# US 12,110,500 B2 · Universal donor stem cells and related methods

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,110,500 B2
- Document ID: US12110500B2
- Status: Granted
- Kind: B2
- Application number: 17/993,667
- Filed: 2022-11-23
- Published: 2023-10-12
- Granted: 2024-10-08
- Earliest claimed: 2015-05-08
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Torsten B. Meissner; Leonardo M. R. Ferreira; Jack L. Strominger; Chad A. Cowan
- Web page: https://patentgrep.com/patent/US12110500B2

## CPC

- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- A61K 39/001: {Preparations to induce tolerance to non-self, e.g. prior to transplantation}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2501/50: Cell markers; Cell surface determinants
- C12N 2501/599: with CD designations not provided for elsewhere
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2510/00: Genetically modified cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

Disclosed herein are universal donor stem cells and related methods of their use and production. The universal donor stem cells disclosed herein are useful for overcoming the immune rejection in cell-based transplantation therapies. In certain embodiments, the universal donor stem cells disclosed herein do not express one or more MHC-I and MHC-II human leukocyte antigens. Similarly, in certain embodiments, the universal donor stem cells disclosed herein do not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B and/or HLA-C) corresponding to MHC-I and MHC-II human leukocyte antigens, thereby rendering such cells hypoimmunogenic.

## Claims

### Claim 1

A genetically modified cell comprising:
reduced cell surface expression of one or more MHC-I human leukocyte antigen molecules and/or one or more MHC-II human leukocyte antigen molecules relative to an unmodified cell of the same type; and
increased cell surface expression of one or more tolerogenic factors relative to an unmodified cell of the same type, wherein the one or more tolerogenic factors comprise CD47.

### Claim 2 (depends on 1)

The genetically modified cell of claim 1, wherein a nucleic acid encoding the one or more tolerogenic factors is inserted into at least one allele of a safe harbor locus of the genetically modified cell.

### Claim 3 (depends on 2)

The genetically modified cell of claim 2, wherein the safe harbor locus comprises an AAVS1 locus.

### Claim 4 (depends on 1)

The genetically modified cell of claim 1, wherein the one or more tolerogenic factors inhibit an immune response when the genetically modified cell is administered to a subject.

### Claim 5 (depends on 1)

The genetically modified cell of claim 1, wherein the one or more tolerogenic factors further comprise HLA-C.

### Claim 6 (depends on 1)

The genetically modified cell of claim 1, wherein the one or more tolerogenic factors further comprise HLA-E.

### Claim 7 (depends on 1)

The genetically modified cell of claim 1, wherein the one or more tolerogenic factors further comprise HLA-G.

### Claim 8 (depends on 1)

The genetically modified cell of claim 1, wherein the one or more tolerogenic factors further comprise PD-L1.

### Claim 9 (depends on 1)

The genetically modified cell of claim 1, wherein the one or more tolerogenic factors further comprise CTLA-4-Ig.

### Claim 10 (depends on 1)

The genetically modified cell of claim 1, wherein the one or more tolerogenic factors further comprise C1-inhibitor.

### Claim 11 (depends on 1)

The genetically modified cell of claim 1, wherein the one or more tolerogenic factors further comprise IL-35.

### Claim 12 (depends on 1)

The genetically modified cell of claim 1, comprising one or more indels in one or more genes encoding an MHC-I human leukocyte antigen molecule, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules.

### Claim 13 (depends on 12)

The genetically modified cell of claim 12, comprising one or more indels in an HLA-A gene, an HLA-B gene, an HLA-C gene, or a combination thereof in the genome of the genetically modified cell, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules.

### Claim 14 (depends on 12)

The genetically modified cell of claim 12, further comprising one or more indels in a class II major histocompatibility complex transactivator (CIITA) gene in the genome of the genetically modified cell, thereby resulting in the reduced cell surface expression of the one or more MHC-II human leukocyte antigen molecules.

### Claim 15 (depends on 1)

The genetically modified cell of claim 1, comprising one or more indels in one or more genes encoding a transcriptional regulator of an MHC-I human leukocyte antigen molecule, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules.

### Claim 16 (depends on 1)

The genetically modified cell of claim 1, comprising one or more indels in a CIITA gene, a β2M gene, a TAP1 gene, an NLRC5 gene, an RFX5 gene, an RFXAP gene, an RFXANK gene, an NFY-A gene, an NFY-B gene, an NFY-C gene, an IRF-1 gene, or a combination thereof, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules and/or the one or more MHC-II human leukocyte antigen molecules.

### Claim 17 (depends on 16)

The genetically modified cell of claim 16, comprising one or more indels in a class II major histocompatibility complex transactivator (CIITA) gene in the genome of the genetically modified cell, thereby resulting in the reduced cell surface expression of the one or more MHC-II human leukocyte antigen molecules.

### Claim 18 (depends on 17)

The genetically modified cell of claim 17, wherein the one or more indels comprises a CIITA gene knock out.

### Claim 19 (depends on 16)

The genetically modified cell of claim 16, comprising one or more indels in a β2M gene in the genome of the genetically modified cell, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules.

### Claim 20 (depends on 19)

The genetically modified cell of claim 19, wherein the one or more indels comprises a β2M gene knock out.

### Claim 21 (depends on 1)

The genetically modified cell of claim 1, wherein the genetically modified cell is a CIITA−/− genetically modified cell.

### Claim 22 (depends on 1)

The genetically modified cell of claim 1, wherein the genetically modified cell is a β2M−/− genetically modified cell.

### Claim 23 (depends on 1)

The genetically modified cell of claim 1, wherein the genetically modified cell is a β2M−/− CIITA−/− genetically modified cell.

### Claim 24 (depends on 1)

The genetically modified cell of claim 1, wherein the genetically modified cell is a cardiomyocyte, an endothelial cell, a hepatocyte, a hepatocyte-like cell, a beta cell, a mesenchymal progenitor cell, a neural progenitor cell, a macrophage or a T cell.

### Claim 25 (depends on 1)

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

### Claim 26 (depends on 25)

The genetically modified cell of claim 25, wherein the stem cell is an embryonic stem cell.

### Claim 27 (depends on 25)

The genetically modified cell of claim 25, wherein the stem cell is an induced pluripotent stem cell.

### Claim 28 (depends on 1)

A composition comprising the genetically modified cell of claim 1.

### Claim 29 (depends on 1)

The genetically modified cell of claim 1, wherein cell surface expression of the one or more MHC-I human leukocyte antigen molecules and/or the one or more MHC-II human leukocyte antigen molecules is eliminated.

### Claim 30 (depends on 28)

The composition of claim 28, wherein cell surface expression of the one or more MHC-I human leukocyte antigen molecules and/or the one or more MHC-II human leukocyte antigen molecules is eliminated.

### Claim 31 (depends on 1)

The genetically modified cell of claim 1, comprising one or more indels in one or more genes encoding an MHC-II human leukocyte antigen molecule in the genome of the genetically modified cell, thereby resulting in the reduced cell surface expression of the one or more MHC-II human leukocyte antigen molecules.

### Claim 32 (depends on 1)

The genetically modified cell of claim 1, comprising one or more indels in one or more genes encoding a transcriptional regulator of an MHC-II human leukocyte antigen molecule, thereby resulting in the reduced cell surface expression of the one or more MHC-II human leukocyte antigen molecules.

### Claim 33 (depends on 28)

The composition of claim 28, wherein the genetically modified cell is a β2M−/− CIITA−/− genetically modified cell.