Humanized heart muscle

Abstract

Described herein is a method for producing a chimeric non-human animal expressing a human NKX2-5, HANDII, TBX5 gene or a combination thereof gene comprising: a) generating a NKX2-5, HANDII, TBX5 or combination thereof null non-human animal cell, wherein both copies of the non-human NKX2-5, HANDII, TBX5 gene or combination thereof carry a mutation that prevents production of functional NKX2-5, HANDII, TBX5 protein or combination thereof in said non-human animal; b) creating a NKX2-5, HANDII, TBX5 or combination thereof null non-human blastocyst by somatic cell nuclear transfer comprising fusing a nucleus from said NKX2-5, HANDII, TBX5 or combination thereof null non-human animal cell of a) into an enucleated non-human oocyte and activating said oocyte to divide so as to form an NKX2-5, HANDII, TBX5 or combination thereof null non-human blastocyst; c) introducing human stem cells into the NKX2-5, HANDII, TBX5 or combination null non-human blastocyst of b); and d) implanting said blastocyst from c) into a pseudopregnant surrogate non-human animal to generate a chimeric non-human animal expressing human NKX2-5, HANDII, TBX5 or combination thereof.

1 . A non-human animal cell, morula or blastocyst wherein the genome carries a mutation in both alleles of the NKX2-5 gene, HANDII gene, TBX5 gene or a combination thereof such that the non-human animal cell or blastocyst lacks functional NKX2-5 protein, HANDII protein, TBX5 protein or a combination thereof. 
     
     
         2 . The non-human animal cell, morula or blastocyst of  claim 1 , wherein the mutation is a deletion of the NKX2-5 gene, HANDII gene, TBX5 gene or a combination thereof. 
     
     
         3 . The non-human animal cell, morula or blastocyst of  claim 1 , wherein the non-human animal cell or blastocyst is a porcine, bovine, equine or goat. 
     
     
         4 - 7 . (canceled) 
     
     
         8 . A method for producing a chimeric non-human animal expressing a human NKX2-5 gene, HANDII gene, TBX5 gene or a combination thereof comprising:
 a) generating a NKX2-5, HANDII, TBX5 or a combination thereof null non-human animal cell, wherein both copies of the non-human NKX2-5 gene, HANDII gene, TBX5 gene or a combination thereof gene carry a mutation that prevents production of functional NKX2-5 protein, HANDII protein, TBX5 protein or combination thereof;   b) creating a NKX2-5, HANDII, TBX5 or a combination thereof null non-human morula blastocyst by somatic cell nuclear transfer comprising fusing a nucleus from said NKX2-5, HANDI, TBX5 or a combination thereof null non-human cell of a) into an enucleated non-human oocyte and activating said oocyte to divide so as to form a NKX2-5, HANDII, TBX5 or a combination thereof null non-human morula or blastocyst;   c) introducing human stem cells into the non-human NKX2-5, HANDII, TBX5 or a combination thereof null morula or blastocyst of b); and   d) implanting said morula or blastocyst from c) into a pseudopregnant surrogate non-human animal to generate a chimeric non-human animal expressing human NKX2-5, HANDII, TBX5 or a combination thereof.   
     
     
         9 . (canceled) 
     
     
         10 . A method of producing human or humanized cardiac cells in a non-human animal comprising:
 a) generating a NKX2-5, HANDII, TBX5 or a combination thereof null non-human cell, wherein both alleles of the non-human NKX2-5 gene, HANDII gene, TBX5 gene or a combination thereof carry a mutation that prevents production of functional the non-human NKX2-5 protein, HANDII protein, TBX5 protein or combination thereof;   b) creating a NKX2-5, HANDII, TBX5 or a combination thereof null non-human morula or blastocyst by somatic cell nuclear transfer comprising fusing a nucleus from said MYF5, MYOD, MRF4 or a combination thereof null non-human cell of a) into an enucleated non-human animal oocyte and activating said oocyte to divide so as to form a NKX2-5, HANDII, TBX5 or a combination thereof null non-human animal blastocyst;   c) introducing human stem cells into the NKX2-5, HANDII, TBX5 or a combination thereof null non-human animal blastocyst or morula of b); and   d) implanting said blastocyst or morula from c) into a pseudopregnant surrogate non-human animal so as to generate a non-human animal expressing human or humanized cardiac cells.   
     
     
         11 . The method of  claim 8 , wherein the non-human animal is a porcine, bovine, equine or goat. 
     
     
         12 . The method of  claim 8 , wherein the human stem cell is a tissue specific stem cell, pluripotent stem cell, multipotent adult stem cell, induced pluripotent stem cell or umbilical cord blood stem cell (UCBSC). 
     
     
         13 . The method of  claim 8 , wherein the induced pluripotent cell is formed from a fibroblast cell. 
     
     
         14 . A non-human animal produced by the method of  claim 8 . 
     
     
         15 . Chimeric heart, chimeric heart tissue and/or chimeric heart cells made by a process comprising:
 a) generating an NKX2-5 gene, HANDII gene, TBX5 gene or combination thereof null non-human animal cell, wherein both alleles of the non-human NKX2-5, HANDII, TBX5 genes or the combination thereof carry a mutation and/or deletion that prevents production of functional NKX2-5, HANDII, TBX5 protein or a combination thereof;   b) creating an NKX2-5 gene, HANDII gene, TBX5 gene or the combination thereof null non-human blastocyst or morula by somatic cell nuclear transfer comprising fusing a nucleus from said NKX2-5 gene, HANDII gene, TBX5 gene or the combination thereof null non-human animal cell of a) into an enucleated non-human oocyte and activating said oocyte to divide so as to form a NKX2-5 gene, HANDII gene, TBX5 gene or the combination thereof null non-human blastocyst or using gene editing to generate a NKX2-5, HANDII, TBX5 or combination thereof null non-human blastocyst;   c) introducing human donor stem cells into the NKX2-5 gene, HANDII gene, TBX5 gene or combination thereof null non-human blastocyst of b); and   d) implanting said blastocyst from c) into a pseudopregnant surrogate non-human animal so as to generate a non-human animal expressing chimeric heart, chimeric heart tissue and/or chimeric heart cells.   
     
     
         16 - 18 . (canceled) 
     
     
         19 . The chimeric heart, chimeric heart tissue and/or chimeric heart cells of  claim 15 , wherein the non-human animal is swine. 
     
     
         20 . (canceled)