Methods and compositions for treatment of a genetic condition

• US 10,196,652 B2
• Filed: 01/16/2018
• Issued: 02/05/2019
• Est. Priority Date: 05/15/2013
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Conway, Anthony, Cost, Gregory J., Dekelver, Russell, Rebar, Edward J., Reik, Andreas, Urnov, Fyodor, Wang, Jianbin, Zhang, H. Steve
• Primary Examiner(s): Gonzalez, Antonio Galisteo
• Application Number:
  US15/872,644
• Publication Number:
  US 20180148740A1
• Time in Patent Office: 385 Days
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C07K 2/00 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• 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 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21004 Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

Methods and compositions comprising a CRISPR/Cas nuclease that cleaves a T cell receptor (TCR) gene and/or human leukocyte (HLA) gene, the nuclease comprising a cleavage domain and a single guide RNA that binds to a target site in the TCR and/or HLA gene.

Claims

1. A CRISPR/Cas nuclease comprising a cleavage domain and a single guide RNA (sgRNA), wherein the sgRNA binds to a target site as shown in any of SEQ ID NO:

96-108 in a T cell receptor (TCR) gene or a target site as shown in any of SEQ ID NO;

118-148 in a human leukocyte (HLA) gene or HLA accessory gene;

wherein the nuclease cleaves and inactivates the TCR and/or HLA gene.

2. The nuclease of claim 1, wherein the TCR gene is a T-cell receptor alpha (TRAC) gene or a T-cell receptor beta (TRBC) gene.

3. The nuclease of claim 1, wherein the HLA gene is an HLA-A gene, an HLA B gene, an HLA C gene, an HLA-DPA gene, an HLA-DQ gene, an HLA-DRA gene and/or a beta-2 microglobulin gene.

4. The nuclease of claim 1, wherein the cleavage domain comprises a Type IIS restriction enzyme nuclease domain or a Cas protein.

5. A mammalian cell comprising the nuclease of claim 1.

6. The mammalian cell of claim 5, wherein the mammalian cell is a stem cell.

7. The mammalian cell of claim 6, wherein the stem cell is a hematopoietic stem cell.

8. The mammalian cell of claim 5, further comprising a transgene encoding a chimeric antigen receptor (CAR).

9. The mammalian cell of claim 5, further wherein one or more additional genes are inactivated in the cell.

10. The mammalian cell of claim 9, wherein the one or more additional inactivated genes comprise a PD1 gene, a CTLA-4 gene, a Tap1 gene, a Tap2 gene, a tapasin gene, a CIITA gene and/or an RFX5 gene.

11. A method of inactivating an endogenous TCR and/or HLA gene in a cell, the method comprising the steps of:

administering to the cell one or more CRISPR/Cas nucleases according to claim 1, wherein the nucleases cleave and inactivate the TCR and/or HLA genes.

12. The method of claim 11, comprising a Type IIS restriction enzyme nuclease domain or a Cas protein.

13. The method of claim 11, wherein the cell is a mammalian cell.

14. The method of claim 13, wherein the mammalian cell is a stem cell.

15. The method of claim 14, wherein the stem cell is a hematopoietic stem cell.

16. The method of claim 11, further comprising integrating one or more CAR transgenes into the cell.

17. A cell made by method of claim 11.

18. A method of treating a disorder in a subject in need thereof, the method comprising

administering to the subject one or more CRISPR/Cas nucleases according to claim 1, wherein the nucleases cleave and inactivate the TCR and/or HLA genes in a cell of the subject, such that the disorder is treated.

19. The method of claim 18, wherein the disorder is a cancer.

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

21. The method of claim 20, wherein the stem cell is a hematopoietic stem cell.