UNIVERSAL DONOR CELLS
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.
1 .- 29 . (canceled)
30 . A genetically modified human pluripotent stem cell, comprising:
a nucleotide sequence encoding programmed death-ligand 1 (PD-L1) operably linked to an exogenous promoter and inserted near or within a gene encoding beta-2 microglobulin (B2M); and
wherein the genetically modified human pluripotent stem cell expresses PD-L1 and has disrupted expression of B2M.
31 . The genetically modified human pluripotent stem cell of claim 30 , wherein insertion of the nucleotide sequence encoding PD-L1 disrupts a translational start site of the B2M gene.
32 . The genetically modified human pluripotent stem cell of claim 30 , wherein the exogenous promoter is selected from the group consisting of a CMV promoter, an EF1α promoter, a PGK promoter, a CAG promoter, and a UBC promoter.
33 . The genetically modified human pluripotent stem cell of claim 30 , wherein the exogenous promoter is a CAG promoter.
34 . The genetically modified human pluripotent stem cell of claim 33 , wherein the CAG promoter comprises a CMV enhancer, a 3-actin promoter, and a chimeric intron.
35 . The genetically modified human pluripotent stem cell of claim 30 , wherein the nucleotide sequence encoding PD-L1 is operably linked to a poly(A) signal.
36 . The genetically modified human pluripotent stem cell of claim 35 , wherein the poly(A) signal comprises SEQ ID NO: 12.
37 . The genetically modified human pluripotent stem cell of claim 30 , wherein the nucleotide sequence encoding PD-L1 comprises SEQ ID NO: 11.
38 . The genetically modified human pluripotent stem cell of claim 30 , wherein the human pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
39 . The genetically modified human pluripotent stem cell of claim 30 , wherein the genetically modified human pluripotent stem cell is capable of being differentiated into an endoderm cell.
40 . The genetically modified human pluripotent stem cell, further comprising a second nucleotide sequence operably linked to an exogenous CAG promoter and inserted within a gene encoding thioredoxin interacting protein (TXNIP), wherein the second nucleotide sequence encodes an HLA-E trimer comprising 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, wherein the genetically modified human pluripotent stem cell expresses the HLA-E trimer and does not express TXNIP.
41 . A genetically modified cell differentiated from the genetically modified human pluripotent stem cell of claim 30 .
42 . A genetically modified cell differentiated from the genetically modified human pluripotent stem cell of claim 40 .
43 . The genetically modified cells of claim 41 , wherein the genetically modified cell is a primitive gut tube cell, a posterior foregut cell, a pancreatic endoderm progenitor, or a pancreatic endocrine progenitor.
44 . The genetically modified cells of claim 42 , wherein the genetically modified cell is a primitive gut tube cell, a posterior foregut cell, a pancreatic endoderm progenitor, or a pancreatic endocrine progenitor.
45 . A plurality of genetically modified human pluripotent stem cells, comprising the genetically modified human pluripotent stem cell of claim 30 .
46 . A plurality of genetically modified human pluripotent stem cells, comprising the genetically modified human pluripotent stem cell of claim 40 .
47 . A population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from the plurality of genetically modified human pluripotent stem cells of claim 42 .
48 . The population of lineage-restricted progenitor cells or fully differentiated somatic cells of claim 47 , wherein the lineage-restricted progenitor cells are pancreatic endoderm progenitors or pancreatic endocrine progenitors.
49 . The population of lineage-restricted progenitor cells or fully differentiated somatic cells of claim 47 , wherein the fully differentiated somatic cells are immature beta cells or mature beta cells.