Mateo, CA.) directly conjugated to FITC.
  • the Gal o.(l,3) Gal cell surface epitope was visualized by staining control and transfected cells with the FITC-conjugated lectin, IB4 (EY Laboratories, Inc., San Mateo, CA) .
  • transfected LLC-P ⁇ cells were also stained with the anti-SLA class I (anti-pig major histocompatibility antigen class I) mAb, PT85A (VMRD, Inc., Pullman WA) , as a positive control.
  • Goat anti-mouse IgG antisera monoclonal sera, Zymed Laboratories, South San Francisco, CA
  • FITC fluorescent microparticle-associated fluorescent protein
  • G418 resistant control LLC-PK ⁇ cells (clone PKl:neo #B6) normally express low levels of both the Gal c.(l,3) Gal epitope and the H epitope (FIG. 5B) compared to staining with secondary antibody alone (FIG. 5D; 2° curve) .
  • cells transfected with the human H transferase vector (clone #A3) express high levels of the H epitope (FIG. 5A) and reduced levels of the Gal 0.(1,3) Gal epitope (FIG. 5A) .
  • Transfection of these cells with H transferase did not alter the cell surface expression of the SLA class I gene product (FIG. 5C) relative to G418 resistant control cells (FIG. 5D) .
  • LLC-PK X cells stably transfected with pHT demonstrate little to no reactivity to either human IgG (FIG. 6A) or IgM (FIG. 6B) relative to G418 resistant control cells which demonstrate significant binding to human IgG (FIG. 6C) and IgM (FIG. 6D) present in 20% human serum.
  • the binding of human IgG and IgM present in 20% human serum to H transferase-expressing LLC-PK X cells is similar to the binding observed with 0% whole human serum.
  • the functional significance of recombinant H transferase expression by LLC-PK X cells was assessed by neasuring the efflux of the trapped cytoplasmic indicator iye, Calcein AM (Molecular Probes, Inc.), from cells subjected to human complement-mediated damage by human erum.
  • Transfected cells expressing the human H ransferase and the neomycin resistance gene (clone #A3; ee Examples 3 and 4 above) or the neomycin resistance ene alone (clone #C6 ; prepared in the same manner as clone B6 described above in Examples 3 and 4) were grown o confluence in 96-well plates.
  • HBSS/BSA HBSS/BSA
  • calcein AM was added (lOmM final) and the plates were incubated at 37°C for 30 minutes. Subsequently, the cells were incubated at 37°C for 30 minutes in the jresence of increasing concentrations of human whole serum.
  • Dye released from the cells was determined by the fluorescence in the supernatant. Total cell associated dye was determined from a 1% SDS cell lysate. The dye release was calculated as a percent of total, correcting fpr non-specific dye release and background fluorescence measured for identically matched controls without the addition of serum. Fluorescence was measured using a Millipore Cytofluor 2350 fluorescence plate reader (490nm excitation, 530nm emission) . As shown in FIG.
  • LLC-PK X cells stably transfected with pHT were significantly less sensitive to the lytic activity of human complement relative to control LLC-PK X cells (clone #C6; closed circles) at all concentrations of human serum tested between 1% and 40%.
  • pGT porcine galactose ⁇ (l,3) galactosyltransferase cDNA subcloned into CMV-based expression plasmid pCDNAI (Invitrogen, Sand Diego, CA) .
  • pHT human H transferase cDNA subcloned into CMV-based expression plasmid pAPEX-1.
  • IB4 binds to the Gal 0.(1,3) Gal epitope.
  • ASH-1952 binds to the H epitope.
  • Robertson 1987, in Robertson (ed) . Teratocarcinomas and Embryonic Stem Cells a Practical Approach. IRL Press, Eynsham, Oxford, England.
  • TITLE Molecular cloning, sequence, and expression of a human GDP-L-fucose: -D-galactoside 2-alpha-L- fucosyltransferase cDNA that can form the H blood group antigen.
