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 method of generating a genetically modified stem cell, comprising
 (a) introducing an insertion of a polynucleotide encoding programmed death-ligand 1 (PD-L1) and tumor necrosis factor alpha induced protein 3 (TNFAIP3) into the genome of a stem cell; and   (b) introducing an insertion of a polynucleotide encoding mesencephalic astrocyte derived neurotrophic factor (MANF) into the genome of the stem cell, thereby generating a genetically modified stem cell expressing PD-L1, TNFAIP3 and MANF at an increased level relative to an unmodified stem cell.   
     
     
         2 . The method of  claim 1 , comprising introducing a deletion into the beta-2 microglobulin (B2M) gene of the stem cell to result in a disrupted B2M gene, wherein the stem cell has reduced or eliminated expression of B2M. 
     
     
         3 . The method of  claim 2 , wherein the polynucleotide encoding PD-L1 and TNFAIP3 is inserted into the disrupted B2M gene. 
     
     
         4 . The method of  claim 3 , wherein (a) comprises delivering to the stem cell:
 (a) an RNA-guided nuclease;   (b) a guide RNA (gRNA) targeting a target site in the B2M gene locus; and   (c) a vector comprising a nucleic acid, the nucleic acid comprising (i) a nucleotide sequence homologous with a region located left of the target site in the B2M gene locus, (ii) the polynucleotide sequence encoding PD-L1 and TNFAIP3, and (iii) a nucleotide sequence homologous with a region located right of the target site in the B2M gene locus, wherein (ii) is flanked by (i) and (iii);   wherein the B2M gene locus is cleaved at the target site and the nucleic acid is inserted into the B2M gene locus, thereby disrupting the B2M gene.   
     
     
         5 . The method of  claim 4 , wherein the target site in the B2M gene locus comprises a sequence selected from SEQ ID NOs: 1-13. 
     
     
         6 . The method of  claim 4 , wherein the polynucleotide encoding PD-L1 and TNFAIP3 comprises the sequence of SEQ ID NO: 54. 
     
     
         7 . The method of  claim 4 , wherein the nucleotide sequence homologous with a region located left of the target site in the B2M gene locus comprises SEQ ID NO: 15, the nucleotide sequence homologous with a region located right of the target site in the B2M gene locus comprises SEQ ID NO: 22, or both. 
     
     
         8 . The method of  claim 4 , wherein the B2M gene locus is cleaved at the target site and the nucleic acid is inserted into the B2M gene locus within 50 base pairs of the target site. 
     
     
         9 . The method of  claim 1 , comprising introducing a deletion into the thioredoxin interacting protein (TXNIP) gene of the stem cell to result in a disrupted TXNIP gene, wherein the stem cell has reduced or eliminated expression of TXNIP. 
     
     
         10 . The method of  claim 9 , wherein the polynucleotide encoding MANF is inserted into the disrupted TXNIP gene. 
     
     
         11 . The method of  claim 10 , wherein the polynucleotide encoding MANF also encodes HLA class I histocompatibility antigen, alpha chain E (HLA-E). 
     
     
         12 . The method of  claim 11 , wherein (b) comprises delivering to the stem cell:
 (a) an RNA-guided nuclease;   (b) a guide RNA (gRNA) targeting a target site in the TXNIP gene locus; and   (c) a vector comprising a nucleic acid, the nucleic acid comprising (i) a nucleotide sequence homologous with a region located left of the target site in the TXNIP gene locus, (ii) the polynucleotide sequence encoding MANF and HLA-E, and (iii) a nucleotide sequence homologous with a region located right of the target site in the TXNIP gene locus, wherein (ii) is flanked by (i) and (iii);   wherein the TXNIP gene locus is cleaved at the target site and the nucleic acid is inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene.   
     
     
         13 . The method of  claim 12 , wherein the target site in the TXNIP gene locus comprises a sequence selected from SEQ ID NOs: 32-41. 
     
     
         14 . The method of  claim 12 , wherein the polynucleotide encoding MANF and the HLA-E trimer comprises the sequence of SEQ ID NO: 55. 
     
     
         15 . The method of  claim 12 , wherein the nucleotide sequence homologous with a region located left of the target site in the TXNIP gene locus comprises SEQ ID NO: 42, the nucleotide sequence homologous with a region located right of the target site in the TXNIP gene locus comprises SEQ ID NO: 44, or both. 
     
     
         16 . The method of  claim 4 , wherein the RNA-guided nuclease is Cas9 nuclease. 
     
     
         17 . The method of  claim 16 , wherein the Cas9 nuclease is linked to at least one nuclear localization signal (NLS). 
     
     
         18 . The method of  claim 16 , wherein the Cas9 nuclease is a  S. pyogenes  Cas9. 
     
     
         19 . The method of  claim 1 , wherein the polynucleotide encoding PD-L1 and TNFAIP3, the polynucleotide encoding MANF, or both is operably linked to an exogenous promoter. 
     
     
         20 . The method 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 and progenitor cell.