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 method of producing a genetically modified mammalian cell that has reduced immunogenicity and/or improved immunosuppression as compared to a mammalian cell without said genetic modification, the method comprising: stably transfecting an isolated mammalian cell with an exogenous nucleic acid comprising:
a. a nucleic acid sequence encoding a Human leukocyte antigen-G (HLA-G) protein comprising a full length amino acid sequence of SEQ ID NO:2, and operably linked to an Elongation Factor-I alpha (EF-1α) promoter comprising a sequence of SEQ ID NO:6; and
b. a 3′ untranslated region (UTR) comprising a full length nucleotide sequence of SEQ ID NO:3;
wherein the encoded HLA-G protein is expressed by the genetically modified mammalian cell for at least seven weeks, and wherein the reduced immunogenicity and/or improved immunosuppression of the genetically modified cell is determined by either:
(1) a reduction of natural killer cell, NK-92 cytotoxicity of the genetically modified cell as compared to the mammalian cell without said genetic modification,
(2) a reduction of in vitro peripheral blood mononuclear cell proliferation of the genetically modified cell as compared to the mammalian cell without said genetic modification, or
(3) an increase in the size and weight of tumor formation by the genetically modified cell as compared to the mammalian cell without said genetic modification in humanized NOD scid gamma, NSG mice.
2. The method of claim 1 , wherein the genetically modified mammalian cell expresses HLA-G protein for at least 20 weeks.
3. The method of claim 1 , wherein the genetically modified mammalian cell expresses HLA-G protein for at least 50 weeks.
4. The method of claim 1 , wherein the genetically modified mammalian cell expresses HLA-G protein on the cell surface of the genetically modified mammalian cell.
5. The method of claim 1 , wherein the genetically modified mammalian cell is a human cell.
6. The method of claim 1 , wherein the genetically modified cell is selected from the group consisting of a stem cell, a progenitor cell, a cell obtained by in vitro differentiation of the stem cell or the progenitor cell, a fully differentiated cell, an epidermal progenitor cell, a pancreatic progenitor cell, a hematopoietic stem cell, a keratinocyte, a fibroblast, a hepatocyte, a mesenchymal stem cell, a cardiomyocyte, a neural stem cell, a neuron, and an astrocyte.
7. The method of claim 1 , wherein the genetically modified cell is selected from the group consisting of a human embryonic stem cell, a human mesenchymal stem cell, a human embryonic epidermal progenitor cell, and a human dermal fibroblast.
8. The method of claim 1 , wherein the exogenous nucleic acid is an expression vector.
9. The method of claim 8 , wherein the expression vector is a transposon vector.
10. The method of claim 8 , wherein the expression vector further comprises a nucleic acid sequence encoding a reporter protein.
11. A method of producing an artificial tissue, a skin-graft, a skin-repair, or a skin-regeneration composition comprising the genetically modified mammalian cell produced by the method of claim 1 .
12. The method of claim 1 , wherein genetically modifying a mammalian cell with an exogenous nucleic acid comprises stably transfecting the mammalian cell with the exogenous nucleic acid.
13. The method of claim 1 , wherein the genetically modified mammalian cells are selected using antibiotic resistance or flow cytometry.