HLA G-MODIFIED CELLS AND METHODS
Disclosed herein are methods for producing genetically modified cells expressing HLA-G (e.g., cell surface HLA-G) persistently, and nucleic acid compositions useful for generating such genetically modified cells. Also disclosed are cell therapy methods that utilize genetically modified cells that express HLA-G persistently. The HLA-G genetic modifications described herein provide the cells with characteristics of reduced immunogenicity and/or improved immunosuppression, such that these cells have the promise of being universal or improved donor cells for transplants, cellular and tissue regeneration or reconstruction, and other therapies.
1 . A genetically modified mammalian cell that has reduced immunogenicity and/or improved immunosuppression as compared to a mammalian cell without said genetic modification,
wherein the genetically modified mammalian cell comprises in its genome an exogenous nucleic acid comprising:
a nucleic acid sequence encoding a variant Human leukocyte antigen-G (HLA-G) protein, and
wherein the genetically modified mammalian cell is selected from a group consisting of a stem cell and a progenitor cell.
2 . The genetically modified mammalian cell of claim 1 , wherein the genetically modified mammalian cell is an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell (iPSC), or a totipotent stem cell.
3 . The genetically modified mammalian cell of claim 2 , wherein the genetically modified mammalian cell is the iPSC.
4 . The genetically modified mammalian cell of claim 1 , wherein the genetically modified mammalian cell is a pancreatic progenitor cell.
5 . The genetically modified mammalian cell of claim 4 , wherein the genetically modified mammalian cell is a pancreatic islet cell.
6 . The genetically modified mammalian cell of claim 5 , wherein the genetically modified mammalian cell is a pancreatic beta ((3) cell.
7 . The genetically modified mammalian cell of claim 1 , wherein the nucleic acid sequence encodes the variant HLA-G protein set forth in SEQ ID NO:2.
8 . The genetically modified mammalian cell of claim 7 , wherein the nucleic acid sequence is operably linked to one or more of an Elongation Factor-1 alpha (EF-1α) promoter, a 3′ untranslated region (UTR), or a combination thereof.
9 . The genetically modified mammalian cell of claim 8 , wherein the nucleic acid sequence is operably linked to one or more of the EF-1α promoter comprising the sequence of SEQ ID NO:6, the 3′ UTR comprising a nucleotide sequence of SEQ ID NO:3, or a combination thereof
10 . The genetically modified mammalian cell of claim 8 , wherein the nucleic acid sequence is operably linked to the EF-1α promoter comprising the sequence of SEQ ID NO:6, and wherein the nucleic acid sequence is operably linked to the 3′ UTR comprising a nucleotide sequence of SEQ ID NO:3.
11 . The genetically modified mammalian cell of claim 1 , wherein the genetically modified mammalian cell is a genetically modified human cell.
12 . The genetically modified mammalian cell of claim 1 , wherein the reduced immunogenicity and/or improved immunosuppression of the genetically modified cell as compared to a mammalian cell of the same type without said genetic modification is determined by at least one of:
a reduction of natural killer cell, NK-92, induced cytotoxicity of the genetically modified cell as compared to the mammalian cell of the same type without said genetic modification;
a reduction of the in vitro peripheral blood mononuclear cell proliferation of the genetically modified cell as compared to the mammalian cell of the same type without said genetic modification; and/or
an increase in the size and weight of tumor formation by the genetically modified cell as compared to the mammalian cell of the same type without said genetic modification in humanized NOD scid gamma (NSG) mice.
13 . An artificial tissue comprising the genetically modified mammalian cell of claim 1 .
14 . The genetically modified mammalian cell of claim 1 , wherein the exogenous nucleic acid is an expression vector.
15 . A method of treating diabetes in subject in need thereof, the method comprising injecting, implanting, or grafting to the subject a cellular composition or tissue composition comprising a population of the genetically modified mammalian cells of claim 1 .
16 . The method of claim 15 , wherein the genetically modified mammalian cells are pancreatic islet cells.
17 . The method of claim 16 , wherein the genetically modified cells are pancreatic beta ((3) cells.
18 . The method of claim 15 , wherein the diabetes is diabetes mellitus type 1.
19 . The method of claim 15 , wherein the subject has at least one mismatched classical HLA class I or HLA class II molecule as compared to the population of genetically modified mammalian cells, and
wherein the population of genetically modified mammalian cells exhibits reduced immunogenicity and/or improved immunosuppression as compared to mammalian cells of the same type without said genetic modification.
20 . The method of claim 19 , wherein the at least one mismatched classical HLA class I or HLA class II molecule is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR.
21 . The method of claim 15 , wherein the subject has no matches with the genetically modified mammalian cells with respect to HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR.
22 . The genetically modified mammalian cell of claim 1 , wherein a plasmid expression vector, a retroviral vector, or a transposon vector comprises the nucleic acid sequence.