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 one or more MHC-I or MHC-II human leukocyte antigens or component or transcriptional regulator of the MHC-I or MHC-II complex, at least one genetic modification that increases the expression of at least one polynucleotide that encodes a tolerogenic factor, and optionally at least one genetic modification that increases or decreases the expression of at least one gene that encodes a survival factor.
1. An in vitro method for generating a universal donor cell, the method comprising delivering to a stem cell:
(a) an RNA-guided nuclease;
(b) a guide RNA (gRNA) targeting a target site in a beta-2-microglobulin (B2M) gene locus; and
(c) a vector comprising a nucleic acid, the nucleic acid comprising: (i) a nucleotide sequence encoding a tolerogenic factor; (ii) a nucleotide sequence consisting of SEQ ID NO: 13 and having sequence homology with a genomic region located left and within 50 nucleobases of the target site; and (iii) a nucleotide sequence consisting of SEQ ID NO: 19 having sequence homology with a genomic region located right and within 50 nucleobases of the target site, wherein (i) is flanked by (ii) and (iii);
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, thereby disrupting the B2M gene and generating a universal donor cell, wherein the universal donor cell has increased immune evasion and/or cell survival compared to a stem cell that does not comprise the nucleic acid inserted into the B2M gene locus.
2. The method of claim 1 , wherein the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
3. The method of claim 1 , wherein the tolerogenic factor is programmed death ligand 1 (PD-L1).
4. The method of claim 1 , wherein the nucleotide sequence encoding the tolerogenic factor is operably linked to an exogenous promoter.
5. The method of claim 4 , wherein the exogenous promoter is constitutive, cell type-specific, tissue-type specific, or temporally regulated.
6. The method of claim 5 , wherein the exogenous promoter is a CAG promoter.
7. The method of claim 1 , wherein the vector is a plasm id vector.
8. The method of claim 1 , wherein the RNA-guided nuclease is a Cas9 nuclease.
9. The method of claim 8 , wherein the Cas9 nuclease is linked to at least one nuclear localization signal.
10. The method of claim 9 , wherein the Cas9 nuclease is a S. pyogenes Cas9.
11. The method of claim 1 , wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem and progenitor cell.
12. The method of claim 1 , wherein the stem cell is a human stem cell.
13. The method of claim 12 , wherein the human stem cell is a human hematopoietic stem and progenitor cell.
14. An in vitro method for generating a universal donor cell, the method comprising delivering to a stem cell:
(a) an RNA-guided nuclease;
(b) a guide RNA (gRNA) targeting a target site in a beta-2-microglobulin (B2M) gene locus, wherein the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO: 2; and
(c) a vector comprising a nucleic acid, the nucleic acid comprising: (i) a nucleotide sequence encoding a tolerogenic factor; (ii) a nucleotide sequence consisting of SEQ ID NO: 13 and having sequence homology with a genomic region located left and within 50 nucleobases of the target site; and (iii) a nucleotide sequence consisting of SEQ ID NO: 19 having sequence homology with a genomic region located right and within 50 nucleobases of the target site, wherein (i) is flanked by (ii) and (iii);
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, thereby disrupting the B2M gene and generating a universal donor cell, wherein the universal donor cell has increased immune evasion and/or cell survival compared to a stem cell that does not comprise the nucleic acid inserted into the B2M gene locus.
15. The method of claim 14 , wherein the tolerogenic factor is programmed death ligand 1 (PD-L1).
16. The method of claim 14 , wherein the nucleotide sequence encoding the tolerogenic factor is operably linked to an exogenous promoter.
17. The method of claim 16 , wherein the exogenous promoter is constitutive, cell type-specific, tissue-type specific, or temporally regulated.
18. The method of claim 17 , wherein the exogenous promoter is a CAG promoter.
19. The method of claim 14 , wherein the vector is a plasmid vector.
20. The method of claim 14 , wherein the RNA-guided nuclease is a Cas9 nuclease.
21. The method of claim 20 , wherein the Cas9 nuclease is linked to at least one nuclear localization signal.
22. The method of claim 21 , wherein the Cas9 nuclease is a S. pyogenes Cas9.
23. The method of claim 14 , wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem and progenitor cell.
24. The method of claim 14 , wherein the stem cell is a human stem cell.
25. The method of claim 24 , wherein the human stem cell is a human hematopoietic stem and progenitor cell.