Transgenic Ungulates Expressing CTLA4-IG and Uses Thereof

Abstract

The present invention provides ungulates, including pigs, expressing CTLA4-Ig, as well as tissue, organs, cells and cell lines derived from such animals. Such animals, tissues, organs and cells can be used in research and medical therapy, including xenotransplanation. In addition, methods are provided to prepare organs, tissues and cells expressing the CTLA4-Ig for use in xenotransplantation, and nucleic acid constructs and vectors useful therein.

1 . A transgenic ungulate that expresses a CTLA4 peptide, wherein the CTLA4 peptide is fused to an immunoglobulin (Ig). 
     
     
         2 . The transgenic ungulate of  claim 1 , wherein the CTLA4 peptide is porcine CTLA4. 
     
     
         3 . The transgenic ungulate of  claim 1 , wherein the CTLA4 peptide is human CTLA4. 
     
     
         4 . The transgenic ungulate of  claim 1 , wherein the CTLA4 peptide is truncated. 
     
     
         5 . The transgenic ungulate of  claim 1 , wherein the CTLA4 peptide is mutated. 
     
     
         6 . The transgenic ungulate of  claim 5 , wherein the CTLA4 peptide is mutated by substitution of (a) an alanine at position +29 with a tryptophan, and (b) a leucine at position +104 with a glutamic acid. 
     
     
         7 . The transgenic ungulate of  claim 1 , wherein the CTLA4 peptide is modified by the addition of an intracellular retention signal. 
     
     
         8 . The transgenic ungulate of  claim 1 , wherein the Ig is human Ig. 
     
     
         9 . The transgenic ungulate of  claim 1 , wherein the Ig is IgG. 
     
     
         10 . The transgenic ungulate of  claim 9 , wherein the IgG is IgG1 or IgG4. 
     
     
         11 . The transgenic ungulate of  claim 1 , wherein the CTLA4 is porcine and the Ig is human. 
     
     
         12 . The transgenic ungulate of  claim 1 , wherein the ungulate is porcine. 
     
     
         13 . A tissue derived from the transgenic ungulate of  claim 1 . 
     
     
         14 . An organ derived from the transgenic ungulate of  claim 1 . 
     
     
         15 . A cell derived from the transgenic ungulate of  claim 1 . 
     
     
         16 . A non-human transgenic cell comprising a nucleotide sequence encoding human CTLA4 and a nucleotide sequence encoding porcine CTLA4. 
     
     
         17 . A tissue comprising the non-human transgenic cell of  claim 16 . 
     
     
         18 . An organ comprising the non-human transgenic cell of  claim 16 . 
     
     
         19 . An animal comprising the non-human transgenic cell of  claim 16 . 
     
     
         20 . A method of reducing or eliminating cell mediated rejection of a xenotransplant in a recipient comprising providing xenogenic cells, tissues or organs to a recipient which have been genetically modified to express CTLA4 fused to an immunoglobulin (Ig). 
     
     
         21 . The method of  claim 20 , wherein the xenogenic tissues or organs are provided. 
     
     
         22 . The method of  claim 20 , wherein the CTLA4 is porcine. 
     
     
         23 . The method of  claim 20 , wherein the CTLA4 is human. 
     
     
         24 . The method of  claim 20 , wherein the cells, tissue or organs have been genetically modified to express human CTLA4 and porcine CTLA4. 
     
     
         25 . The method of  claim 20 , further comprising administering soluble CTLA4 to the recipient. 
     
     
         26 . The method of  claim 21 , wherein the soluble CTLA 4 is porcine. 
     
     
         27 . The method of  claim 21 , wherein the soluble CTLA4 is human. 
     
     
         28 . A transgenic ungulate that expresses a CTLA4 peptide, wherein expression of the CTLA4 peptide is under the control of a tissue-specific promoter. 
     
     
         29 . The transgenic ungulate of  claim 28 , wherein the tissue specific promoter is selected from the group consisting of liver-specific promoters, lymphoid-specific promoters, T-cell receptor and immunoglobulin promoters, endothelial promoters, pancreas-specific promoters, and mammary gland-specific promoters. 
     
     
         30 . The transgenic ungulate of  claim 28 , wherein the CTLA4 peptide is human CTLA4 or porcine CLTA4. 
     
     
         31 . The transgenic ungulate of  claim 28 , wherein the ungulate is porcine. 
     
     
         32 . A transgenic animal that expresses a CTLA4 peptide, wherein expression of the CTLA4 peptide is under the control of a regulatable promoter. 
     
     
         33 . The transgenic animal of  claim 32 , wherein the animal is an ungulate. 
     
