Double knockout (gt/cmah-ko) pigs, organs and tissues

Abstract

The invention provides double knockout transgenic pigs (GT/CMAH-KO pigs) lacking expression of any functional αGAL and CMAH. Double knockout GT/CMAH-KO transgenic organs, tissues and cells are also provided. Methods of making and using the GT/CMAH-KO pigs and tissue are also provided.

We claim: 
     
         1 . A knockout pig comprising disrupted α(1,3)-galactosyltransferase and CMAH genes, wherein expression of functional α(1,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and when tissue from said pig is transplanted into a human, hyperacute rejection is decreased as compared to when tissue from a wild-type pig is transplanted into a human. 
     
     
         2 . Porcine organs, tissue or cells for transplantation into a human having reduced expression of αGal and Neu5Gc on the porcine organs, tissue or cells. 
     
     
         3 . A method for modifying a porcine organs, tissue or cells for transplantation into a human, the method comprising removing or reducing expression of αGal and Neu5Gc on the porcine organs, tissue or cells. 
     
     
         4 . The method of  claim 3  wherein the porcine organs, tissue, or cells are selected from the group consisting of skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, and components thereof. 
     
     
         5 . A method for making porcine organs, tissue or cells for transplantation into a human, the method comprising reducing expression of αGal and CMAH on the porcine organs, tissue or cells. 
     
     
         6 . The method of  claim 5  wherein said porcine organs, tissue, or cells are selected from the group consisting of skin, heart, liver, kidneys, lung, pancreas, small bowel, and components thereof. 
     
     
         7 . A knockout pig comprising disrupted α(1,3)-galactosyltransferase and CMAH genes, wherein expression of functional α(1,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and wherein when tissue from said knockout pig is transplanted into a human, thrombocytopenia is decreased as compared to when tissue from a wild-type pig is transplanted into a human. 
     
     
         8 . A knockout pig comprising disrupted α(1,3)-galactosyltransferase and CMAH genes, wherein expression of functional α(1,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and wherein a liver from said pig exhibits reduced uptake of human platelets when said liver is exposed to said human platelets. 
     
     
         9 . A method of increasing the duration of the period between when a human subject is identified as a subject in need of human liver transplant and when said human liver transplant occurs, said method comprising providing a liver from a knockout pig comprising disrupted α(1,3)-galactosyltransferase and CMAH genes, wherein expression of functional α(1,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig, and surgically attaching said liver from said knockout pig to said human subject in a therapeutically effective manner. 
     
     
         10 . The method of  claim 9 , wherein said liver from said knockout pig is internal to said human subject. 
     
     
         11 . The method of  claim 9 , wherein said liver from said knockout pig is external to said human subject. 
     
     
         12 . The method of  claim 9 , wherein said liver is directly or indirectly attached to said subject. 
     
     
         13 . A method of preparing organs, tissues, or cells for xenotransplantation into human patients with reduced rejection, the method comprising providing a transgenic pig as a source of transplant material wherein the transplant material is selected from the group consisting of organs, tissues, or cells, and wherein the pig masks or reduces the expression of at least two xenoreactive antigens on the transplant material. 
     
     
         14 . The method of  claim 13 , wherein the at least two xenoreactive antigens are αGal and Neu5Gc. 
     
     
         15 . A knockout pig comprising disrupted α(1,3)-galactosyltransferase and CMAH genes, wherein the disruption of said α(1,3)-galactosyltransferase gene is a 3 base pair deletion adjacent to a G to A substitution, wherein the disruption of said CMAH gene is a four base pair insertion and wherein expression of functional α(1,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig and when tissue from said knockout pig is transplanted into a human, hyperacute rejection is decreased as compared to when tissue from a wild-type pig is transplanted into a human. 
     
     
         16 . A method of improving symptoms of hyperacute rejection in a patient comprising transplanting porcine organs, tissue or cells having reduced expression of αGal and CMAH into a human, wherein the symptoms of hyperacute rejection are improved as compared to when tissue from a wild-type swine is transplanted into a human. 
     
     
         17 . A cell culture reagent derived from a knockout pig comprising disrupted α(1,3)-galactosyltransferase and CMAH genes, wherein the disruption of said α(1,3)-galactosyltransferase gene is a 3 base pair deletion adjacent to a G to A substitution, wherein the disruption of said CMAH gene is a four base pair insertion and wherein expression of functional α(1,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig, wherein said cell culture reagent is selected from the group comprising cell culture media, cell culture serum, cell culture additive and an isolated cell capable of proliferation. 
     
     
         18 . A method of producing a glycoprotein of interest, said method comprising the step of incubating an isolated cell capable of expressing said glycoprotein of interest with a cell culture reagent derived from a knockout pig comprising disrupted α(1,3)-galactosyltransferase and CMAH genes, wherein the amount of Neu5Gc or alphaGal epitopes on said glycoprotein of interest is lower than the amount of Neu5Gc or alphaGal epitopes on said glycoprotein of interest when said isolated cell capable of expressing said glycoprotein of interest is incubated with a cell culture reagent derived from a wild-type pig. 
     
     
         19 . The method of  claim 18  wherein said glycoprotein of interest is a glycoprotein selected from the group comprising an antibody, growth factor, cytokine, hormone and clotting factor. 
     
     
         20 . The method of  claim 18 , wherein the disruption of said α(1,3)-galactosyltransferase gene is a 3 base pair deletion adjacent to a G to A substitution, wherein the disruption of said CMAH gene is a four base pair insertion and wherein expression of functional α(1,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig.