WO2002081688A1

WO2002081688A1 - Dna molecules encoding igb3 synthase, and uses thereof for the disruption of glycosyltransferase genes in xenotransplantation tissues and organs

Info

Publication number: WO2002081688A1
Application number: PCT/AU2002/000429
Authority: WO
Inventors: Mauro Sergio Sandrin
Original assignee: The Austin Research Institute
Priority date: 2001-04-03
Filing date: 2002-04-03
Publication date: 2002-10-17
Also published as: AUPR414701A0

Abstract

Nucleic acid molecules encoding iGb3 synthase are disclosed, as well as fragments and mutants thereof. IGb3 synthase is, in part, responsible for the production of a Gal?a(1,3)Gal epitope in xenograft cells, tissues and organs which in humans provokes hyperacute rejection (HAR). The nucleic acid molecules of the invention may be used to generate animals in which the iGb3 synthase gene is disrupted. The cells, tissues and organs harvested from such animals are therefore useful in xenotransplantation.

Description

DNA MOLECULES ENCODING iGb3 SYNTHASE. AND USES THEREOF FOR THE DISRUPTION OF GLYCOSYLTRANSFERASE GENES IN XENOTRANSPLANTATION

TISSUES AND ORGANS

FIELD OF THE INVENTION:

The present invention relates to a substantially pure or isolated nucleic acid molecule comprising a nucleotide sequence according to any one of SEQ ID NOS: 1 to 7 and to uses thereof to produce cells in which a gene is disrupted. In a particular application, the invention relates to the use of the nucleic acid molecule, or fragments or mutants thereof, to the generation of animals in which the gene encoding iGb3 synthase is disrupted or otherwise rendered non-functional, and to the use of tissues and organs harvested from such animals in xenotransplantation.

BACKGROUND OF THE INVENTION: Glycosyltranferases are a group of about 100 enzymes involved in biosynthesis of glycoproteins and glycolipids by addition of glycans on to different aglycone protein and lipid substrates respectively. The resulting glycoproteins and glycosphingolipids (GSL) in turn comprise a multitude of family members based on the carbohydrate structure. For example, the GSL can be divided into six major series based on the carbohydrate - gangliosides, lacto-, muco-, neolacto-, isoglobo- and globo-series. The common aglycone precursor to these GSL is LacCer (Galβl,4GlcβlCer). The significance of the individual glycan structures is unknown.

Rat iGb3 synthase is a relatively new member of the ABO blood group gene family. It belongs to the same class of glycosyltransferases as αl,3-galactosyltransferase (αl,3GT), initiating the synthesis of isoglobo-GSL by catalysing the transfer of a galactose (gal) from UDP-galactose to LacCer. Rat iGb3 synthase has recently been cloned (Keusch JJ, etal. "Expression cloning of a new member of the ABO blood group glycosyltransferases, iGb3 synthase, that directs the synthesis of isoglobo- glycosphingolipids", J Biol Chem, 275 (33), pp 25308-25314 (2000)), but until now, it has

141314414 been unclear if homologues exist in other species, or if in the rat, iGb3 synthase is the equivalent gene product as αl,3GT. αl,3GT is an example of a glycosyltransferase that plays a role in the synthesis of glycoproteins. It catalyses the synthesis of the glycoprotein, galα(l,3)galβl,4GlcNAcR (Galα(l,3)Gal) by transfering galactose from UDP-galactose to the acceptor,

Galβl,4GlcNAc (LacNAc). αl,3GT is expressed in New World primates and many non- primate animals but only very poorly in Old World primates, apes and man due to a frame shift mutation in the gene. The Galα(l,3)Gal has been identified as an epitope responsible for the hyperacute rejection (HAR) of xenografted tissues in humans who, in the absence of Galα(l,3)Gal, produce antibodies reactive with this epitope on the cell surface of animal organs.

The inactivation or deletion of the gene encoding αl,3GT in animals such as mice, rats and porcine cells have produced cells that do not react with anti-Galα(l,3)Gal antibodies, and which initially do not manifest symptoms of HAR when exposed to human serum. However, cell lysis eventually occurs. The inactivation or deletion of the αl,3GT gene has therefore not completely resolved the problem of rejection of transplanted tissues from a discordant species by a recipient. The reasons or causes for the persistent immune reaction remain unknown. In addition, there remains a need for xenografts that will not be rejected by a recipient. In particular, there is a need for animal cells, tissues or organs that can be transplanted into humans because of the extreme shortage of human donor tissues.

The present invention relates to a novel gene encoding an epitope reactive with anti-Galαl,3Gal antibodies.

SUMMARY OF THE INVENTION:

In a first aspect, the invention provides a substantially pure or isolated nucleic acid molecule comprising the nucleotide sequence according to SEQ ID NO: 1, or homologous, variant or derivative sequences thereof.

In a second aspect, the invention provides a nucleic acid molecule consisting of a nucleotide sequence according to a fragment of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4, or homologous, variant or derivative sequences thereof. In a third aspect, the invention provides a mutant of the nucleotide sequence of any one of SEQ ID NOS: 1 to 7, wherein the mutant comprises one or more nucleotide substitutions, insertions, additions or deletions.

In a fourth aspect, the invention provides a nucleic acid construct for disrupting a gene encoding a glycosyltransferase such as an iGb3 synthase, said construct comprising the nucleotide sequence of any one of SEQ ID NOS: 5 to 7, or a fragment thereof.

In a fifth aspect, the invention provides a nucleic acid construct for disrupting a gene encoding a glycosyltransferase such as an iGb3 synthase gene, said construct comprising a mutant of the nucleotide sequence of any one of SEQ ID NOS: 5 to 7, or a fragment thereof.

In a sixth aspect of the invention, there is provided a mammalian cell comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted, such that expression of functional iGb3 synthase is disrupted.

In a seventh aspect, the invention provides a method of disrupting a glycosyltransferase gene such as an iGb3 synthase gene in a mammalian cell, the method comprising the step of disrupting the expression of said gene using a nucleic acid construct according to the fourth or fifth aspect of the present invention.

In an eighth aspect, the invention provides a method of xenotransplantation, comprising the step of transplanting a donor-cell, -tissue or -organ into a recipient, the cell, tissue or organ comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted.

In a ninth aspect, the invention relates to a non-human, transgenic donor animal comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted. In a tenth aspect, the present invention provides a cell in which both the iGb3 synthase and αl,3GT genes have been disrupted or deleted.

In an eleventh aspect , the invention provides a cell comprising a gene encoding αl,2 fucosyltransferase (FT), and wherein the iGb3 synthase gene has been disrupted or deleted. In a twelfth aspect, the present invention provides a cell comprising a chimeric gene, said chimeric gene comprising a nucleotide sequence encoding a localisation signal, wherein the localisation signal comprises a cytoplasmic domain from a first kind of glycosyltransferase and a catalytic site from a second kind of glycosyltransferase, and wherein the iGb3 synthase gene has been disrupted or deleted.

In a further aspect, the invention provides a nucleic acid construct for reducing expression of Galα(l,3)Gal in a mammalian cell, said construct comprising a first nucleotide sequence from a gene selected from the group consisting of the iGb3 synthase and αl,3GT genes, and a second nucleotide sequence from a gene selected from the group consisting of the FT and Se genes, wherein said first nucleotide sequence is capable of disrupting the iGb3 synthase and /or αl,3GT genes. In a still further aspect, the present invention provides cells, tissues or organs harvested from a transgenic animal, wherein said cells, tissues or organs comprise disrupted or deleted iGb3 synthase and αl,3GT genes.

