Universal donor cells
AssigneeCrispr Therapeutics AG
InventorsAlireza Rezania, Rebeca Ramos-Zayas
Life of this patent
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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 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.
Claims25
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 essentially 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 essentially 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.
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 essentially 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 essentially 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.
Dependencies are inferred from the claim text.