Nuclease-mediated regulation of gene expression

• US 9,957,501 B2
• Filed: 06/03/2016
• Issued: 05/01/2018
• Est. Priority Date: 06/18/2015
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: Sangamo Therapeutics, Inc., University of Washington
• Inventors: Reik, Andreas, Stamatoyannopoulos, John A., Vierstra, Jeff
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): Leonard, Arthur S
• Application Number:
  US15/172,626
• Publication Number:
  US 20160369262A1
• Time in Patent Office: 697 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. An isolated genetically modified cell comprising a genomic modification at one cleavage site made by a zinc finger nuclease, (ZFN) or TAL-effector domain nuclease (TALEN), which ZFN or TALEN binds to a target site as shown in any of SEQ ID Nos:

58-62, 65 or 74-77, wherein the genomic modification comprises insertions and/or deletions within the one cleavage site.

2. The genetically modified cell of claim 1, wherein the cell is a stem cell.

3. The genetically modified cell of claim 2, wherein the stem cell is a hematopoictic stem cell.

4. The genetically modified cell of claim 3, wherein the hematopoietic stem cell is a CD34+ cell.

5. A genetically modified differentiated cell descended from the stem cell of claim 2.

6. The genetically modified cell of claim 5, wherein the cell is a red blood cell (RBC).

7. The genetically modified cell of claim 1, wherein the nuclease is introduced into the cell as a polynucleotide.

8. The genetically modified cell of claim 1, wherein the insertion comprises integration of a donor polynucleotide encoding a transgene.

9. A pharmaceutical composition comprising the genetically modified cell of claim 1.

10. A DNA binding molecule comprising a TALE-effector protein (TALE) or a single guide RNA that binds to a target site as shown in any of SEQ ID Nos:

58-62, 65 or 74-77.

11. A polynucleotide encoding one or more DNA-binding molecule of claim 10.

12. An isolated cell comprising one or more DNA-binding molecules according to claim 10.

13. A kit comprising a polynucleotide according to claim 11.