Nuclease-mediated regulation of gene expression

• US 10,619,154 B2
• Filed: 03/21/2018
• Issued: 04/14/2020
• Est. Priority Date: 06/18/2015
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Reik, Andreas, Stamatoyannopoulos, John A., Vierstra, Jeff
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): Leonard, Arthur S
• Application Number:
  US15/927,357
• Publication Number:
  US 20180237768A1
• Time in Patent Office: 755 Days
• Field of Search: None
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic cell.

Claims

1. A method of altering globin gene expression in a cell, the method comprising:

introducing, into the cell, one or more polynucleotides encoding a zinc finger nuclease (ZFN) or a TAL-effector domain nuclease (TALEN) that binds to a target site in a BCL11A gene as shown in any of SEQ ID Nos;

58-61, 65, 74 or 77, under conditions such that the nuclease cleaves and genetically modifies the BCL11A gene and expression of the globin gene is altered.

2. The method of claim 1, wherein expression of the globin gene is increased.

3. The method of claim 2, wherein the globin gene is a gamma globin or beta globin gene.

4. The method of claim 1, further comprising introducing at least one donor sequence into the cell such that the donor sequence is integrated into the BCL11A gene following cleavage by the nuclease.

5. The method of claim 4, wherein the donor sequence is introduced to the cell using a viral vector, as an oligonucleotide or on a plasmid.

6. The method of claim 1, wherein the cell is selected from the group consisting of a red blood cell (RBC) precursor cell and a hematopoietic stem cell.

7. An isolated cell with altered globin expression as generated by the method of claim 1.

8. A pharmaceutical composition comprising one or more cells of claim 7.

9. A method of treating a patient in need of an increase in globin gene expression, the method comprising administering to the patient the pharmaceutical composition of claim 8.

10. The method of claim 9, wherein the patient is known to have, is suspected of having, or is at risk of developing a hemoglobinopathy.

11. The method of claim 10, wherein the hemoglobinopathy is a thalassemia or sickle cell disease.

12. The method of claim 11, wherein the thalassemia is β

-thalassemia.