# US 10,865,424 B2 · Universal donor cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,424 B2 - Document ID: US10865424B2 - Status: Granted - Kind: B2 - Application number: 16/928,151 - Filed: 2020-07-14 - Published: 2020-11-05 - Granted: 2020-12-15 - Earliest claimed: 2018-09-07 - Assignees: Crispr Therapeutics AG - Applicants: Crispr Therapeutics AG - Inventors: Alireza Rezania; Rebeca Ramos-Zayas - Web page: https://patentgrep.com/patent/US10865424B2 ## CPC - C12N 15/85: for animal cells - C07K 14/70532: {B7 molecules, e.g. CD80, CD86} - C07K 14/70539: {MHC-molecules, e.g. HLA-molecules} - C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)} - C12N 5/0607: {Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696)} - C12N 5/0676: {Pancreatic cells} - C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS} - C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein} - C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} - C12N 15/907: {in mammalian cells} - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2501/50: Cell markers; Cell surface determinants - C12N 2506/02: from embryonic cells - C12N 2506/03: from non-embryonic pluripotent stem cells - C12N 2506/45: from artificially induced pluripotent stem cells - C12N 2510/00: Genetically modified cells - C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## 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. ## Claims ### Claim 1 An in vitro method for generating a universal donor cell, the method comprising delivering to a stem cell: (a) a ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in a beta-2-microglobulin (B2M) gene locus; and (b) 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) a nucleotide sequence encoding a tolerogenic factor, and (iii) a nucleotide sequence homologous with a region located right of the target site in the B2M gene locus, wherein the vector comprises a nucleotide sequence consisting of SEQ ID NO: 33, 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) The method of claim 1, wherein the nucleotide sequence of (i) consists essentially of SEQ ID NO: 13, and the nucleotide sequence of (iii) consists essentially of SEQ ID NO: 19. ### Claim 4 (depends on 1) The method of claim 1, wherein the vector is a plasmid vector. ### Claim 5 (depends on 1) The method of claim 1, wherein the RNA-guided nuclease is a Cas9 nuclease. ### Claim 6 (depends on 5) The method of claim 5, wherein the Cas9 nuclease is linked to at least one nuclear localization signal. ### Claim 7 (depends on 6) The method of claim 6, wherein the Cas9 nuclease is a S. pyogenes Cas9. ### Claim 8 (depends on 1) 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. ### Claim 9 (depends on 1) The method of claim 1, wherein the stem cell is a human stem cell. ### Claim 10 An in vitro method for generating a universal donor cell, the method comprising delivering to a stem cell: (a) a ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and 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 (b) 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, wherein the nucleotide sequence consists essentially of SEQ ID NO: 13, (ii) a nucleotide sequence encoding a tolerogenic factor, and (iii) a nucleotide sequence homologous with a region located right of the target site in the B2M gene locus, wherein the nucleotide sequence consists essentially of SEQ ID NO:19, wherein the vector comprises a nucleotide sequence consisting of SEQ ID NO: 33, 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 the 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. ### Claim 11 (depends on 10) The method of claim 10, wherein the vector is a plasmid vector. ### Claim 12 (depends on 10) The method of claim 10, wherein the RNA-guided nuclease is a Cas9 nuclease. ### Claim 13 (depends on 12) The method of claim 12, wherein the Cas9 nuclease is linked to at least one nuclear localization signal. ### Claim 14 (depends on 13) The method of claim 13, wherein the Cas9 nuclease is a S. pyogenes Cas9. ### Claim 15 (depends on 10) The method of claim 10, 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. ### Claim 16 (depends on 10) The method of claim 10, wherein the stem cell is a human stem cell.