Universal donor cells

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.

1 . A genetically modified cell, comprising:
 an insertion of a polynucleotide encoding programmed death-ligand 1 (PD-L1) and tumor necrosis factor alpha induced protein 3 (TNFAIP3); and   an insertion of a polynucleotide encoding mesencephalic astrocyte derived neurotrophic factor (MANF);   wherein the cell expresses PD-L1, TNFAIP3 and MANF at an increased level relative to an unmodified cell.   
     
     
         2 . The genetically modified cell of  claim 1 , comprising a disrupted beta-2 microglobulin (B2M) gene, wherein the cell has reduced or eliminated expression of B2M. 
     
     
         3 . The genetically modified cell of  claim 2 , wherein the polynucleotide encoding PD-L1 and TNFAIP3 is inserted into the disrupted B2M gene. 
     
     
         4 . The genetically modified cell of  claim 3 , wherein the polynucleotide encoding TNFAIP3 and PD-L1 comprises a nucleotide sequence encoding a ribosome skip between the coding sequence of TNFAIP3 and the coding sequence of PD-L1. 
     
     
         5 . The genetically modified cell of  claim 4 , wherein the ribosome skip is a 2A sequence family member. 
     
     
         6 . The genetically modified cell of  claim 5 , wherein the 2A sequence family member is an F2A, T2A, E2A, or P2A peptide. 
     
     
         7 . The genetically modified cell of  claim 3 , wherein the polynucleotide encoding TNFAIP3 and PD-L1 comprises the sequence of SEQ ID NO: 54. 
     
     
         8 . The genetically modified cell of  claim 1 , comprising a disrupted thioredoxin interacting protein (TXNIP) gene, wherein the cell has reduced or eliminated expression of TXNIP. 
     
     
         9 . The genetically modified cell of  claim 8 , wherein the polynucleotide encoding MANF is inserted into the disrupted TXNIP gene. 
     
     
         10 . The genetically modified cell of  claim 9 , wherein the polynucleotide encoding MANF also encodes HLA class I histocompatibility antigen, alpha chain E (HLA-E). 
     
     
         11 . The genetically modified cell of  claim 10 , 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. 
     
     
         12 . The genetically modified cell of  claim 11 , wherein the polynucleotide encoding MANF and the HLA-E trimer comprises a nucleotide sequence encoding a ribosome skip between the coding sequence of MANF and the coding sequence of the HLA-E trimer. 
     
     
         13 . The genetically modified cell of  claim 12 , wherein the ribosome skip is a 2A sequence family member. 
     
     
         14 . The genetically modified cell of  claim 13 , wherein the 2A sequence family member is an F2A, T2A, E2A, or P2A peptide. 
     
     
         15 . The genetically modified cell of  claim 11 , wherein the polynucleotide encoding MANF and the HLA-E trimer comprises the sequence of SEQ ID NO: 55. 
     
     
         16 . The genetically modified cell of  claim 1 , wherein the polynucleotide encoding PD-L1 and TNFAIP3, the polynucleotide encoding MANF, or both is operably linked to an exogenous promoter. 
     
     
         17 . The genetically modified cell of  claim 16 , wherein the exogenous promoter is a constitutive, inducible, temporal-, tissue-, or cell type-specific promoter. 
     
     
         18 . The genetically modified cell of  claim 1 , wherein the cell comprises increased expression of one or more safety switch proteins relative to an unmodified cell. 
     
     
         19 . The genetically modified cell of  claim 1 , wherein the cell is a stem cell. 
     
     
         20 . The genetically modified cell of  claim 19 , 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 and progenitor cell. 
     
     
         21 . A plurality of genetically modified stem cells of  claim 19 . 
     
     
         22 . A plurality of lineage-restricted progenitor cells or fully differentiated somatic cells derived from the plurality of genetically modified stem cells of  claim 21 . 
     
     
         23 . The plurality of lineage-restricted progenitor cells or fully differentiated somatic cells of  claim 21 , comprising definitive endoderm cells, primitive gut tube cells, posterior foregut cells, pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, pancreatic beta cells, or combinations thereof. 
     
     
         24 . A composition, comprising the plurality of lineage-restricted progenitor cells or fully differentiated somatic cells of  claim 22  and at least one pharmaceutically acceptable excipient. 
     
     
         25 . A device, comprising the plurality of lineage-restricted progenitor cells or fully differentiated somatic cells of  claim 22 . 
     
     
         26 . The device of  claim 25 , wherein the device is configured for delivering the plurality of lineage-restricted progenitor cells or fully differentiated somatic cells to a subject. 
     
     
         27 . The device of  claim 25 , wherein the device is an implantable device.