Granted Patent B2

US 12,344,655 · App. 18/068,056 · Granted Jul 1, 2025

Inventors: Valentin Sluch (South Boston, MA); Alireza Rezania (South Boston, MA); Jason Sagert (South Boston, MA); Danielle Swain (South Boston, MA)

Assignee: CRISPR THERAPEUTICS AG

C07K14/70539A61K40/15A61K40/31A61K40/4215C07K14/5443C07K14/7051C07K14/7155C07K14/8121C07K16/2878C12N5/0606C12N5/0646C12N5/0696C12N5/10C12N15/66C12N15/85C12N15/907A61K2239/31A61K2239/38A61K2239/48C07K2319/00C12N2310/20C12N2506/11C12N2510/00

Abstract

Claims (33)

1. An in vitro method of generating a genetically modified cell, comprising delivering to a cell:

(a) a first CRISPR associated endonuclease;

(b) a first guide RNA (gRNA) targeting a target site in the Class II major histocompatibility complex transactivator (CIITA) gene locus; and

(c) a first vector comprising a nucleic acid, wherein the 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, (ii) a polynucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E), and (iii) a nucleotide sequence having sequence homology with a genomic region located right of the target site in the CIITA gene locus, wherein (ii) is flanked by (i) and (iii), wherein the polynucleotide encoding HLA-E encodes an HLA-E trimer, wherein the HLA-E trimer comprises a beta-2-microglobulin (B2M) signal peptide fused to an HLA-G presentation peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide;

wherein the CIITA gene locus is cleaved at the target site and the nucleic acid is inserted into the CIITA gene locus, thereby disrupting the CIITA gene.

2. The method of claim 1 , wherein the genetically modified cell has reduced or eliminated expression of CIITA.

3. The method of claim 1 , wherein the polynucleotide encoding HLA-E comprises a coding sequence of a chimeric antigen receptor (CAR).

4. The method of claim 3 , wherein the CAR is an anti-CD30 CAR, anti-CD19 CAR, anti-CD70 CAR, anti-CD33 CAR, or anti-BCMA CAR.

5. The method of claim 3 , wherein the polynucleotide encoding HLA-E and CAR comprises a nucleotide sequence encoding a self-cleaving peptide between the coding sequence of CAR and the coding sequence of HLA-E.

6. The method of claim 5 , wherein the self-cleaving peptide is P2A, E2A, F2A or T2A.

7. The method of claim 1 , wherein the nucleotide sequence having sequence homology with a genomic region located left of the target site in the CIITA gene locus comprises SEQ ID NO: 22, the nucleotide sequence having sequence homology with a genomic region located right of the target site in the CIITA gene locus comprises SEQ ID NO: 32, or both.

8. The method of claim 1 , wherein the first CRISPR associated endonuclease is a Cas9 nuclease and the first gRNA comprises a spacer sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 13-17.

9. The method of claim 1 , comprising delivering to the cell:

a second CRISPR associated endonuclease;

a second guide RNA (gRNA) targeting a target site in the B2M gene locus; and

a second vector comprising a nucleic acid, wherein the nucleic acid comprises (i) a nucleotide sequence having sequence homology with a genomic region located left of the target site in the B2M gene locus, (ii) a polynucleotide sequence encoding SERPINB9, and (iii) a nucleotide sequence having sequence homology with a genomic region located right of the target site in the B2M gene locus, wherein (ii) is flanked by (i) and (iii);

wherein the B2M gene locus is cleaved at the target site and the nucleic acid is inserted into the B2M gene locus, thereby disrupting the B2M gene to result in reduced or eliminated expression of B2M.

10. The method of claim 9 , wherein the polynucleotide sequence encoding SERPINB9 comprises a coding sequence of a fusion protein of interleukin 15 (IL15) and interleukin 15 receptor subunit alpha (IL15Rα).

11. The method of claim 10 , wherein the polynucleotide sequence encoding SERPINB9 and the fusion protein of IL15 and IL15Rα comprises a self-cleaving peptide between the coding sequence of the SERPINB9 protein and the coding sequence of the fusion protein.

12. The method of claim 11 , wherein the polynucleotide sequence encoding SERPINB9 and the fusion protein of IL15 and IL15Rα comprises a nucleotide sequence of SEQ ID NO: 137.

13. The method of claim 10 , wherein the polynucleotide sequence encoding SERPINB9 and the fusion protein of IL15 and IL15Rα is operably linked to an exogenous promoter.

14. The method of claim 9 , wherein the second-RNA guided CRISPR associated endonuclease is a Cas9 nuclease.

15. The method of claim 9 , wherein the nucleotide sequence having sequence homology with a genomic region located left of the target site in the B2M gene locus comprises SEQ ID NO: 36, the nucleotide sequence having sequence homology with a genomic region located right of the target site in the B2M gene locus comprises SEQ ID NO: 54, or both.

16. The method of claim 1 , comprising delivering to the cell

a third CRISPR associated endonuclease;

a third guide RNA (gRNA) targeting a target site in the cytokine-inducible SH2-containing protein (CISH) gene locus;

wherein the CISH gene locus is cleaved at the target site and at least one insertion or deletion mutation is introduced into the CISH gene, thereby disrupting the CISH gene.

17. The method of claim 1 , comprising delivering to the cell

a fourth CRISPR associated endonuclease;

a fourth guide RNA (gRNA) targeting a target site in the Fas cell surface death receptor (FAS) gene locus;

wherein the FAS gene locus is cleaved at the target site and at least one insertion or deletion mutation is introduced into the FAS gene, thereby disrupting the FAS gene.

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

19. The method of claim 18 , 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 and progenitor cell.

Assignments (1)

Continuity (5)

References Cited (395)