Universal donor cells

Patent metadata

Field	Value
Publication number	US-12559726-B2
Application number	US-202318151369-A
Country	US
Kind code	B2
Filing date	Jan 6, 2023
Priority date	Dec 31, 2020
Publication date	Feb 24, 2026
Grant date	Feb 24, 2026

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 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 genetic modification comprises an insertion of a polynucleotide encoding a tolerogenic factor and/or survival factor. The universal donor cells may further comprise at least one genetic modification within or near a gene that encodes a survival factor, wherein said genetic modification comprises an insertion of a polynucleotide encoding a second tolerogenic factor and/or a different survival factor.

First claim

The invention claimed is:

1 . An isolated genetically modified stem cell, comprising:
a disrupted thioredoxin interacting protein (TXNIP) gene and an insertion of a polynucleotide encoding mesencephalic astrocyte derived neurotrophic factor (MANF) and HLA class I histocompatibility antigen, alpha chain E (HLA-E) into the disrupted TXNIP gene, wherein the cell expresses MANF and HLA-E and has disrupted expression of TXNIP.

2 . The genetically modified stem cell of claim 1 , wherein the polynucleotide encoding MANF and HLA-E comprises a nucleotide sequence encoding an HLA-E trimer, wherein the HLA-E trimer comprises a B2M signal peptide fused to an HLA-G presentation peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

3 . The genetically modified stem cell of claim 2 , wherein the polynucleotide encoding MANF and the HLA-E trimer comprises a nucleotide sequence encoding a P2A peptide between the coding sequence of MANF and the coding sequence of the HLA-E trimer.

4 . The genetically modified stem cell of claim 2 , wherein the polynucleotide encoding MANF and the HLA-E trimer comprises the sequence of SEQ ID NO: 55.

5 . The genetically modified stem cell of claim 2 , wherein the polynucleotide encoding MANF and the HLA-E trimer is operably linked to an exogenous promoter comprising a CMV, EF1α, PGK, CAG, or UBC promoter.

6 . The genetically modified stem cell of claim 5 , wherein the cell expression of TXNIP is reduced or eliminated.

7 . The genetically modified stem cell of claim 1 , comprising:
a disrupted beta-2 microglobulin (B2M) gene and an insertion of a polynucleotide encoding tumor necrosis factor alpha induced protein 27 (TNFAIP3) and programmed death-ligand 1 (PD-L1) into the disrupted B2M gene, wherein the cell expresses TNFAIP3 and PD-L1 and has reduced or eliminated expression of B2M.

8 . The genetically modified stem cell of claim 7 , wherein the polynucleotide encoding TNFAIP3 and PD-L1 is operably linked to an exogenous promoter comprising a CMV, EF1α, PGK, CAG, or UBC promoter.

9 . The genetically modified stem cell of claim 7 , wherein the polynucleotide encoding TNFAIP3 and PD-L1 comprises a nucleotide sequence encoding a P2A peptide between the coding sequence of TNFAIP3 and the coding sequence of PD-L1.

10 . The genetically modified stem cell of claim 7 , wherein the polynucleotide encoding TNFAIP3 and PD-L1 comprises the sequence of SEQ ID NO: 54.

11 . The genetically modified stem cell of claim 3 , comprising a disrupted B2M gene and an insertion of a polynucleotide encoding TNFAIP3 and PD-L1 into the disrupted B2M gene, wherein the cell expresses TNFAIP3 and PD-L1 and has reduced or eliminated expression of B2M, and wherein the polynucleotide encoding TNFAIP3 and PD-L1 comprises a nucleotide sequence encoding a P2A peptide between the coding sequence of TNFAIP3 and the coding sequence of PD-L1.

12 . The genetically modified stem cell of claim 11 , wherein the polynucleotide encoding MANF and the HLA-E trimer comprises the sequence of SEQ ID NO: 55.

13 . The genetically modified stem cell of claim 12 , wherein the polynucleotide encoding TNFAIP3 and PD-L1 comprises the sequence of SEQ ID NO: 54.

14 . The genetically modified stem cell of claim 1 , comprising:
a disrupted class II transactivator (CIITA) gene and an insertion of a polynucleotide encoding cluster of differentiation 39 (CD39) into the disrupted CIITA gene, wherein the cell expresses CD39 and has disrupted expression of CIITA.

15 . The genetically modified stem cell of claim 1 , wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, a pluripotent stem cell, or a hematopoietic stem cell.

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

17 . A plurality of the isolated genetically modified stem cell of claim 1 .

18 . An isolated plurality of lineage-restricted progenitor cells or fully differentiated somatic cells derived from the plurality of genetically modified stem cells of claim 17 .

19 . The plurality of lineage-restricted progenitor cells or fully differentiated somatic cells of claim 18 , comprising definitive endoderm cells, primitive gut tube cells, posterior foregut cells, pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and/or pancreatic beta cells.

20 . A composition, comprising the plurality of lineage-restricted progenitor cells or fully differentiated somatic cells of claim 18 and at least one pharmaceutically acceptable excipient.

Assignees

• Crispr Therapeutics Ag

Inventors

• Rezania Alireza
• Sluch Valentin

Classifications

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12N15/85

  for animal cells · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title