Genetic modification of pigs for xenotransplantation

Abstract

The invention provides for transgenic donor animals (e.g., pigs) whose cells, tissues and organs have a better long-term survival when transplanted into a human patient. The transgenic donor animal carries one or more human transgenes which is expressed only when the endogenous gene of the donor animal is knocked out shortly before a graft is harvested for transplantation. This “genetic switch” allows the donor animal to remain healthy during the majority of its lifetime, while still allowing expression of the human transgene for optimal transplant tolerance in a human recipient. The transgene may encode a cytokine receptor, an adhesion molecule, or a complement regulatory protein.

What is claimed is: 
     
         1 . A method of supplying a graft from a donor mammal of a first species for transplant in a recipient mammal of a second species, the method comprising the steps of:
 (a) inducing knockout of at least one conditional knockout allele of a target gene in the donor mammal, wherein the knockout of the target gene results in expression of a transgene of the second species, the transgene being a homolog of the target gene of the first species, and wherein the transgene is expressed under control of native regulatory sequence of the target gene when the target gene is knocked out; and   (b) providing a graft from the donor mammal for transplant.   
     
     
         2 . The method of  claim 1 , wherein the first species is swine. 
     
     
         3 . The method of  claim 2 , wherein the swine is a miniature swine. 
     
     
         4 . The method of  claim 1 , wherein the second species is human. 
     
     
         5 . The method of  claim 1 , wherein the transgene encodes a cytokine receptor, or an adhesion molecule. 
     
     
         6 . The method of  claim 1 , wherein the transgene encodes a complement regulatory protein. 
     
     
         7 . The method of  claim 1 , wherein the transgene encodes IL-3 receptor, CD47, thrombomodulin, CD39, CD200, a ligand for paired Ig-like receptor (PIR)-B, a ligand for immunoglobulin-like transcript (ILT)3, a ligand for CD33-related receptors, CD46 (MCP), CD55 (DAF), CD59, MSP and the HLA-E/beta2 microglobulin/human leader peptide trimer. 
     
     
         8 . The method of  claim 1 , wherein the conditional knockout allele is flanked by two LoxP sequences and is knocked out by an inducible Cre recombinase. 
     
     
         9 . The method of  claim 8 , wherein the LoxP sequences are SEQ ID NO:1. 
     
     
         10 . The method of  claim 8 , wherein expression of the inducible Cre recombinase is under control of an inducible promoter. 
     
     
         11 . The method of  claim 10 , wherein the inducible promoter is induced by a drug. 
     
     
         12 . The method of  claim 8 , wherein the Cre recombinase is induced by translocating to the nucleus of a cell upon addition of a drug. 
     
     
         13 . The method of  claim 11  or  12 , wherein the drug is selected from the group consisting of doxycycline, tetracycline, RU486, and tamoxifen. 
     
     
         14 . The method of  claim 1 , wherein in step (a) homozygous conditional knockout alleles of the target gene are knocked out. 
     
     
         15 . The method of  claim 1 , wherein the conditional knockout allele of the target gene comprises a nucleic acid sequence at least 90% identical to the sequence of wildtype allele of the target gene of the first species. 
     
     
         16 . The method of  claim 1 , wherein the graft comprises cells, a tissue or an organ. 
     
     
         17 . The method of  claim 1 , wherein the graft comprises hematopoietic stem cells. 
     
     
         18 . The method of  claim 1 , wherein the graft comprises bone marrow. 
     
     
         19 . The method of  claim 1 , wherein the graft comprises a heart, a kidney, a liver, a pancreas, a lung, an intestine, skin, a small bowel, a trachea, a cornea, or combinations thereof. 
     
     
         20 . A method of supplying a graft from a donor swine for transplant in a human recipient, the method comprising the steps of:
 (a) inducing knockout of at least one conditional knockout allele of a target gene in the donor swine, wherein the knockout of the target gene results in expression of a human transgene, the transgene being a homolog of the swine target gene, and wherein the transgene is expressed under control of native regulatory sequence of the target gene when the target gene is knocked out; and   (b) providing a graft from the donor swine for transplant.   
     
