Genetically engineered animals for use as organ donors

Abstract

Methods to manipulate animals such as pigs, and the animals and tissues thereby derived, to reduce their immunogenicity following implantation into humans, are described. These methods are based on the discovery that certain carbohydrate structures on pig tissues, which require expression of the gene encoding the alpha 1->3 galactosyl transferase enzyme, are targets for natural preformed antibodies of humans and elicit further antibody production in humans, while other carbohydrate structures do not or do so in a reduced amount. In the preferred embodiment, animals are produced by homologous recombination of the gene encoding alpha 1->3 galactosyl transferase in embryonic stem cells or by microinjection into embryos of sequences eliminating or decreasing expression of alpha 1->3 galactosyl transferase. In alternative embodiments, animals are produced having reduced amounts of ->1->3 galactosyl epitopes or epitopes which are masked by sialylation or fucosylation.

We claim:  
     
         1 . A method for making an organ for implantation into a human comprising 
 genetically engineering a non-human animal having α 1→3 galactosyl epitopes on its cells to decrease or prevent expression of α 1→3 galactosyl epitopes on the cells of the animal.    
     
     
         2 . The method of  claim 1  wherein the animal is engineered to decrease or prevent expression of an α 1→3 transferase enzyme.  
     
     
         3 . The method of  claim 1  wherein the α 1→3 galactosyl epitopes are masked.  
     
     
         4 . The method of  claim 1  wherein the animal is genetically engineered by homologous recombination in embryonic stem cells to insert or delete a gene selected from the group encoding the α 1→3 galactosyl transferase enzyme, sialyltransferase enzyme, and α 1→3 fucosyltransferase.  
     
     
         5 . The method of  claim 1  wherein the animal is genetically engineered by manipulation of the embryo to alter expression of an enzyme selected from the group consisting of the α 1→3 galactosyl transferase enzyme, sialyltransferase enzyme, and α 1→3 fucosyltransferase.  
     
     
         6 . The method of  claim 2  wherein the embryo is altered by introduction of sequences which inhibit expression of the α 1→3 galactosyl transferase enzyme gene.  
     
     
         7 . A non-primate animal, and tissues derived therefrom, which is deficient in expression of α 1→3 galactosyl epitopes on its cells.  
     
     
         8 . The animal of  claim 7  wherein the animal is engineered to decrease or prevent expression of an α 1→3 transferase enzyme.  
     
     
         9 . The animal of  claim 7  wherein the α 1→3 galactosyl epitopes are masked.  
     
     
         10 . The animal of  claim 7  wherein the animal is genetically engineered by homologous recombination in embryonic stem cells of a gene selected from the group encoding the α 1→3 galactosyl transferase enzyme, sialyltransferase enzyme, and α 1→3 fucosyltransferase.  
     
     
         11 . The animal of  claim 7  wherein the animal is genetically engineered by manipulation of the embryo to alter expression of an enzyme selected from the group consisting of the α 1→3 galactosyl transferase enzyme, sialyltransferase enzyme, and α 1→3 fucosyltransferase.  
     
     
         12 . The animal of  claim 8  wherein the embryo is altered by introduction of sequences which inhibit expression of the α 1→3 galactosyl transferase enzyme gene.  
     
     
         13 . The animal of  claim 8  not expressing carbohydrate structures selected from the group consisting of α Gal(1→3) β Gal(1→4) β GlcNac (linear B type 2), α Gal (1→3) β Gal (1→4) β Glc (linear B type 6), α Gal(1→3) β Gal (B disaccharide), and α Gal (α-D-galactose).  
     
     
         14 . The animal of  claim 8  wherein expression of the transferase gene is decreased by alteration of the regulatory sequences required for expression of the transferase gene.  
     
     
         15 . The animal of  claim 7  wherein the animal is a pig.  
     
     
         16 . The tissues of  claim 7  selected from the group consisting of skin, heart, liver, kidney, lung, pancreas, small bowel, and components thereof.  
     
     
         17 . The tissues of  claim 16  wherein the animal also does not express, or expresses in reduced amounts, on its cell surfaces, carbohydrate structures selected from the group consisting of N-acetyl-β-D-glucosaminide (β GlcNac) and other structures containing a terminal β GlcNac, α-L-Rhamnose and Rhamnose-containing structures, Forssman disaccharides, Forssman trisaccharides, and A or A-like carbohydrates.  
     
     
         18 . The tissues of  claim 7  wherein the tissue is selected from the group consisting of kidney, heart, liver, skin, lung, pancreas, small bowel, and components thereof.  
     
     
         19 . The animal of  claim 9  wherein the α 1→3 galactose residues are capped with a carbohydrate selected from the group consisting of sialic acid and fucose.  
     
     
         20 . A method for decreasing rejection of a xenotransplant comprising isolating tissues from non-human animals, which normally have α 1→3 galactosyl epitopes on their cells, which have been genetically engineered to decrease or prevent expression of α 1→3 galactosyl epitopes on the cells of the animal.  
     
     
         21 . The method of  claim 20  wherein the animal is engineered to decrease or prevent expression of an α 1→3transferase enzyme.  
     
     
         22 . The method of  claim 20  wherein the animal is genetically engineered in embryonic stem cells to alter expression of a gene selected from the group encoding the α 1→3 galactosyl transferase enzyme, sialyltransferase enzyme, and α 1→3 fucosyltransferase.  
     
     
         23 . The method of  claim 20  wherein the animal is genetically engineered by manipulation of the embryo to alter expression of an enzyme selected from the group consisting of the α 1→3 galactosyl transferase enzyme, sialyltransferase enzyme, and α 1→3 fucosyltransferase.  
     
     
         24 . The method of  claim 20  wherein the α 1→3 galactosyl epitopes are masked.  
     
     
         25 . The method of  claim 20  wherein the α 1→3 galactosyl epitopes are not produced or are decreased by elimination of or decrease in expression of α 1→3 galactosyl transferase in the animal.  
     
     
         26 . The method of  claim 20  further comprising transplanting the tissues into a human.  
     
     
         27 . The method of  claim 20  wherein the animal is a pig and the tissue is selected from the group consisting of liver, heart, skin, kidney, and components thereof.