In yet a still further aspect, the invention provides a method of treating a human cancer or Graves' disease in a subject, comprising administering to said subject an agent to reduce or eliminate the expression of functional iGb3 synthase by diseased cells.

DETAILED DISCLOSURE OF THE INVENTION:

The nucleic acid molecule of the first aspect of the invention comprises a nucleotide sequence according to SEQ ID NO: 1, or homologous, variant or derivative sequences thereof. Preferably, homologous, variant or derivative sequences of the sequence of SEQ ID NO: 1 are those which hybridise to the sequence of SEQ ID NO: 1 under stringent conditions of hybridisation.

"Stringent conditions of hybridisation" are those conditions generally understood by those skilled in the art and which may be found in, for example, Sambrook etal, "Molecular Cloning: A Laboratory Manual ", 2^nd edition, Cold Spring Harbor Laboratory Press (1989). One specific example of stringent conditions of hybridisation is 65°C and 0.1 x SSC (where 1 x SSC is 0.15 M NaCl, 0.015 M Na₃Citrate, pH 7.0).

Preferably, the nucleic acid molecule of the invention encodes an iGb3 synthase. Most preferably, it encodes porcine iGb3 synthase. In the case where the nucleic acid molecule of the invention comprises a nucleotide sequence which is homologous to the sequence of SEQ ID NO: 1, the nucleic acid molecule preferably comprises a homologous nucleotide sequence of murine or human origin and, more preferably, comprises a homologous nucleotide sequence selected from the group consisting of SEQ ID NOS: 2 to 4.

In the case where the nucleic acid molecule of the invention comprises a nucleotide sequence which is a variant sequence of SEQ ID NO: 1, the nucleic acid molecule preferably comprises a naturally occurring variant nucleotide sequence which varies from the sequence of SEQ ID NO: 1 only by the inclusion of one or more nucleotide substitution(s) (eg a nucleotide substitution which effects a conservative amino acid substitution in the encoded product), insertion(s), addition(s) or deletion(s) which do not substantially alter the biological function of the nucleotide sequence of SEQ ID NO: 1. In the case where the nucleic acid molecule of the invention comprises a nucleotide sequence which is a derivative sequence of SEQ ID NO: 1, the nucleic acid molecule may comprise SEQ ID NO: 1 together with another sequence upstream or downstream of SEQ ID NO: 1. Preferably, such upstream or downstream sequences comprise an exon of porcine iGb3 synthase gene. More preferably, these sequences exhibit at least 30% nucleotide sequence homology with the rat, murine or human iGb3 synthase gene or at least 30% amino acid sequence homology with the amino acid sequence of the rat, murine or human iGb3 synthase.

Nucleic acid molecules consisting of a nucleotide sequence according to a fragment, and particularly a biologically functional fragment, of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4, or a homologous, variant or derivative sequence thereof, comprise the second aspect of the present invention.

The fragment may comprise about 50 to 100 % of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4, preferably about 55 to 95%, more preferably about 60 to 85% and most preferably about 65 to 80%.

In a particular embodiment of the invention of the second aspect, the fragment comprises sub-sequences selected from the group consisting of nucleotide no. 1 to 480, no. 355-1027 and no. 3800-4450 of SEQ ID NOS: 1, 2 and 3 respectively. These fragments are designated hereinafter as SEQ ID NOS: 5, 6 and 7 respectively. In a third aspect, the invention provides a mutant of the nucleotide sequences of any one of SEQ ID NOS: 1 to 7 or a fragment thereof, wherein the mutant comprises one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s).

The mutant may comprises a missense or nonsense mutation. Preferably, the mutant comprises one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s) in at least one exon of the nucleotide sequences of any one of SEQ ID NOS: 1 to 7. For example, exon 5 and /or exon 9 may comprise one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s).

In a particular embodiment of the invention of the third aspect, the one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s) is/ are located within a sub-sequence of SEQ ID NOS: 1 to 3, said sub-sequence being selected from the group consisting of SEQ ID NOS: 5 to 7. If desired, an exon from SEQ ID NO: 4 may be mutated by one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s).

The mutant may also consist of a fragment of the nucleotide sequence of any one of SEQ ID NOS: 1 to 7, provided that it is able to be employed so as to disrupt a glycosyltransferase gene or otherwise render a glycosyltransferase gene as nonfunctional. Preferably, the gene encodes iGb3 synthase, particularly porcine iGb3 synthase.

A peptide encoded by a fragment of any one of SEQ ID NOS: 1 to 7, or homologous, variant or derivative sequences thereof are also contemplated by the invention, as is a derivative peptide thereof which comprises one or more amino acid substitution(s) by a modified amino acid or non-codable amino acid (ie an amino acid not naturally found in proteins or peptides), and a peptidomimetic based on the peptide. Modified amino acids may include but are not limited to o-Phosphoserine, 4- Hydroxproline, δ-Hydroxylysine or γ-Carboxyglutamic acid. Non-codable amino acids may include but are not limited to β-Alanine, D-Alanine, γ-Aminobutyric acid, D- Glumatic acid, Homoserine, Ornithine, Sarcosine and Thyroxine.

Polyclonal and monoclonal antibodies, and fragments thereof, which are reactive with the above peptide are also contemplated as forming part of the present invention. In a fourth aspect, the invention provides a nucleic acid construct for disrupting a gene encoding a glycosyltransferase such as an iGb3 synthase, said construct comprising the nucleotide sequence of any one of SEQ ID NOS: 5 to 7, or a fragment thereof.

Suitable fragments of SEQ ID NOS: 5 to 7 are fragments which are capable of disrupting a glycosyltransferase gene. Generally, these fragments will comprise at least 10 contiguous nucleotides, more preferably at least 20 contiguous nucleotides and most preferably at least 50 contiguous nucleotides. These fragment sequences may optionally be linked to flanking sequences which are preferably l-4kb long. They may also form part of a cassette comprising other components such as promoters, linkers, marker sequences or tracers including green fluorescent dye.

Preferably, the wild type glycosyltransferase gene to be disrupted is a mammalian iGb3 synthase gene including but not limited to porcine, bovine, murine, ovine, equine and primate iGb3 synthase gene. More preferably, the wild type glycosyltransferase gene to be disrupted is the porcine iGb3 synthase gene. Most preferably, the wild type glycosyltransferase gene to be disrupted is the porcine iGb3 synthase comprising a nucleotide sequence according to SEQ ID No.l.

Preferably, the wild type glycosyltransferase gene to be disrupted is a mammalian iGb3 synthase gene including but not limited to porcine, bovine, murine, ovine, equine and primate iGb3 synthase gene. More preferably, the wild type glycosyltransferase gene to be disrupted is the porcine iGb3 synthase gene. Most preferably, the wild type glycosyltransferase gene to be disrupted is the porcine iGb3 synthase comprising a nucleotide sequence according to SEQ ID NO: 1.

Thus, in a sixth aspect of the invention, there is provided a mammalian cell comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted, such that expression of functional iGb3 synthase is disrupted. The mammalian cell in which the wild type iGb3 synthase gene has been disrupted include but are not limited to porcine, bovine, murine, ovine, equine, and primate cells. More preferably, the cells are of porcine, ovine or primate origin. Most preferably, the cells are of porcine origin.

The wild type iGb3 synthase gene is preferably disrupted, such that the disruption inhibits the expression of the gene, decreases the expression of the gene and /or inhibits or decreases the formation of a functional gene product (eg an attenuated gene product), preferably such that there is substantially no expression of functional iGb3 synthase enzyme by said mammalian cell.

Preferably, the method results in an iGb3 synthase gene being disrupted in a manner whereby the disruption inhibits the expression of the gene, decreases the expression of the gene and /or inhibits or decreases the formation of a functional gene product (eg an attenuated gene product), preferably such that there is substantially no expression of functional iGb3 synthase enzyme by said mammalian cell.

Preferably, the gene being disrupted is the porcine iGb3 synthase gene.

The gene may be disrupted by homologous recombination between the exogenous iGb3 synthase nucleotide sequence, or fragment or mutant thereof, contained in the nucleic acid construct, and the wild type iGb3 synthase gene (see, for example, Capecchi MR, "Altering the genome by homologous recombination", Science, 244, pp 1288-1292 (1989); and Merlino GT, "Transgenic animals in Biomedical Research", FASEB J, 5, pp 2996-3001 (1991), the disclosures of which are to be regarded as incorporated herein by reference). In a preferred embodiment, markers such as the neomycin resistance gene (ned or hygromycin resistance gene ( yg) may be used to assist in subsequent determination of successful homologous recombination and identification of cells carrying a disrupted iGb3 synthase gene. Other conventional markers that can be used will be known to those skilled in the art.

In other preferred embodiments of the seventh aspect of the invention, the iGb3 synthase gene may be disrupted by other means well known to those skilled in the art. These include but are not limited to selective deletion of the gene encoding iGb3 synthase by Cre/lox as described in Saure B, "Inducible gene targetting in mice using the Cre/lox system", Methods, 14(4), pp 381-392 (1998), the disclosure of which is to be regarded as incorporated herein by reference). The disruption may also be achieved by targetting the promoter of the gene to be inactivated. For example, the promoter or promoter region of the gene may be methylated, as described in Chomet PD, "Cytosine methylation in gene- silencing mechanisms", Curr Opin Cell Biol, 3, pp 438-443 (1991); and Doerfler W, etal. "Promoter inactivation or inhibition by sequence-specific methylation and mechanisms of reactivation", Cell Biophys, 15, pp 21-27 (1989), the disclosures of which are to be regarded as incorporated herein by reference). Anti-sense sequences of any one of SEQ ID NOS: 1 to 7 may also be used to disrupt an iGb3 synthase gene. For example, the promoter from any one of SEQ ID NOS: 1 to 4 may be deactivated so that transcription of the gene is inhibited. Nucleotide sequences that are anti-sense polynucleotides of any one of SEQ ID NOS: 5 to 7 may also be used in the gene disruption. The gene may also be activated using the promoter trap methodology as described in McCreath etal. "Production of gene-targeted sheep by nuclear transfer from cultured somatic cells", Nature, 405, pp 1066-1069 (2000).

Cells in which the iGb3 synthase gene has been disrupted or deleted will be useful for xenotransplantation, or for generation of animals producing cells, tissue or organs in which the function of the iGb3 synthase gene product has been disrupted as a result of inactivation, deletion or attenuation of the gene product's function.

Thus, in an eighth aspect, the invention provides a method of xenotransplantation, comprising the step of transplanting a donor-cell, -tissue or -organ into a recipient, the cell, tissue or organ comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted. And, in a ninth aspect, the invention relates to a non-human, transgenic donor animal comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted.

The donor animal of the ninth aspect may be produced as described in further detail below. The donor animal may be used as a source of cells, tissues or organs which do not produce functional iGb3 synthase or only a low level of iGb3 synthase from a disrupted gene, for xenotransplantation in a recipient. Preferably, the donor animal is selected from the group consisting of porcine, bovine, equine, murine, ovine and primate (other than human) animals. More preferably, the donor animal is a pig and the recipient animal is a human.

In a tenth aspect, the present invention provides a cell in which both the iGb3 synthase and αl,3GT genes have been disrupted or deleted.

Disruption of the ocl,3GT gene may be undertaken in the same manner as that for iGb3 synthase (ie by using mutated αl,3GT nucleotide sequences or fragments of the αl,3GT gene, in accordance with International Patent Specification No. WO 94/21799, the disclosure of which is to be regarded as incorporated herein in its entirety by reference; by the Cre/lox method as described above, by targetting the promoter as described above or by using anti-sense nucleotide sequences). αl,2 fucosyltransferase (FT or H-transferase) is an enzyme that competes with l,3GT for the same acceptor substrate, LacNAc, to form fucosylated LacNAc or the H- antigen (ie the O blood group), which is universally tolerated. In cells that have been fransfected with the gene for FT, a reduction in production of galα(l,3)gal is observed.

Thus, in an eleventh aspect , the invention provides a cell comprising a gene encoding αl,2 fucosyltransferase (FT), and wherein the iGb3 synthase gene has been disrupted or deleted.

In one embodiment of the cell of the eleventh aspect, the ocl,3GT gene, which may also be present in the cell, has also been disrupted or deleted.

Cells in accordance with the invention which comprise a disrupted iGb3 synthase gene, may further comprise a chimeric gene, said chimeric gene comprising a nucleotide sequence encoding a localisation signal, wherein the localisation signal comprises a cytoplasmic domain from a first kind of glycosyltransferase and a catalytic site from a second kind of glycosyltransferase. For example, the chimeric gene may encode the cytoplasmic domain of αl,3GT and the catalytic domain from another glycosyltransferase selected from the group consisting of FT and Se (Secretor or pig secretory FT). The chimeric gene may be made in accordance with the description provided in International Patent Specification No. WO 98/05768, the disclosure of which is to be regarded as incorporated herein in its entirety by reference. Thus, in a twelfth aspect, the present invention provides a cell comprising a chimeric gene, said chimeric gene comprising a nucleotide sequence encoding a localisation signal, wherein the localisation signal comprises a cytoplasmic domain from a first kind of glycosyltransferase and a catalytic site from a second kind of glycosyltransferase, and wherein the iGb3 synthase gene has been disrupted or deleted. In a further aspect, the invention provides a nucleic acid construct for reducing expression of Galα(l,3)Gal in a mammalian cell, said construct comprising a first nucleotide sequence from a gene selected from the group consisting of the iGb3 synthase and αl,3GT genes, and a second nucleotide sequence from a gene selected from the group consisting of the FT and Se genes, wherein said first nucleotide sequence is capable of disrupting the iGb3 synthase and /or αl,3GT genes.

Nucleic acid constructs made in accordance with the present invention may be used for generating cells as well as transgenic animals that do not express the Galα(l,3)Gal epitope thereby rendering such cells, or the cells, tissues or organs of such transgenic animals more suitable for xenotransplantation into humans. Preferably, the transgenic animals are selected from the group consisting of porcine, bovine, equine, murine, ovine and primate (other than human) animals. Most preferably, the transgenic animals are pigs.