     
         34 . The transgenic animal of  claim 33 , wherein the ungulate is porcine. 
     
     
         35 . The transgenic animal of  claim 32 , wherein the inducible promoter is selected from the group consisting of metallothionein promoters, tetracycline-regulated promoters, ecdysone-inducible promoter, cytochrome P450 inducible promoters, CYP1 A1 promoters, and mifepristone promoters. 
     
     
         36 . The transgenic animal of  claim 1 , further comprising a second genetic modification. 
     
     
         37 . The transgenic animal of  claim 36 , wherein the second genetic modification eliminates or reduces the functional expression of a gene. 
     
     
         38 . The transgenic animal of  claim 37 , wherein the gene is alpha-1,3-galactosyltransferase (α(1,3)GT) gene. 
     
     
         39 . The transgenic animal of  claim 36 , wherein the second genetic modification adds functional expression of a gene. 
     
     
         40 . The transgenic animal of  claim 39 , wherein the gene is tissue factor pathway inhibitor (TFPI) gene or a complement inhibitor gene. 
     
     
         41 . The transgenic animal of  claim 40 , wherein the complement inhibitor gene is DAF, MCP or CD59. 
     
     
         42 . A tissue or organ derived from the transgenic animal of  claim 36 . 
     
     
         43 . A cell derived from the transgenic animal of  claim 36 . 
     
     
         44 . A method for producing a transgenic ungulate that expresses CTLA4 peptide fused to an immunoglobulin (Ig), comprising introducing a nucleic acid construct or vector encoding the CTLA4 peptide fused to an immunoglobulin molecule into the genome of an ungulate cell. 
     
     
         45 . The method of  claim 44 , wherein the construct or vector is introduced by transfection. 
     
     
         46 . The method of  claim 45 , wherein transfection is accomplished by electroporation or lipofection. 
     
     
         47 . The method of  claim 40 , wherein the construct integrates into the genome. 
     
     
         48 . The-method of  claim 47 , wherein the integration is random. 
     
     
         49 . The method of  claim 47 , wherein the integration is targeted. 
     
     
         50 . The method of  claim 44 , wherein the expression of the CTLA4 peptide is under the control of a tissue-specific promoter. 
     
     
         51 . The method of  claim 44 , wherein the expression of the CTLA4 peptide is under the control of a regulatable promoter. 
     
     
         52 . A nucleic acid construct comprising a nucleotide sequence encoding a CTLA4 peptide operably linked to a tissue specific promoter, wherein the promoter is not a mammary-specific promoter. 
     
     
         53 . The nucleic acid construct of  claim 52 , wherein the promoter is not a neuron-specific promoter. 
     
     
         54 . The nucleic acid construct of  claim 52 , wherein the CTLA4 peptide is porcine CTLA4. 
     
     
         55 . The nucleic acid construct of  claim 52 , wherein the CTLA4 peptide is human CTLA4. 
     
     
         56 . The nucleic acid construct of  claim 52 , wherein the promoter is a pancreas-specific promoter. 
     
     
         57 . A method of reducing or eliminating cell mediated rejection of a xenotransplant in a recipient comprising providing xenogenic cells, tissues or organs to a recipient which have been genetically modified to express a CTLA4 peptide, wherein the expression of CTLA4 peptide is under the control of a tissue-specific promoter. 
     
     
         58 . The method of  claim 57 , wherein the xenogenic tissues or organs are provided. 
     
     
         59 . The method of  claim 57 , wherein the CTLA4 peptide is porcine. 
     
     
         60 . The method of  claim 57 , wherein the CTLA4 peptide is human. 
     
     
         61 . The method of  claim 57 , wherein the cells, tissues or organs further comprise a second genetic modification. 
     
     
         62 . The method of  claim 61 , wherein the second genetic modification eliminates or reduces functional expression of a gene. 
     
     
         63 . The method of  claim 61 , wherein the gene is alpha-1,3-galactosyltransferase (α(1,3)GT) gene. 
     
     
         64 . The method of  claim 61 , wherein the second genetic modification adds functional expression of a gene. 
     
     
         65 . The method of  59 , wherein the gene is tissue factor- pathway inhibitor (TFPI) gene or complement inhibitor gene. 
     
     
         66 . The method of  claim 57 , wherein the cells, tissue or organs have been genetically modified to expression human CTLA4 and porcine CTLA4. 
     
     
         67 . The method of  claim 57 , further comprising administering soluble CTLA4 to the recipient. 
     
     
         68 . The method of  claim 57 , wherein the soluble CTLA 4 is porcine. 
     
     
         69 . The method of  claim 57 , wherein the soluble CTLA4 is human.