Universal donor cells

An in vitro method for generating a universal donor cell, the method comprising delivering to a stem cell:

• (a) a first RNA-guided nuclease and a first guide RNA (gRNA) targeting a target site in a beta-2 microglobulin (B2M) gene locus;
• (b) a first vector comprising a nucleic acid, the nucleic acid comprising:
• (i) a nucleotide sequence comprising SEQ ID NO:
• 54 and encoding tumor necrosis factor alpha induced protein 3 (TNFAIP3) and a nucleotide sequence encoding programmed death-ligand 1 (PD-L1);
• (ii) a nucleotide sequence having sequence homology with a genomic region located left of the target site in the B2M gene locus;
• and (iii) a nucleotide sequence having sequence homology with a genomic region located right of the target site in the B2M gene locus, wherein (i) is flanked by (ii) and (iii);
• wherein the B2M gene locus is cleaved at the target site and the nucleic acid comprising the nucleotide sequences encoding TNFAIP3 and PD-L1 is inserted into the B2M gene locus, thereby disrupting the B2M gene;
• and;
• (c) a second RNA-guided nuclease and a second gRNA targeting a target site in a thioredoxin interacting protein (TXNIP) gene locus;
• and
• (d) a second vector comprising a nucleic acid, the nucleic acid comprising:
• (i) a nucleotide sequence encoding mesencephalic astrocyte derived neurotrophic factor (MANF) and a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E);
• (ii) a nucleotide sequence having sequence homology with a genomic region located left of the target site in the TXNIP gene locus;
• and (iii) a nucleotide sequence having sequence homology with a genomic region located right of the target site in the TXNIP gene locus, wherein (i) is flanked by (ii) and (iii);
• wherein the TXNIP gene locus is cleaved at the target site and the nucleic acid comprising the nucleotide sequences encoding MANF and HLA-E is inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene;
• wherein the universal donor cell expresses TNFAIP3, PD-L1, MANF and HLA-E and has disrupted expression of B2M and TXNIP.