Using the nucleic acid constructs of the present invention, transgenic animals may be produced in accordance with, for example, "Manipulating the mouse embryo - a laboratory manual", Cold Spring Harbor Laboratory, 1986, B Hogan, F Constantini and B Lacy, the disclosure of which is to be incorporated in its entirety herein by reference. Preferably, the transgenic animals are produced by nuclear transfer, for example, as described in Onishi A, etal. "Pig Cloning by Microinjection of Fetal Fibroblast Nuclei", Science, 289, pp 1722-1727 (2000); Polejaeva IA, etal. "Cloned pigs produced by nuclear transfer from adult somatic cells", Nature, 407, pp 86-90 (2000); or Betthauser J, etal. "Production of cloned pigs from in vitro systems", Nat. Biotechnol., 18, pp 1055-1059 (2000) (each of which is incorporated herein by this reference), or by generation of totipotent cells into which a nucleic acid construct according to the present invention has been introduced in order to inactivate the iGb3 synthase and /or αl,3GT, and to induce expression of FT. The fransfected totipotent cells, which may be embryonic stem (ES) cells, primordial germ cells (PGC), fertilised or unfertilised eggs, may then be used for generation of transgenic animals in accordance with the methods described in "Manipulating the mouse embryo- a laboratory manual" (supra).

In a still further aspect, the present invention provides cells, tissues or organs harvested from a transgenic animal, wherein said cells, tissues or organs comprise disrupted or deleted iGb3 synthase and αl,3GT genes.

In one preferred embodiment of the invention of this still further aspect, the cells, tissues or organs may further comprise a chimeric gene selected from the group encoding GT-HT and GT-Se. The anomalous expression of the Galα(l,3)Gal epitope has been described in human cancer cells (La Temple DC, etal. "increased Immunogenicity of Tumor Vaccines Complexed with Anti-Gal: Studies in Knockout mice for 1,3 Galactosyltransferase", Cancer Res., 59, pp 3417-3423 (1999)), and in thyroid cells from Graves' disease patients (Winand RJ, etal. "Specific stimulation of Grave's disease thyrocytes by the natural anti- Gal antibody from normal and autologous serum", J. Immunol., 53, pp 1386-1395 (1994)), and it has been suggested that the presence of the Galα(l,3)Gal epitope on these cells may reflect the activity of iGb3 synthase (Keusch JJ, supra). These diseases may therefore benefit from therapies designed to reduce or eliminate the level of expression of functional iGb3 synthase and thereby reduce or eliminate the level of the Galα(l,3)Gal epitope present on the diseased cells. Such therapies may employ nucleic acid constructs in accordance with the present invention which are capable of disrupting an iGb3 synthase gene (wherein the constructs may be introduced into a suitable virus or other vector designed to target the nucleic acid construct to the diseased cells), or otherwise involve the administration of an agent which binds to iGb3 synthase in a manner whereby the iGb3 synthase is rendered non-functional. Such an agent may be an anti-iGb3 synthase monoclonal antibody or a fragment thereof (eg scFv fragments).

Thus, in yet a still further aspect, the invention provides a method of treating a human cancer or Graves' disease in a subject, comprising administering to said subject an agent to reduce or eliminate the expression of functional iGb3 synthase by diseased cells. Throughout this specification, the word "comprise" or grammatical equivalents thereof will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

The invention will hereinafter be further described by way of the following, non- limiting examples and accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES:

Figure 1 provides the genomic nucleotide sequence and amino acid sequence for porcine iGb3 synthase.

Figure 2 provides the nucleotide sequence and amino acid sequence for a murine iGb3 synthase cDNA clone.

Figure 3 provides a partial genomic nucleotide sequence for the murine iGb3 synthase gene.

Figure 4 provides a partial genomic nucleotide sequence, and putative amino acid for human iGb3 synthase. Figure 5 shows a flow cytometry profile for CHOP cells fransfected with a porcine iGb3 cDNA clone.

Figure 6 shows a Southern blot analysis of genomic DNA for porcine, murine and ovine iGb3 synthase and αl,3GT.

Figure 7 shows the peptide sequence identity of rat, murine, human and porcine iGb3 synthase.

Figure 8 provides the nucleotide sequence of a rat α(l,3)GT cDNA clone. The predicted amino acid sequence is designated above the nucleotide sequence as single letter code. The putitive transmembrane domain (^— ) and the theoretical N- glycosylation sites(^Λ). The DXD motif is marked (*****) and the errors in the 3' region due to murine α(l,3)GT primers are circled. Figure 9 provides a Southern blot comparing rat α(l,3)GT and iGb3 synthase. Rat (Lanes 1 & 3) or murine (Lane 2) genomic DNA was digested with PvuII and hybridised with either the cDNA of rat α(l,3)GT (Lane 1 & 2) or iGb3 synthase (Lane 3).

Figure 10 shows a comparison of exon boundaries between rat α(l,3)GT and murine α(l,3)GT. Comparing the coding exon boundary sequences (exons 4, 5, 6, 7, 8 and 9) between the genomic sequences from rat and murine α(l,3)GT. Nucleotide differences are circled between the two sequences.

Figure 11 provides an alignment of the putative amino acid sequences of the rat α(l,3)GT with murine, pig, marmoset and ox α(l,3)GT and rat iGb3 synthase. The identical residues between species are boxed.

Figure 12 shows a multi-tissue Northern blot comparing rat α(l,3)GT and iGb3 synthase expression. Poly A+ RNA purified from 8 rat tissues : heart (Lane 1), brain (Lane 2), spleen (Lane 3), lung (Lane 4), liver (Lane 5), skeletal muscle (Lane 6), kidney (Lane 7), testis (Lane 8) was hybridized with either the cDNA of rat αl,3GT, (A), or iGb3 synthase, (B). Figure 13 shows cell surface Gal(l,3)Gal expression comparing rat αl,3GT and iGb3 synthase. CHOP cells were transfected with 1 ug of DNA of the following constructs: r αl,3GT, m αl,3GT, r iGb3 synthase, Mock (pCDNAl); 48 hrs post transfection they were cell surfaced stained with IB4-FITC and fluorescence measured by FACS analysis. Transfected cells ( "~~) and Mock transfected ( ).

Figure 14 shows cell surface Forssman expression for rat αl,3GT and iGb3 synthase. CHOP cells were co-transfected with equal ratios of each construct of either; pCDNAl with either rat αl,3GT, iGb3 synthase; or dog Forssman synthase with either rat αl,3GT or iGb3 synthase. 48 hrs post tranfection they were cell surfaced stained with IB4-FITC and fluorescence measured by FACS analysis. Transfected cells and non-transfected cells ( ), pCDNAl alone ( ). Figure 15 shows immunoprecitation of cell surface Gal(l,3)Gal expressing glycoproteins. CHOP cells were transfected with either pCDNAl (Lane 1 & 5), murine αl,3GT (Lane 2 & 6), rat αl,3GT (Lane 3 & 7) or iGb3 synthase (Lane 4 & 8) and then immunoprecipitated with IB4 conjugated Sepharose beads. Binding was determined to be Gal(l,3)Gal specific by competitive binding with 100 mM melibiose (Lanes 5-8). Figure 16 provides the results of glycolipid analysis of iGb3 synthase. CHOP cells were transfected with either pCDNAl (Lane 1) or iGb3S (Lane 2). The cells were metabolically radiolabeled with ^H-Gal and the glycolipids extracted. The extracted lipids were resolved on HPTLC plates in Chloroform:Methanol:Water (65:35:8). To determine the number of neutral sugars present, purified rabbit red blood cell glycolipids containing Ceramide Tri-Hexose (Gal(l-4)Lac-Cer, (CTH) and Ceramide Penta-Hexose (Gal(l,3)Gal(l-4)GlcNAc(l-3)Lac-Cer (CPH) were used. The iGb3 synthase band was extremely faint on the autoradiograph an its position on the plate has been marked (^*).