  • TGG TGT AAA GAA AAC ATC GAC ACC TCC CAG GGC GAT GTG ACG 854 Trp Cys Lys Glu Asn lie Asp Thr Ser Gin Gly Asp Val Thr 270 275 280
  • GTAATTTCGC CATCAAGGGC AGCGAGGGCT TCTCCAGATA AAATAGCTTC 1550
  • TAGTATTAAG CAGAGGCCGG GGACCCCTGG GCCCGCTTAC TCTGGAGAAA 1950
  • GAGCTATTCC AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG 2250
  • GAC GCT ATA GGC AAC GAA- AAG GAA CAA AGA AAA GAA GAC AAC 259 Asp Ala lie Gly Asn Glu Lys Glu Gin Arg Lys Glu Asp Asn 60 65 70 AGA GGA GAG CTT CCG CTA GTG GAC TGG TTT AAT CCT GAG AAA 301 Arg Gly Glu Leu Pro Leu Val Asp Trp Phe Asn Pro Glu Lys
  • CAG GTT CTA AAC ATC ACT CAG GAG TGC TTC AAG GGA ATC CTC 931 Gin Val Leu Asn He Thr Gin Glu Cys Phe Lys Gly He Leu
  • AGC CAT CTA AAC AAG TAT TTC CTT CTC
  • AAC AAA CCC ACT AAA 1015 Ser His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys 310 315 320

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US6166288A (en) * 1995-09-27 2000-12-26 Nextran Inc. Method of producing transgenic animals for xenotransplantation expressing both an enzyme masking or reducing the level of the gal epitope and a complement inhibitor
EP0877549A4 (de) * 1995-11-03 2002-01-02 Mount Sinai Medical Ct Verfahren und zusammenstellungen zur verminderung der xenotransplantation-abstossung
CA2233705A1 (en) * 1996-08-02 1998-02-12 The Austin Research Institute Improved nucleic acids encoding a chimeric glycosyltransferase
AUPO182396A0 (en) * 1996-08-23 1996-09-12 Austin Research Institute, The Improved nucleic acids for reducing carbohydrate epitopes
US6455037B1 (en) 1996-11-01 2002-09-24 Mount Sinai School Of Medicine Of The City University Of New York Cells expressing an αgala nucleic acid and methods of xenotransplantation
JP4451933B2 (ja) 1996-12-27 2010-04-14 住友化学株式会社 遺伝子操作による植物へのｐｐｏ阻害性除草剤耐性付与法
CA2279542A1 (en) * 1997-02-05 1998-08-06 The General Hospital Corporation Tolerance to natural antibody antigens
US6498285B1 (en) 1997-08-06 2002-12-24 Alexion Pharmaceuticals, Inc. Methods for producing transgenic pigs by microinjecting a blastomere
DE10025920A1 (de) * 2000-05-27 2001-12-06 Mologen Forschungs Entwicklung Verfahren und Mittel zur Behandlung von Immunreaktionen am Auge
US7485769B2 (en) 2000-08-25 2009-02-03 Nippon Meat Packers, Inc. Transgenic mammals
US7126039B2 (en) 2001-03-21 2006-10-24 Geron Corporation Animal tissue with carbohydrate antigens compatible for human transplantation
AU2002242538B2 (en) * 2001-03-21 2007-06-28 Geron Corporation Animal tissue with carbohydrate antigens compatible for human transplantation
US7033790B2 (en) 2001-04-03 2006-04-25 Curagen Corporation Proteins and nucleic acids encoding same
ATE451448T1 (de) 2002-08-21 2009-12-15 Revivicor Inc Schweine ohne jegliche expression funktioneller alpha-1,3-galactosyltransferase
EP1651050B1 (de) 2003-07-21 2012-08-22 Lifecell Corporation Zellfreie gewebsmatrize aus galactose-alpha-1,3-galactose-freiem gewebe
WO2005047469A2 (en) 2003-11-05 2005-05-26 University Of Pittsburgh PORCINE ISOGLOBOSIDE 3 SYNTHASE PROTEIN, cDNA, GENOMIC ORGANIZATION, AND REGULATORY REGION
ES2537030T3 (es) 2004-03-17 2015-06-01 Revivicor, Inc. Productos de tejidos derivados de animales que carecen de cualquier expresión de alfa-1,3-galactosiltransferasa funcional
US9420770B2 (en) 2009-12-01 2016-08-23 Indiana University Research & Technology Corporation Methods of modulating thrombocytopenia and modified transgenic pigs
CN105682697B (zh) 2013-11-04 2022-10-14 生命细胞公司 去除α-半乳糖的方法
US11905549B2 (en) 2016-08-10 2024-02-20 Genahead Bio, Inc. Method for modifying target site in genome of eukaryotic cell, and method for detecting presence or absence of nucleic acid sequence to be detected at target site

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