     
         21 . The method of  claim 20 , wherein the transgene encodes a cytokine receptor, or an adhesion molecule. 
     
     
         22 . The method of  claim 20 , wherein the transgene encodes a complement regulatory protein. 
     
     
         23 . The method of  claim 20 , wherein the transgene encodes IL-3 receptor, CD47, thrombomodulin, CD39, CD200, a ligand for paired Ig-like receptor (PIR)-B, a ligand for immunoglobulin-like transcript (ILT)3, a ligand for CD33-related receptors, CD46 (MCP), CD55 (DAF), CD59, MSP and the HLA-E/beta2 microglobulin/human leader peptide trimer. 
     
     
         24 . The method of  claim 20 , wherein the conditional knockout allele is flanked by two LoxP sequences and is knocked out by an inducible Cre recombinase. 
     
     
         25 . The method of  claim 24 , wherein expression of the inducible Cre recombinase is under control of an inducible promoter. 
     
     
         26 . The method of  claim 25 , wherein the inducible promoter is induced by a drug. 
     
     
         27 . The method of  claim 24 , wherein the Cre recombinase is induced by translocating to the nucleus of a cell upon addition of a drug. 
     
     
         28 . The method of  claim 26  or  27 , wherein the drug is selected from the group consisting of doxycycline, tetracycline, RU486, and tamoxifen. 
     
     
         29 . The method of  claim 20 , wherein in step (a) homozygous conditional knockout alleles of the target gene are knocked out. 
     
     
         30 . The method of  claim 20 , wherein the conditional knockout allele of the target gene comprises a nucleic acid sequence at least 90% identical to the sequence of wildtype allele of the target gene of the first species. 
     
     
         31 . The method of  claim 20 , wherein the graft comprises hematopoietic stem cells. 
     
     
         32 . The method of  claim 20 , wherein the graft comprises bone marrow. 
     
     
         33 . The method of  claim 20 , wherein the graft comprises a heart, a kidney, a liver, a pancreas, a lung, an intestine, skin, a small bowel, a trachea, a cornea, or combinations thereof. 
     
     
         34 . A transgenic mammal of a first mammalian species, comprising:
 a) at least one conditional knockout allele of a target gene of the first mammalian species, at its natural genomic locus of the mammal, and   b) a transgene of a second mammalian species, wherein the transgene is a homolog of the target gene of the first mammalian species, wherein the transgene is located downstream from the conditional knockout allele of the target gene, and wherein the transgene is expressed under control of native regulatory sequence of the target gene when the target gene is knocked out.   
     
     
         35 . The transgenic mammal of  claim 34 , wherein the first mammalian species is swine. 
     
     
         36 . The transgenic mammal of  claim 35 , wherein the swine is a miniature swine. 
     
     
         37 . The transgenic mammal of  claim 34 , wherein the second mammalian species is human. 
     
     
         38 . The transgenic mammal of  claim 34 , wherein the transgene encodes a cytokine receptor, or an adhesion molecule. 
     
     
         39 . The transgenic mammal of  claim 34 , wherein the transgene encodes a complement regulatory protein. 
     
     
         40 . The transgenic mammal of  claim 34 , wherein the transgene encodes IL-3 receptor, CD47, thrombomodulin, CD39, CD200, a ligand for paired Ig-like receptor (PIR)-B, a ligand for immunoglobulin-like transcript (ILT)3, a ligand for CD33-related receptors, CD46 (MCP), CD55 (DAF), CD59, MSP and the HLA-E/beta2 microglobulin/human leader peptide trimer. 
     
     
         41 . The transgenic mammal of  claim 34 , wherein the conditional knock-out allele is flanked by two LoxP sequences and is knocked out by an inducible Cre recombinase. 
     
     
         42 . The transgenic mammal of  claim 41 , wherein the LoxP sequences are SEQ ID NO:1. 
     