Figure 17 provides the results of staining Gal o/o thymocytes for Galα(l,3)Gal after treatment with αgalactoside, pepsin, and αgalactoside after pepsin digestion.

Figure 18 shows a Southern blot analysis of genomic DNA for C57BL/6 and Gal o/o mice.

EXAMPLES:

Example 1: Isolation and sequencing of a genomic clone encoding porcine iGb3 synthase.

A part of a pig genomic clone was isloated from a genomic library in the lambda vector (CLONTECH Laboratories, Inc). The library was screened using a cDNA probe containing the rat coding sequence (Genbank, Accession # AF248543). This sequence represents part of coding exon 5 of the iGb3 synthase gene (Fig 1).

The complete coding sequence of the mouse iGb3 synthase gene was obtained by reverse PCR from a mouse thymus library using 5' and 3' primers from rat sequence [5'- primer: 5'-cccaagcttatggctctggagggactcagg-3' (SEQ ID NO: 8); 3' primer: 5'- cgggatccctaggttcgcaccagtgcgta-3' (SEQ ID NO: 9)]. The sequence was confirmed by comparison with genomic data retrieved from NCBI, BCM, CSH and ENSEMBLE databases (Fig 2).

The mouse iGb3 synthase gene consists of 5 coding exons (upper case in Fig 3; nnnn represents unidentified number of nucleotides). The sequence was obtained by comparison with genomic data retrieved from NCBI, BCM, CSH and ENSEMBLE databases.

The human iGb3 synthase gene consists of 5 coding exons (upper case in Fig 4; nnnn represents unidentified number of nucleotides). The sequence was obtained by comparison with genomic data retrieved from NCBI, BCM, CSH and ENSEMBLE databases.

Example 2: Effects of transfecting cells with iGb3 DNA clone.

CHOPS or COS cells were transfected with clones encoding iGb3 synthase, αl,3GT, αl,2 fucosyltransferase (FT) in the following manner: iGb3 synthase with FT or with αl,3GT and FT. Expression of Galαl,3Gal was measured by IB4 lectin, purified human anti-Gal antibodies, or murine anti-Gal monoclonal antibody. H-antigen was measured using UAE1 lectin. This was done in accordance with Sandrin MS, etal. "Enzymatic remodeling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement-mediated cytolysis", Nature Medicine, 1:1261-1267 (1995). The results are shown in Figures 5a to d. Expression of Galα(l,3)Gal was detected using the 1B4 lectin (Fig 5a), purified human anti-Galα(l,3)Gal (Fig 5b) or the mouse anti- Galα(l,3)Gal monoclonal antibody. In contrast to αl,3GT, no decrease in Galα(l,3)Gal was seen using IB4 or purified human anti-Galα(l,3)Gal (Fig 5c) after co-transfection with FT. However, using the mouse monoclonal anti-Galα(l,3)Gal antibodies, a decrease in Gal α(l,3)Gal was seen after co- transfection with FT(Fig 5d), suggesting that the epitope produced in this transfected cell is a complex epitope.

Similar results were seen in COS cells (Table 1).

The results show that in the cells transfected with iGb3 synthase + FT, expression of the gal transferase was higher than that for the ccl,3GT + FT transfectants. This suggested that in the mouse, the iGb3 synthase gene product is not the same as that of αl,3GT.

TABLE 1: Expression of Galα(l,3)Gal after transfection.

Example 3: Gene product of the murine iGb3 synthase DNA molecule.

Northern blots of murine iGb3 synthase were produced in accordance with instructions supplied with the probe (CLONTECH Laboratories, Inc). The results indicated that the iGb3 synthase was expressed in selected tissues: brain, eye, heart, skeletal muscle, pancreas, thyroid, thymus, submaxillary gland, testis, ovary, prostate, epididymus and uterus were positive, whereas liver, lung, kidney, smooth muscle and spleen were weak (Table 2). The results may be compared with ocl,3GT which is a ubiquitous enzyme.

TABLE 2: Comparison of mRNA expression in the mouse.

Dot blot of polyA⁺ RNA probed with iGb3 synthase and αl,3GT cDNA inserts, washed and autoradiographed. += binging of probe; - = no binding of probe; +/- = weak binding of probe. Note under the conditions used the two probes do not cross hybridise.

Immunohistochemical analysis of Gal o/o mice using anti-Galα(l,3)Gal monoclonal antibodies mAbl and mAb2 (Austin Research Institute, Heidelberg, Victoria, Australia), showed some residual Galα(l,3)Gal in certain tissues (ie thymus, heart liver and kidney), which suggested a second αl,3 galactosyltransferase (Table 3).

TABLE 3: Expressionof Galα(l,3)Gal using monoclonal antibodies.

These results suggested that iGb3 synthase may be responsible for the production of an epitope for anti-galα(l,3)gal antibodies.

Example 4: Southern blots of αl,3GT and iGb3 synthase.

Southern blot analysis of genomic DNA from mouse, pig and sheep show that the genes encoding the iGb3 synthase and αl,3GT are distinct. That is, murine genomic DNA digested with BamHI shows a 7 kb band when probed with GT2 cDNA, compared with a 3.1 kb band when probed with GTl cDNA, EcoRI digested DNA shows a 3.6 kb band with GT2 probe and 2.8 kb with GTl probe. Pig genomic DNA digested with BamHI shows a 4.9 kb band when probed with GT2 cDNA, compared with a 2.8 kb band when probed with GTl cDNA, EcoRI digested DNA shows a 4.9 kb band with GT2 probe and 5.5 kb with GTl probe. Sheep genomic DNA digested with BamHI shows a 2.7 kb band when probed with GT2 cDNA, compared with a 4.9 kb band when probed with GTl cDNA, EcoRI digested DNA shows a 4.9 kb band with GT2 probe and 7.5 kb with GTl probe (Fig 6). A Southern blot of a λ clone of genomic DNA gave a band of ~50kb. On sequencing, this appeared to correlate with exon 9 of porcine αl,3GT (SEQ ID NO: 1).

Example 5: Porcine iGb3 synthase from Pig Endothelial PIEC cells.

Using reverse transcribed mRNA from the PIEC (pig endothelial) cell line as a template for PCR, a band of ~lkb was obtained. This band hybridised with the iGb3 synthase cDNA insert, and using internal primers specific from SEQ ID NO: 1

(nucleotides 1-1023), a 480 bp fragment identical in sequence to SEQ ID NO: 1 was obtained. The results suggested iGb3 synthase to be the product of a gene distinct from that encoding αl,3GT.

No hybridisation was observed using the iGb3 synthase cDNA insert, even at low stringency, on a panel of human tissue mRNA. This suggests that the human iGb3 synthase is a non-transcribed pseudogene.

Example 6: Production of iGb3 synthase knockout cells.

An iGb3 synthase knockout cell line is produced by inserting a selectable marker gene such as neo^r in the antisense orientation of coding exon 5 of the pig, mouse or human DNA. A convenient restriction site is used to introduce the selectable gene, but unique sites may be engineered into this coding exon to provide nonsense or missense mutations into the exon. The knockout construct is then engineered into any common plasmid vector (eg pBluescript) to produce sufficient insert DNA for DNA sequencing and targeted disruption in cells. The knockout insert is then purified from the plasmid DNA and introduced into cell lines and fibroblasts from pig and mice by standard methods for introduction of foreign DNA into cells (eg lipofectamine, DEAE-Dextran, electroporation or other methods known to those skilled in the art), and the transfected cell cultured in the presence of the drug for selection (eg G418 for neo^r) to select for stable integration of the knockout DNA. Specific integration in the iGb3 synthase gene is determined by PCR and genomic Southern blots. Nuclei from cells with homologous recombination will be used to produce live animals (mice or pigs) by the well known nuclear transfer cloning technique.

