Universal donor cells

• US 10,724,052 B2
• Filed: 09/06/2019
• Issued: 07/28/2020
• Est. Priority Date: 09/07/2018
• Status: Active Grant

• Current Assignee: CRISPR Therapeutics AG
• Original Assignee: CRISPR Therapeutics AG
• Inventors: Rezania, Alireza, Ramos-Zayas, Rebeca
• Primary Examiner(s): Chen, Shin Lin
• Application Number:
  US16/563,553
• Publication Number:
  US 20200080114A1
• Time in Patent Office: 326 Days
• Field of Search: 4353201, 435455, 424 9321, 536 231, 536 235
• CPC Class Codes: C07K 14/70532 B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2501/50 Cell markers; Cell surface determinants
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

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

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 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.

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 nucleotide sequence of (i) consists essentially of SEQ ID NO:

13, and the nucleotide sequence of (iii) consists essentially of SEQ ID NO;

19.

4. The method of claim 1, wherein the vector is a plasmid vector.

5. The method of claim 1, wherein the RNA-guided nuclease is a Cas9 nuclease.

6. The method of claim 5, wherein the Cas9 nuclease is linked to at least one nuclear localization signal (NLS).

7. The method of claim 6, wherein the Cas9 nuclease is a S. pyogenes Cas9.

8. The method of claim 1, wherein the stem cell is an embryonic stem cell (ESC), an adult stem cell (ASC), an induced pluripotent stem cell (iPSC), or a hematopoietic stem and progenitor cell (HSPC).

9. The method of claim 1, wherein the stem cell is a human stem cell.

10. 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 homologous with a region located left of the target site in the B2M gene locus, wherein the nucleotide sequence that 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 that 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.

11. The method of claim 10, wherein the vector is a plasmid vector.

12. The method of claim 10, wherein the RNA-guided nuclease is a Cas9 nuclease.

13. The method of claim 12, wherein the Cas9 nuclease is linked to at least one nuclear localization signal (NLS).

14. The method of claim 13, wherein the Cas9 nuclease is a S. pyogenes Cas9.

15. The method of claim 10, wherein the stem cell is an embryonic stem cell (ESC), an adult stem cell (ASC), an induced pluripotent stem cell (iPSC), or a hematopoietic stem and progenitor cell (HSPC).

16. The method of claim 10, wherein the stem cell is a human stem cell.