HLA G-modified cells and methods

• US 10,502,738 B2
• Filed: 06/16/2017
• Issued: 12/10/2019
• Est. Priority Date: 07/31/2012
• Status: Active Grant

• Current Assignee: AgeX Therapeutics, Inc.
• Original Assignee: AgeX Therapeutics, Inc.
• Inventors: Hantash, Basil M.
• Primary Examiner(s): Leavitt, Maria G
• Application Number:
  US15/625,964
• Publication Number:
  US 20170292952A1
• Time in Patent Office: 907 Days
• Field of Search: 435366, 435325, 435372
• CPC Class Codes: A01K 2207/12 Animals modified by administration of exogenous cells
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/025 Animal producing cells or organs for transplantation
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/33 Fibroblasts
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 2333/71 for growth factors; for growth regulators
• CPC Class Codes: G01N 2800/245 Transplantation related diseases, e.g. graft versus host disease
• CPC Class Codes: G01N 33/566 : using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/56977 : HLA or MHC typing
• CPC Class Codes: G01N 33/6893 : related to diseases not provided for elsewhere

Abstract

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.

Claims

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.