Example 7: Isolation of a cDNA clone encoding rat αl,3GT and comparison against rat iGb3 synthase clone.

To clone a rat (1,3)GT, the structure of the mouse αl,3GT (Joziasse DH, etal. "Murine alpha 1,3-galactosyltransferase. A single gene locus specifies four isoforms of the enzyme by alternative splicing." J. Biol. Chem., 267, pp5534-5541 (1992)) was used with oligonucleotide primers designed to the 5' and 3' ends of the mouse αl,3GT coding sequence [5' primer: 5'-cccaagcttatgaatgtcaagggaaaagtaatc-3' (Hindlll site underlined)

(SEQ ID NO: 10); 3' primer: 5'-gctctagatcagacattatttctaaccaaattata-3' (Xbal site underlined) (SEQ ID NO: 11)], the rationale for using mouse αl,3GT was that it is likely to have homology to a putative rat αl,3GT. PCR using the oligonucleotide primers and rat spleen cDNA was used to isolate a ~lkb which was amplified and subcloned into the expression vector pCDNAl (invitrogen). Two independent clones were sequenced in both the 3' and 5' directions (Fig 8). The nucleotide sequence of the rat αl,3GT contains an open reading frame of 1014 bp encoding a protein of 337 amino acids.

A DNA blot of genomic DNA isolated from rat spleen cells probed with rat αl,3GT cDNA (Fig 9, Lane 1) shows 3 bands after digestion with PvuII: 3.8 Kb, 2.5 Kb and 1.7 Kb. These bands were of similar sizes to mouse DNA digested with the same enzyme (Fig 9, Lane 2). In contrast probing with iGb3 synthase cDNA showed 4 bands: 1.1 Kb, 0.6 Kb, 0.3 Kb and 0.2 Kb (Fig 9, Lane 3), which further indicates that the rat iGb3 synthase and the art αl,3GT genes are distinct.

The rat genomic sequence was obtained by searching a rat genomic database (http:/ /www.ncbi.n/m.nih.gov/BLAST/) using the cloned αl,3GT cDNA as a reference, and the retrieved sequence confirmed the rat αl,3GT cDNA sequence isolated by PCR. The only exception was for the 3' end, precisely where the mouse αl,3GT designed primer annealed to the template during PCR amplification which had introduced three errors in the nucleotide sequence T- G (999nt), A-G (1004nt), and T-C (1005nt) (Fig 8). From the genomic sequence, the gene organization of the rat αl,3GT was deduced and shows a preservation and arrangement of exons identical to that of murine αl,3GT.

The predicted amino acid sequence (including exons 5 and 6) was aligned with other known αl,3GT and the iGb3 synthase (Fig 10). The alignment shows that rat αl,3GT has very high amino acid identity when compared with mouse, pig and ox ocl,3GT:- 90 %, 76%, and 75 % respectively (Fig 11). However, the αl,3GT cloned here differs to the rat iGb3 synthase (ie with an amino acid identity of only 42 % between the two), clearly demonstrating that rat αl,3GT has a much higher amino acid identity to αl,3GT from other species than to its intra-species homologue iGb3 synthase.

The greatest identity between rat ccl,3GT and iGb3 synthase is their catalytic domains, exons 8 and 9, with an identity of 51 %. Their greatest divergence is at the N- terminal end of the proteins: the cytoplasmic tail, transmembrane and stem regions with no amino acid identity able to be determined between the two transferases. Therefore, based on amino acid alignments alone the evidence is strong that two distinct, Gal(l,3)Gal synthesising genes exist in the rat, one, the gene encoding rat αl,3GT and the other, the gene for rat iGb3 synthase. The transcripts of αl,3GT and iGb3 synthase were determined on a multi- tissue Northern (Fig 12). The rat αl,3GT showed a single band of ~3.1kb (Fig 12A). This compares with the mRNA sizes of murine αl,3GT 3.6kb (Joziasse DH, 1992 supra), bovine αl,3GT 3.6-3.4kb (Joziasse DH, etal. "Bovine alpha 1.3-galactosyltransf erase: isolation and characterisation of a cDNA clone. Identification of homologous sequences in a human genomic DNA." J. Biol. Chem., 264, pp 14290-14297 (1989)) and pig αl,3GT 3.9kb (Sandrin MS, etal. "Characterisation of cDNA clones for porcine αl,3galactosyltransferase: the enzyme generating the Gaα(l,3)Gal epitope." Xenotransplantation, 1, pp 81 - 88 (1994)) and suggests that, like other species, the rat αl,3GT contains a large 3' untranslated region. The iGb3 synthase shows two bands one of ~4.0kb and the other ~1.8kb (Fig 12B). The reason for two transcripts of the iGb3 synthase gene is unclear but is most likely a consequence of the two transcripts possessing different 3' untranslated regions. Thus, as predicted from the DNA sequence, the two rat transferases are different.

A further difference was found in the tissue distribution in that rat αl,3GT and iGb3S were both expressed in heart, brain, spleen, lung, kidney (Fig 11); rat ocl,3GT expression is higher in spleen tissue, whereas iGb3 synthase expression is higher in lung tissue. Liver was the only tissue which expressed rat αl,3GT and not iGb3 synthase. In skeletal muscle and testis, no expression of either transferase was present. Therefore, from the Nothern blot analysis, it is apparent that the rat αl,3GT and iGb3 synthase differ in their transcript sizes and in their pattern of tissue expression.

Example 8: Expression studies with CHOP cells expressing rat αl,3GT, rat iGb3 synthase and murine l,3GT.

Expression studies were conducted to demonstrate that the rat αl,3GT cDNA is functional and that this cDNA along with the cDNA clone for iGb3 synthase can both lead to the expression of Gal(l,3)Gal.

CHOP cells, a CHO cell line expressing the polyoma large T antigen were maintained in Dulbecco's Modified Eagles Medium (DMEM) (Cytosystems) with 10% fetal bovine serum. The cells were seeded in 6 well plates (Linbro) at 3 x 10⁵ cells and 24 hrs later transfected using Lipofectamine Plus Reagent (Gibco BRL Life Technologies). The following cDNA's were transfected, murine αl,3GT, rat αl,3GT and rat iGb3 synthase, using 1 ug of each per well. After 48 hrs the cells were analysed for expression.

The Gal(l,3)Gal epitope was detected on the CHOP cells by binding of the IB4 lectin (Fig 13A) and furthermore this level of expression was similar to that seen after transfection with the murine ocl,3GT (Fig 13B). These studies indicate that the rat αl,3GT is similar to the previously described mouse, bovine and pig transferases in function. The expression of iGb3 synthase showed weak binding with IB4 compared to the expression of rat and mouse αl,3GT (Fig 13C). Thus, both genes can separately synthesise IB4 binding structures.

