Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1999-12-08
2003-05-06
Wehbe', Anne M. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093200, C435S325000, C435S320100, C800S008000, C800S013000, C800S017000, C800S021000
Reexamination Certificate
active
06558663
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the field of organ transplantation.
Technical advances in allogeneic organ transplantation and the availability of nonspecific immunosuppressive agents have revolutionized the field of organ transplantation. This progress has, however, resulted in a shortage of essential organs of suitable size and match.
The shortage of allograft-organs has led to an increased interest in xenogeneic transplantation. It was demonstrated more than twenty-five years ago that transplants from chimpanzee to man could provide long-term life-supporting function. However, the use of non-human primates as an organ source is of limited applicability. Many primate species are scarce and protected, and those that are more plentiful, such as the baboon, often do not grow to a size which allows the use of their organs in adults. Moreover, in some cultures, the use of primates as a source of organs is ethically unacceptable.
Some of these difficulties could be resolved by use of ungulate organs, especially pig organs. Pigs are domesticated, easy to breed, have large litters, and grow rapidly to the size which allow the use of their organs in the very largest human beings. In addition, pig and man have many anatomical and physiological similarities. However, transplantation of a pig organ into a human results in a vigorous rejection of the graft-organ.
SUMMARY OF THE INVENTION
In general, the invention features, a genetically engineered swine cell, e.g., a cultured swine cell, a retrovirally transformed swine cell, or a cell derived from a transgenic swine. The cell includes a transgene which encodes a xenogeneic, e.g., a human, class I MHC protein, e.g., an HLA A, B, C or G gene.
In preferred embodiments the transgene includes an &agr; subunit, e.g., an HLA class I gene, e.g., an HLA C gene.
Where the transgene includes an HLA C gene, the allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. As is discussed below, alleles of HLA class I genes can often be classed into reactivity groups wherein an allele from a reactivity group can confer protection against NK cells specific to other alleles in the reactivity group. Thus, in preferred embodiments, the transgene includes an allele which is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has: an Asn at residue 77 and a Lys at residue 80; or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments the transgene includes an HLA A gene. In other preferred embodiments the transgene includes an HLA B gene.
In other preferred embodiments the transgene includes an HLA G gene, e.g, any of alleles I-IV of HLA G.
In preferred embodiments: the cell includes a second transgene which includes a class I MHC protein. In preferred embodiments the second transgene includes an HLA class I gene, e.g., an HLA A, B, C or G gene. In preferred embodiments the first transgene includes an allele from a first reactivity group and the second transgene includes an allele from a second reactivity group. For example, the first transgene includes a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, and the second transgene includes a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In preferred embodiments the first transgene encodes an allele which has an Asn at residue 77 and a Lys at residue 80 and the second transgene encodes an allele which has a Ser at residue 77 and an Asn at residue 80. In other preferred embodiments the second transgene encodes a human &bgr; subunit, e.g., a &bgr;-2 microglobulin gene.
In preferred embodiments the transgene includes a chimeric class I gene, e.g., a chimeric HLA A, B, C, or G gene. The chimeric transgene can include a first portion derived from a first allele of a gene encoding a class I protein and a second portion derived from a second allele of the gene encoding the class I protein. In other embodiments, the class I gene is a synthetic sequence selected for the ability to produce a protein which protects a target cell from attack from more than one class of NK cells. In preferred embodiments the transgene includes a gene, e.g., a chimeric or mutated HLA C gene, which confers protection to more than one class of NK cells, e.g., an allele of HLA C having serine at position 77 and lysine at position 80, see e.g., Biassoni, 1995, J. Exp. Med. Vol. 182: 605-609, hereby incorporated by reference. See also Moretta et al., 1996, Ann. Rev. Immunol. 14: 619-648, hereby incorporated by reference, which together with the disclosure herein, provides guidance for altering critical residues in the HLA C genes.
In yet other preferred embodiments the genetically engineered swine cell is: a swine hematopoietic stem cell, e.g., a cord blood hematopoietic stem cell, a bone marrow hematopoietic stem cell, or a fetal or neonatal liver or spleen hematopoietic stem cell; derived from differentiated blood cells, e.g. a myeloid cell, such as a megakaryocyte, monocyte, granulocyte, or an eosinophil; an erythroid cell, such as a red blood cell, e.g. a lymphoid cell, such as B lymphocytes and T lymphocytes; derived from a pluripotent hematopoietic stem cell, e.g. a hematopoietic precursor, e.g. a burst-forming units-erythroid (BFU-E), a colony forming unit-erythroid (CFU-E), a colony forming unit-megakaryocyte (CFU-Meg), a colony forming unit-granulocyte-monocyte (CFU-GM), a colony forming unit-eosinophil (CFU-Eo), or a colony forming unit-granulocyte-erythrocyte-megakaryocyte-monocyte (CFU-GEMM); a swine cell other than a hematopoietic stem cell, or other blood cell; a swine thymic cell, e.g., a swine thymic stromal cell; a bone marrow stromal cell; a swine liver cell; a swine kidney cell; a swine epithelial cell; a swine hematopoietic progenitor cell; a swine muscle cell, e.g., a heart cell; an endothelial cell; or a dendritic cell or precursor thereof.
In yet other preferred embodiments the cell is: isolated or derived from cultured cells, e.g., a primary culture, e.g., a primary cell culture of hematopoietic stem cells; isolated or derived from a transgenic animal.
In yet other preferred embodiments: the swine cell is homozygous for the transgene; the swine cell is heterozygous for the transgene; the swine cell is homozygous for the transgene (heterozygous transgenic swine can be bred to produce offspring that are homozygous for the transgene); the swine cell includes two or more transgenes.
In yet other preferred embodiments the cell includes a one or more, or all of, of a transgene which encodes an HLA A gene, a transgene which encodes an HLA B gene, a transgene which encodes an HLA C gene, and a transgene which encodes an HLA G gene.
In another aspect, the invention features a nucleic acid, e.g., a transgene, including a swine promoter operably linked to a xenogeneic, e.g., human, nucleic acid which encodes a class I MHC protein. The swine promoter can be, e.g., a swine hematopoietic epithelial gene promoter, or a heterologous inducible or developmentally regulated promoter.
In preferred embodiments the nucleic acid includes, e.g., a gene which encodes an a subunit, e.g., an HLA class I gene, e.g., an HLA A, B, C, or G gene, or a gene which encodes a human &bgr; subunit, e.g., a &bgr;-2 microglobulin gene
Where the nucleic acid includes an HLA C gene, the allele, by way of example, can be any Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw7/8v, or Cw10 allele. In preferred embodiments the nucleic acid encodes an allele which is a member of a reactivity group, e.g., a Group 1 allele, e.g., any of an HLA C Cw2, Cw4, Cw5, or Cw6 allele, or a Group 2 allele, e.g., any of an HLA C Cw1, Cw3, Cw7, or Cw8 allele. In other preferred embodiments the allele has an Asn at residue 77 and a Lys at residue 80 or a Ser at residue 77 and an Asn at residue 80.
In preferred embodiments, the nucleic acid or transgene further includes transcriptional regulatory s
DerSimonian Harout
LeGuern Christian
Sachs David H.
Seebach Joerg
Fish & Richardson P.C.
The General Hospital Corporation
Wehbe' Anne M.
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