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 .- 101 . (canceled) 
     
     
         102 . An in vitro method for generating a genetically modified 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 class II transactivator (CIITA) gene locus;   (b) a first nucleic acid comprising a first nucleotide sequence encoding cluster of differentiation 39 (CD39), whereby the CIITA gene locus is cleaved at the target site to disrupt the CIITA gene and the first nucleic acid is inserted into the CIITA gene locus.   
     
     
         103 . The in vitro method of  claim 102 , wherein the first nucleotide sequence encoding CD39 is operably linked to an exogenous promoter. 
     
     
         104 . The in vitro method of  claim 103 , wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter. 
     
     
         105 . The in vitro method of  claim 102 , wherein the first nucleic acid comprises: (i) a nucleotide sequence having sequence homology with a genomic region located left of the target site in the CIITA gene locus and (ii) a nucleotide sequence having sequence homology with a genomic region located right of the target site in the CIITA gene locus; and wherein the first nucleotide sequence encoding CD39 is flanked by (i) and (ii). 
     
     
         106 . The in vitro method of  claim 105 , wherein the first nucleotide sequence encoding CD39 comprises the sequence of SEQ ID NO: 27, the nucleotide sequence of (i) comprises the sequence of SEQ ID NO: 26, and/or the nucleotide sequence of (ii) comprises the sequence of SEQ ID NO: 28. 
     
     
         107 . The in vitro method of  claim 102 , wherein the expression of CIITA in the genetically modified cell is reduced or eliminated. 
     
     
         108 . The in vitro method of  claim 102 , comprising delivering to the stem cell:
 (c) a second RNA guided nuclease and a second gRNA targeting a target site in a transforming growth factor beta 2 (TGF-β2) gene locus, whereby the TGF-β2 gene locus is cleaved at the target site to disrupt the TGF-β2 gene.   
     
     
         109 . The in vitro method of  claim 108 , wherein the second gRNA targeting a target site in the TGF-β2 gene comprises the sequence of SEQ ID NO: 57. 
     
     
         110 . The in vitro method of  claim 102 , wherein the stem cell comprises:
 (a) a disrupted thioredoxin interacting protein (TXNIP) gene and an insertion of a polynucleotide encoding mesencephalic astrocyte derived neurotrophic factor (MANF) into the disrupted TXNIP gene; and/or   (b) a disrupted beta-2 microglobulin (B2M) gene and an insertion of a polynucleotide encoding tumor necrosis factor alpha induced protein 3 (TNFAIP3) into the disrupted B2M gene.   
     
     
         111 . The in vitro method of  claim 102 , wherein the stem cell comprises:
 (a) a disrupted TXNIP gene and an insertion of a polynucleotide encoding MANF and HLA class I histocompatability antigen, alpha chain E (HLA-E) into the disrupted TXNIP gene; and/or   (b) a disrupted B2M gene and an insertion of a polynucleotide encoding TNFAIP3 and programmed death-ligand 1 (PD-L1) into the disrupted B2M gene.   
     
     
         112 . The in vitro method of  claim 102 , wherein the genetically modified cell comprises (a) a disrupted TXNIP gene and an insertion of a polynucleotide encoding MANF and HLA-E into the disrupted TXNIP gene; and/or (b) a disrupted B2M gene and an insertion of a polynucleotide encoding TNFAIP3 and PD-L1 into the disrupted B2M gene. 
     
     
         113 . The in vitro method of  claim 112 , wherein 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. 
     
     
         114 . The in vitro method of  claim 112 , wherein the polynucleotide encoding MANF and HLA-E trimer comprises a nucleotide sequence encoding a P2A peptide between the coding sequence of MANF and the coding sequence of HLA-E trimer. 
     
     
         115 . The in vitro method of  claim 112 , 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. 
     
     
         116 . The in vitro method of  claim 112 , wherein the polynucleotide encoding MANF and HLA-E trimer comprises the sequence of SEQ ID NO: 55, and/or the polynucleotide encoding TNFAIP3 and PD-L1 comprises the sequence of SEQ ID NO: 54. 
     
     
         117 . The in vitro method of  claim 112 , wherein the expression of TXNIP and/or B2M gene in the genetically modified cell is reduced or eliminated. 
     
     
         118 . The in vitro method of  claim 102 , wherein the first RNA guided nuclease is a Cas9 nuclease. 
     
     
         119 . The in vitro method of  claim 102 , 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. 
     
     
         120 . The in vitro method of  claim 102 , wherein the stem cell is a human stem cell. 
     
     
         121 . The in vitro method of  claim 102 , wherein the genetically modified cell is a definitive endoderm cell, primitive gut tube cell, posterior foregut cell, pancreatic endoderm cell, pancreatic endocrine precursor cell, immature beta cell, and/or pancreatic beta cell.