To determine whether rat αl,3GT and iGb3synthase were functionally different, the ability of iGb3 synthase to cause the down stream expression of the globo-series pathway was exploited (Keusch JJ, supra). The transferases were co-expressed with the Forssman synthase and detected using an anti-Forssman antibody. Forssman expression only occurred when iGb3 synthase was present, due to the synthesis of the iGb3 structure and initiation of the globo-series pathway (Fig 14). The lack of Forssman expression for rat c ,3GT (Fig 14) was not surprising and supported the sequence data, that this is the αl,3GT homologue, a distinct transferase to iGb3 synthase as similar evidence for the lack of Forssman expression has been shown previously when mouse αl,3GT was expressed in CHO cells (Keusch JJ, supra).

Moreover, it was found that rat αl,3GT and iGb3 synthase have clear differences in their ability to galactosylate glycolipids for the globo-series pathway with iGb3 synthase only having the capability of forming iGb3. To determine whether their glycosylation of glycoproteins was also different, immunoprecipitated proteins from CHOP cells expressing either transferase were analysed by SDS-PAGE and Western blotted. The rat αl,3GT and mouse αl,3GT showed patterns with protein bands at approximately 120KDa, 90KDa and 52KDa (Fig 15, lanes 2 & 3). The interaction was specific for Gal(l,3)Gal as determined by inhibiting with Melibiose (Fig 15, lanes 5-8), a carbohydrate structure capable of blocking the Gal(l,3)Gal-IB4 interaction. However, Gal(l,3)Gal containing proteins could not be immunoprecipitated from iGb3 synthase transfected cell lysates (Fig 15, lane 4). Thus ,the rat αl,3GT, like other αl,3GT, was able to synthesise Gal(l,3)Gal onto glycoproteins destined for the cell surface whereas iGb3 synthase is only capable of synthesising Gal(l,3)Gal onto glycolipids, distinguishing two different glycosylation pathways for the two transferases.

In further studies, designed to compare the synthesis of oc-Gal glycolipids by the two rat transferases, transfected CHOP cells were metabolically labeled with ^H-Gal and the glycolipids extracted and size separated by HPTLC (Fig 16). Mock transfected cells show three doublet bands corresponding to 1, 2 and 5 Neutral sugars (Fig 16, Lane 1). Glycolipids with identical carbohydrate structures can migrate as doublets as different lipid tail compositions subtly influence their of migration properties on the HPTLC plates and is a common observation of cellular glycolipid extracts. The two doublets migrating further than ceramide tri-hexose (CTH) corresponds to Gal-Cer and Lac-Cer. The third doublet migrating with ceramide penta-hexose (CPH) was resistance to α-galactosidase, β-galactosidase, α-N-acetylgalactosaminidase (data not shown) and is most likely GM3. This is in agreement with other investigators who have shown that CHO cells synthesise simple glycolipids up to Lac-Cer and also GM3. When CHOP cells were transfected with iGb3 synthase, a very weak doublet migrating with CTH corresponding to iGb3 synthase was found together with a stronger doublet below CTH corresponding to iGb4 and a number of bands migrating with CPH and below (Fig 16, Lane 2). The strong iGb4 doublet is caused by the endogenous expression of the Gb4 synthase (Gb4S), converting synthesised iGb3 into iGb4 by the addition of a GalNAc. It is the endogenous expression of Gb4S which completes the globo-series pathway allowing the expression of iso- Forssman (iGb5) when iGb3 synthase and Forssman synthase are co-transfected in CHOP cells (Figure 16). When rat αl,3GT was transfected, the same banding pattern as mock transfected cells was observed as did pig αl,3GT (data not shown). Thus, these αl,3GTs are not capable of forming iGb3 nor can they synthesis the downstream glycolipids as observed for iGb3 synthase. Hence, at least in CHOP cells, αl,3GT is restricted to glycosylating proteins while iGb3 synthase is restricted to the glycosylation of glycolipids. However, it should be noted that αl,3GT transferases are considered able to glycosylate glycolipids, the simplest of which is CPH, Gal(l,3)Gal(l-4)GlcNAc(l-3)Lac-Cer (Galilu U, etal. "A unique natural human IgG antibody with anti-α-galactosyl specificity." J. Exp Med., 160, pp 1519-1531 (1984)). This structure was not synthesised in αl,3GT transfected CHOP cells, probably due to the limited synthesis of glycolipid structures in these cells and therefore the lack of the precursor glycolipids which αl,3GT can use as an acceptor.

Example 9: Masking of αGal epitope on thymocytes of Gal knock-out mice.

Thymocytes were isolated from Gal knock-out mice and 5xl0⁶ cells/ ml were treated with a protease, trypsin (or pepsin) or a glycosidase, α-galactosidase (which removes the terminal galactose from the aglycone backbone). In cells treated with trypsin or pepsin, the concentration of the enzyme was 0.25% wt/v. The cells were incubated for 20 minutes at 37 C after which the enzymic reaction was quenched with FCS (10% final concentration) at 4 C. The cells were then washed in PBS /BSA. For those cells treated with galactosidase, about 1 unit of the enzyme in citrate phosphate buffer was added. Cells were incubated for 60 minutes at 37 C, and washed as described above. Washed cells were then stained for binding to IB4 as described above and examined by FACS. Figure 17 shows an increase in IB4 binding by Gal knock-out thymocytes treated with either a proteolytic or a glycolytic enzyme. Treatment with a protease resulted in a substantial increase in the number of cells binding to IB4, compared with cells that were untreated with enzymes, or treated with the galactosidase only. Cells treated with both types of enzymes bound IB4 at a level intermediate to those cells treated with both the protease and the glycosidase.

The results suggest that Gal knock-out mice have an α Gal epitope that is masked on the surface of thymocytes. That this epitope is produced by iGb3 was confirmed by Northern blot analysis. Figure 18 (right panel) is a Southern blot showing presence of the iGb3 synthase gene in αGal wild type (WT), αGal knock-out and hemizygote (+/-) mice. The left panel shows the presence of GT in WT and hemizygotes, and an inactivated gene of about ~7kb.

Example 10: Susceptibility of iGb3 synthase expressing cells to undergo hyperacute rejection.

Human 293 cells were transfected with the iGb3 synthase cDNA and the selectable gene Neo, and selected for growth in media containing G418. Resistant cells were cloned by limiting dilution and examined for expression of Galα(l,3)Gal epitope using the IB4 lectin. Galα(l,3)Gal epitope expressing cells were transplanted to SCID mice under the kidney capsule, and 7 days later the mice were challenged with normal human serum (1.5 ml ip). 72 hours later the animals were sacrificed, the kidneys removed and the grafts examined histologically. These examinations showed that the iGb3 synthase expressing 293 cells were destroyed, in contrast 293 cells were not. These results suggest that the Galα(l,3)Gal epitope produced by iGb3 synthase can bind human antibodies and such cells undergo hyperacute rejection (HAR) as do cells with Galα(l,3)Gal epitope produced b GT.

DISCUSSION:

The results presented herein show two distinct Gal(l,3)Gal synthesising pathways with two transferases, one contributing to each pathway. Until now the central dogma for many animal species, was that Gal(l,3)Gal was the sole synthesis of one transferase usually the αl,3GT. Already an iGb3 structure has been identified in hog stomach mucosa, in a fucosylated form (Fucl,2)Gal(l,3)LacCer) (Barker AE, etal. "Biochemical and enzymatic characterisation of blood group ABH and related histo-blood group glycosphingolipids in the epithelial cells of porcine small intestine." Glycobiology, 7, pp 943-953 (1997)), providing evidence that iGb3 synthase is expressed in pig. If iGb3 synthase is expressed in other tissues of the pig, as is anticipated, and if Gal(l,3)Gal glycolipid structures are involved in hyperacute rejection, it is likely to present a problem for xenotransplantation. Thus strategies to remove Gal(l,3)Gal in pigs, which have assumed the presence of a single transferase and thereby focus on the removal of the αl,3GT or its product, may be improved by also focussing on the removal of iGb3 synthase or its product.

