# US 10,502,738 B2 · HLA G-modified cells and methods

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- Number: US 10,502,738 B2
- Document ID: US10502738B2
- Status: Granted
- Kind: B2
- Application number: 15/625,964
- Filed: 2017-06-16
- Published: 2017-10-12
- Granted: 2019-12-10
- Earliest claimed: 2012-07-31
- Assignees: AgeX Therapeutics, Inc.
- Applicants: AgeX Therapeutics, Inc.
- Inventors: Basil M. Hantash
- Web page: https://patentgrep.com/patent/US10502738B2

## CPC

- G01N 33/56977: {HLA or MHC typing}
- A01K 67/0271: Chimeric vertebrates, e.g. comprising exogenous cells
- 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)
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 35/33: Fibroblasts
- A61K 35/407: Liver; Hepatocytes
- A61K 35/44: Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 17/00: Drugs for dermatological disorders
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C12N 5/0603: {Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies}
- 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}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2227/105: Murine
- A01K 2267/025: Animal producing cells or organs for transplantation
- C12N 2510/00: Genetically modified cells
- G01N 2333/705: Assays involving receptors, cell surface antigens or cell surface determinants
- G01N 2333/71: for growth factors; for growth regulators
- G01N 2800/245: Transplantation related diseases, e.g. graft versus host disease
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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

### Claim 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the genetically modified mammalian cell expresses HLA-G protein for at least 20 weeks.

### Claim 3 (depends on 1)

The method of claim 1, wherein the genetically modified mammalian cell expresses HLA-G protein for at least 50 weeks.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

The method of claim 1, wherein the genetically modified mammalian cell is a human cell.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

The method of claim 1, wherein the exogenous nucleic acid is an expression vector.

### Claim 9 (depends on 8)

The method of claim 8, wherein the expression vector is a transposon vector.

### Claim 10 (depends on 8)

The method of claim 8, wherein the expression vector further comprises a nucleic acid sequence encoding a reporter protein.

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 1)

The method of claim 1, wherein the genetically modified mammalian cells are selected using antibiotic resistance or flow cytometry.