# US 11,433,103 B2 · Universal donor cells

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- Number: US 11,433,103 B2
- Document ID: US11433103B2
- Status: Granted
- Kind: B2
- Application number: 17/383,760
- Filed: 2021-07-23
- Published: 2022-01-20
- Granted: 2022-09-06
- Earliest claimed: 2019-09-05
- Assignees: Crispr Therapeutics AG
- Applicants: Crispr Therapeutics AG
- Inventors: Alireza Rezania; Rebeca Ramos-Zayas
- Web page: https://patentgrep.com/patent/US11433103B2

## CPC

- A61K 35/39: Pancreas; Islets of Langerhans (Langerhans cells of epidermis A61K35/36)
- 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 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- A61K 38/1709: {from mammals}
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/0607: {Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696)}
- C12N 5/0678: {Stem cells; Progenitor cells; Precursor cells}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 5/10: Cells modified by introduction of foreign genetic material
- C12N 5/16: Animal cells
- C12N 15/85: for animal cells
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C12N 2506/02: from embryonic cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2510/00: Genetically modified cells
- 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)
- 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

Genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and methods of generating said genetic modified cells are provided herein. The universal donor cells comprise at least one genetic modification within or near at least one gene that encodes a survival factor, wherein the genetic modification comprises an insertion of a polynucleotide encoding a tolerogenic factor. The universal donor cells may further comprise at least one genetic modification within or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or a transcriptional regulator of a MHC-I or MHC-II complex, wherein said genetic modification comprises an insertion of a polynucleotide encoding a second tolerogenic factor.

## Claims

### Claim 1

A genetically modified cell comprising a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E) inserted within a thioredoxin interacting protein (TXNIP) gene locus, the nucleotide sequence inserted in the TXNIP gene locus comprising SEQ ID NO: 55, wherein the genetically modified cell expresses HLA-E and does not express TXNIP.

### Claim 2 (depends on 1)

The genetically modified cell of claim 1, wherein SEQ ID NO: 55 comprises a sequence encoding a B2M signal peptide, a sequence encoding an HLA-G presentation peptide, a sequence encoding a B2M membrane protein, and a sequence encoding HLA-E without its signal peptide.

### Claim 3 (depends on 2)

The genetically modified cell of claim 2, wherein the sequence encoding the B2M signal peptide consists essentially of SEQ ID NO: 26, the sequence encoding the HLA-G presentation peptide consists essentially of SEQ ID NO: 27, the sequence encoding the B2M membrane protein consists essentially of SEQ ID NO: 29, and the sequence encoding HLA-E without its signal peptide consists essentially of SEQ ID NO: 31.

### Claim 4 (depends on 1)

The genetically modified cell of claim 1, wherein SEQ ID NO: 55 is operably linked to an exogenous promoter.

### Claim 5 (depends on 4)

The genetically modified cell of claim 4, wherein the exogenous promoter is a CAG promoter comprising a CMV enhancer, a β-actin promoter, and a chimeric intron.

### Claim 6 (depends on 5)

The genetically modified cell of claim 5, wherein the CMV enhancer consists essentially of SEQ ID NO: 8, the β-actin promoter consists essentially of SEQ ID NO: 9, and the chimeric intron consists essentially of SEQ ID NO: 10 or SEQ ID NO: 57.

### Claim 7 (depends on 1)

The genetically modified cell of claim 1, wherein SEQ ID NO: 55 is operably linked to a poly(A) signal.

### Claim 8 (depends on 7)

The genetically modified cell of claim 7, wherein the poly(A) signal consists essentially of SEQ ID NO: 12.

### Claim 9 (depends on 1)

The genetically modified cell of claim 1, wherein the cell is a stem cell.

### Claim 10 (depends on 9)

The genetically modified cell of claim 9, wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

### Claim 11 (depends on 1)

The genetically modified cell of claim 1, wherein the cell is a differentiated cell or a somatic cell.

### Claim 12 (depends on 1)

The genetically modified cell of claim 1, wherein the cell is capable of being differentiated into lineage-restricted progenitor cells or fully differentiated somatic cells.

### Claim 13 (depends on 12)

The genetically modified cell of claim 12, wherein the lineage-restricted progenitor cells are definitive endoderm cells, primitive gut tube cells, posterior foregut cells, pancreatic endoderm progenitors, or pancreatic endocrine progenitors, and the fully differentiated somatic cells are immature beta cells or mature beta cells.

### Claim 14 (depends on 1)

A population of cells comprising one or more genetically modified cells according to claim 1.

### Claim 15 (depends on 14)

The population of cells of claim 14, wherein at least 50% of the cells express HLA-E.

### Claim 16 (depends on 14)

The population of cells of claim 14, wherein at least 70% of the cells express HLA-E.

### Claim 17 (depends on 14)

The population of cells of claim 14, wherein at least 90% of the cells express HLA-E.

### Claim 18 (depends on 1)

A population of cells comprising lineage-restricted progenitor cells or fully differentiated somatic cells derived from one or more genetically modified cells of claim 1, wherein at least one of the cells in the population comprises a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E) inserted within a thioredoxin interacting protein (TXNIP) gene locus, the nucleotide sequence inserted in the TXNIP gene locus comprising SEQ ID NO: 55, wherein the genetically modified cell expresses HLA-E and does not express TXNIP.

### Claim 19 (depends on 18)

The population of cells of claim 18, wherein the lineage-restricted progenitor cells are definitive endoderm cells, primitive gut tube cells, posterior foregut cells, pancreatic endoderm progenitors, or pancreatic endocrine progenitors, and the fully differentiated somatic cells are immature beta cells or mature beta cells.

### Claim 20 (depends on 19)

The population of cells of claim 19, wherein at least 50% of the cells express HLA-E.

### Claim 21 (depends on 19)

The population of cells of claim 19, wherein at least 70% of the cells express HLA-E.

### Claim 22 (depends on 19)

The population of cells of claim 19, wherein at least 90% of the cells express HLA-E.