It will be understood that the present invention has been described with reference to the most preferred embodiment, and that any other variations or embodiments apparent to a person skilled in the art are contemplated as part thereof.

Claims

CLAIMS:

1. A substantially pure or isolated nucleic acid molecule comprising the nucleotide sequence according to SEQ ID NO: 1, or homologous, variant or derivative sequences thereof.

2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a homologous, variant or derivative sequence of the nucleotide sequence of SEQ ID NO: 1, and wherein said homologous, variant or derivative sequence hybridises to the sequence of SEQ ID NO: 1 under stringent conditions of hybridisation.

3. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes an iGb3 synthase.

4. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes porcine iGb3 synthase.

5. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 1.

6. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2 to 4.

7. A nucleic acid molecule consisting of a nucleotide sequence according to a fragment of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4, or a homologous, variant or derivative sequence thereof.

8. The nucleic acid molecule of claim 7, wherein said fragment comprises about 50 to 100 % of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4.

141314414

9. The nucleic acid molecule of claim 7, wherein said fragment comprises about 55 to 95% of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4.

10. The nucleic acid molecule of claim 7, wherein said fragment comprises about 60 to 85% of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4.

11. The nucleic acid molecule of claim 7, wherein said fragment comprises about 65 to ¹o of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4.

12. The nucleic acid molecule of claim 7, wherein said fragment comprises the nucleotide sequence of any one of SEQ ID NOS: 5 to 7.

13. A mutant of the nucleotide sequence of any one of SEQ ID NOS: 1 to 7 or a fragment thereof, wherein said mutant comprises one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s).

14. The mutant of claim 13, wherein said mutant comprises a missense or nonsense mutation.

15. The mutant of claim 13, wherein said mutant comprises one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s) in at least one exon of the nucleotide sequence of any one of SEQ ID NOS: 1 to 7.

16. The mutant of claim 13, wherein said mutant comprises one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s) in exon 5 and /or exon 9 of the nucleotide sequence of any one of SEQ ID NOS: 1 to 7.

17. The mutant of claim 13, wherein said mutant comprises one or more nucleotide substitution(s), insertion(s), addition(s) or deletion(s) in the nucleotide sequence of any of SEQ ID NOS: 5 to 7.

18. A peptide encoded by a fragment of any one of SEQ ID NOS: 1 to 7, or homologous, variant or derivative sequences thereof.

19. An antibody or fragment thereof which is reactive with the peptide of claim 18.

20. A nucleic acid construct for disrupting a gene encoding a glycosyltransferase such as an iGb3 synthase gene, said construct comprising the nucleotide sequence of any one of SEQ ID NOS: 5 to 7, or a fragment thereof.

21. The nucleic acid construct of claim 20, wherein said construct comprises a fragment of at least 10 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOS: 5 to 7.

22. The nucleic acid construct of claim 20, wherein said construct comprises a fragment of at least 20 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOS: 5 to 7.

23. The nucleic acid construct of claim 20, wherein said construct comprises a fragment of at least 50 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOS: 5 to 7.

24. A nucleic acid construct for disrupting a gene encoding a glycosyltransferase such as an iGb3 synthase gene, said construct comprising a mutant of the nucleotide sequence of any one of SEQ ID NOS: 5 to 7, or a fragment thereof.

25. A mammalian cell comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted, such that expression of functional iGb3 synthase is disrupted.

26. The mammalian cell of claim 25, wherein the cell is a porcine, bovine, murine, ovine, equine, or primate cell.

27. The mammalian cell of claim 25, wherein the cell is a porcine, ovine or primate cell.

28. The mammalian cell of claim 25, wherein the cell is a porcine cell.

29. The mammalian cell of any one of claims 25 to 28, wherein the iGb3 synthase gene is disrupted in a manner whereby the disruption inhibits the expression of the gene, decreases the expression of the gene and /or inhibits or decreases the formation of a functional gene product.

30. The mammalian cell of claim 29, wherein there is substantially no expression of functional iGb3 synthase by said mammalian cell.

31. A method of disrupting a glycosyltransferase gene such as an iGb3 synthase gene in a mammalian cell, the method comprising the step of disrupting the expression of said gene using a nucleic acid construct according to any one of claims 20 to 24.

32. The method of claim 31, wherein the iGb3 synthase gene is disrupted in a manner whereby the disruption inhibits the expression of the gene, decreases the expression of the gene and /or inhibits or decreases the formation of a functional gene product.

33. The method of claim 32, wherein there is substantially no expression of functional iGb3 synthase by said mammalian cell.

34. The method of claim 33, wherein the gene is the porcine iGb3 synthase gene.

35. A method of xenotransplantation, comprising the step of transplanting a donor- cell, -tissue or -organ into a recipient, the cell, tissue or organ comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted.

36. A non-human, transgenic donor animal comprising a disrupted iGb3 synthase gene or wherein the iGb3 synthase gene has been deleted.

37. The donor animal of claim 36, wherein the donor animal is selected from the group consisting of porcine, bovine, equine, murine, ovine and primate (other than human) animals.

38. The donor animal of claim 36, wherein the donor animal is a pig.

39. The donor animal of any one of claims 36 to 38, wherein both the iGb3 synthase and αl,3GT genes have been disrupted or deleted.

40. A mammalian cell comprising a gene encoding αl,2 fucosyltransferase (FT), and wherein the iGb3 synthase gene has been disrupted or deleted.

41. The mammalian cell of claim 40, wherein the αl,3GT gene has also been disrupted or deleted.

42. A mammalian cell comprising a chimeric gene, said chimeric gene comprising a nucleotide sequence encoding a localisation signal, wherein the localisation signal comprises a cytoplasmic domain from a first kind of glycosyltransferase and a catalytic site from a second kind of glycosyltransferase, and wherein the iGb3 synthase gene has been disrupted or deleted.

43. The mammalian cell of claim 42, wherein the chimeric gene comprises the cytoplasmic domain of αl,3GT and the catalytic domain from another glycosyltransferase selected from the group consisting of FT and Se.

44. A nucleic acid construct for reducing expression of Galα(l,3)Gal in a mammalian cell, said construct comprising a first nucleotide sequence from a gene selected from the group consisting of the iGb3 synthase and αl,3GT genes, and a second nucleotide sequence from a gene selected from the group consisting of the FT and Se genes, wherein said first nucleotide sequence is capable of disrupting the iGb3 synthase and /or αl,3GT genes.

45. Cells, tissues or organs harvested from a transgenic animal, wherein said cells, tissues or organs comprise disrupted or deleted iGb3 synthase and αl,3GT genes.

46. The cells, tissues or organs of claim 45, wherein said cells, tissues or organs further comprise a chimeric gene selected from the group encoding GT-HT and GT-Se.

47. The cells, tissues or organs of claim 45 or 46, wherein the transgenic animal is selected from the group consisting of porcine, bovine, equine, murine, ovine and primate (other than human) animals.

48. The cells , tissues or organs of claim 46 or 47, wherein the transgenic animal is a Pig-

49. A method of treating a human cancer or Graves' disease in a subject, comprising administering to said subject an agent to reduce or eliminate the expression of functional iGb3 synthase by diseased cells.