     
         43 . The transgenic mammal of  claim 41 , wherein expression of the inducible Cre recombinase is under control of an inducible promoter. 
     
     
         44 . The transgenic mammal of  claim 43 , wherein the inducible promoter is induced by a drug. 
     
     
         45 . The transgenic mammal of  claim 41 , wherein the Cre recombinase is induced by translocating to the nucleus of a cell upon addition of a drug. 
     
     
         46 . The transgenic mammal of  claim 44  or  45 , wherein the drug is selected from the group consisting of doxycycline, tetracycline, RU486, and tamoxifen. 
     
     
         47 . The transgenic mammal of  claim 34 , comprising homozygous conditional knockout alleles of the target gene. 
     
     
         48 . The transgenic mammal of  claim 34 , wherein the conditional knockout allele of the target gene comprises a nucleic acid sequence at least 90% identical to the sequence of wildtype allele of the target gene of the first mammalian species. 
     
     
         49 . A cell from the transgenic mammal of  claim 34 . 
     
     
         50 . The cell of  claim 49 , wherein the cell is a hematopoietic stem cell. 
     
     
         51 . A tissue from the transgenic mammal of  claim 34 . 
     
     
         52 . The tissue of  claim 51 , wherein the tissue is bone marrow. 
     
     
         53 . An organ from the transgenic mammal of  claim 34 . 
     
     
         54 . The organ of  claim 53 , wherein the organ is a heart, a kidney, a liver, a pancreas, a lung, an intestine, skin, a small bowel, a trachea, a cornea, and combinations thereof. 
     
     
         55 . A method for making a transgenic mammal of a first species, comprising the steps of:
 (a) replacing at least one allele of an endogenous target gene of the first species with a conditional knockout allele of the target gene, and   (b) inserting a transgene from a second species downstream from the conditional knockout allele of the target gene, wherein the transgene is a homolog of the target gene of the first species, and wherein the transgene is expressed under control of native regulatory sequence of the target gene when the target gene is knocked out.   
     
     
         56 . The method of  claim 55 , wherein the first species is swine. 
     
     
         57 . The method of  claim 55 , wherein the second species is human. 
     
     
         58 . The method of  claim 55 , wherein the transgene encodes a cytokine receptor, or an adhesion molecule. 
     
     
         59 . The method of  claim 55 , wherein the transgene encodes a complement regulatory protein. 
     
     
         60 . The method of  claim 55 , wherein the transgene encodes IL-3 receptor, CD47, thrombomodulin, CD39, CD200, a ligand for paired Ig-like receptor (PIR)-B, a ligand for immunoglobulin-like transcript (ILT)3, a ligand for CD33-related receptors, CD46 (MCP), CD55 (DAF), CD59, MSP and the HLA-E/beta2 microglobulin/human leader peptide trimer. 
     
     
         61 . The method of  claim 55 , wherein the conditional knockout allele is flanked by two LoxP sequences and is knocked out by an inducible Cre recombinase. 
     
     
         62 . The method of  claim 61 , wherein expression of the inducible Cre recombinase is under control of an inducible promoter. 
     
     
         63 . The method of  claim 62 , wherein the inducible promoter is induced by a drug. 
     
     
         64 . The method of  claim 61 , wherein the Cre recombinase is induced by translocating to the nucleus of a cell upon addition of a drug. 
     
     
         65 . The method of  claim 63  or  64 , wherein the drug is selected from the group consisting of doxycycline, tetracycline, RU486, and tamoxifen. 
     
     
         66 . The method of  claim 55 , wherein in step (a) both alleles of the endogenous target gene of the first species are replaced with homozygous conditional knockout alleles of the target gene. 
     
     
         67 . The method of  claim 55 , wherein the conditional knockout allele of the target gene comprises a nucleic acid sequence at least 90% identical to the sequence of wildtype allele of the target gene of the first mammalian species. 
     
     
         68 . The method of  claim 55 , further comprising a step (c) of somatic cell nuclear transfer. 
     
     
         69 . The method of  claim 68 , wherein the somatic cell is a fibroblast from the first species.