Genetically modified human cell with a corrected mutant sickle cell mutation

• US 10,435,677 B2
• Filed: 09/16/2015
• Issued: 10/08/2019
• Est. Priority Date: 09/16/2014
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Cost, Gregory J., Miller, Jeffrey C., Zhang, Lei
• Primary Examiner(s): Wilson, Michael C
• Application Number:
  US15/511,858
• Publication Number:
  US 20180187173A1
• Time in Patent Office: 1,483 Days
• Field of Search: 435325, 424 9321
• 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: A61K 39/461 characterised by the cell type used
• CPC Class Codes: A61K 39/464 characterised by the antigen targeted or presented
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• 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 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 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 5/0645 Macrophages, e.g. Kuepfer cells in the liver; Monocytes
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

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

Claims

1. An isolated genetically modified human cell comprising a pair of zinc finger nucleases (ZFNs), the pair comprising the zinc finger proteins (ZFPs) designated 47773 and 47817, wherein 47773 binds to a target site within SEQ ID NO:

23 and 47817 binds to a SEQ ID NO;

24 of an endogenous human beta-hemoglobin (Hbb) gene comprising a sickle cell mutation, wherein the pair of ZFNs makes a genomic modification in the mutant endogenous Hbb gene and the genomic modification comprises insertion of a donor sequence such that the sickle cell mutation is corrected to wild-type Hbb, and the cell expresses wild-type Hbb.

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 hematopoietic stem cell.

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

5. A genetically modified 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 donor sequence encodes a transgene.

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

9. A method of correcting a sickle cell mutation in an isolated human cell, the method comprising:

introducing a pair of zinc finger nucleases (ZFNs) into an isolated human cell with an endogenous human beta-hemoglobin (Hbb) gene comprising a sickle cell mutation, the pair comprising the zinc finger proteins (ZFPs) designated 47773 and 47817, wherein 47773 binds to a target site within SEQ ID NO;

23 and 47817 binds to a SEQ ID NO;

24 of the endogenous human Hbb gene comprising a sickle cell mutation, and

introducing a donor sequence into the isolated human cell,

such that the donor sequence is integrated into a double strand break made by the pair of ZFNs, the sickle cell mutation is corrected to wild-type Hbb, and the cell expresses wild-type Hbb.