Methods and compositions for treatment of a beta thalessemia

• US 10,072,066 B2
• Filed: 02/02/2015
• Issued: 09/11/2018
• Est. Priority Date: 02/03/2014
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Liu, Pei-Qi, Reik, Andreas, Zhang, Lei
• Primary Examiner(s): Pak, Yong D
• Application Number:
  US14/611,880
• Publication Number:
  US 20150218253A1
• Time in Patent Office: 1,317 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: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding

Abstract

Methods and compositions for treatment of a beta thalessemia are provided.

Claims

1. A genetically modified CD34+ stem cell comprising a corrected endogenous aberrant human beta-hemoglobin (Hbb) gene, a donor comprising the sequence of SEQ ID NO:

19 or SEQ ID NO;

21 and a pair of zinc finger nucleases comprising first and second zinc finger nucleases, wherein each zinc finger nuclease comprises 5 or 6 zinc finger domains, each zinc finger domain comprising a recognition helix region ordered F1 to F5 or F1 to F6, wherein the zinc finger nuclease (ZFN) comprises;

(i) a first zinc finger nuclease that binds to a target site as shown in SEQ ID NO;

5, the first ZFN comprising the following recognition helix regions;

F1;

LRHHLTR (SEQ ID NO;

11);

F2;

QSGTRKT (SEQ ID NO;

12);

F3;

RSDNLST (SEQ ID NO;

13);

F4;

DSANRIK (SEQ ID NO;

14);

F5;

LRHHLTR (SEQ ID NO;

11); and

F6;

QSGNLHV (SEQ ID NO;

15); and

(ii) a second zinc finger nuclease that binds to a target site as shown in SEQ ID NO;

4, the second ZFN comprising the following recognition helix regions;

F1;

AMQTLRV (SEQ ID NO;

16);

F2;

DRSHLAR (SEQ ID NO;

7);

F3;

RSDNLSE (SEQ ID NO;

8);

F4;

ASKTRKN (SEQ ID NO;

9); and

F5;

TSSDRKK (SEQ ID NO;

17) or VYEGLKK (SEQ ID NO;

18),

wherein the donor sequence is integrated into an endogenous aberrant human beta-hemoglobin (Hbb) gene following targeted cleavage by the pair of zinc finger nucleases, thereby correcting the sequence of the Hbb gene.

2. The genetically modified cell of claim 1, wherein the aberrant Hbb gene comprises an intervening sequence 1, mutation number 1 (IVS1-1) mutation.

3. The genetically modified cell of claim 2, wherein the aberrant Hbb gene comprises a point mutation.

4. The genetically modified cell of claim 3, wherein the aberrant Hbb gene comprises a genomic modification within one or more of the sequences of SEQ ID NO:

3.

5. The genetically modified cell of claim 4, wherein the genomic modification within SEQ ID NO:

3 disrupts a splice donor motif.

6. The genetically modified cell of claim 5, wherein the GT dinucleotide at the beginning of intron 1 of the human beta-globin gene is changed to an AT dinucleotide.

7. The genetically modified cell of claim 3, wherein the donor corrects a mutation that alters the splice donor site for mRNA processing of the aberrant Hbb gene that leads to beta-thalassemia.

8. The genetically modified cell of claim 3, wherein the donor sequence is integrated within any of the sequences of SEQ ID Nos. 4 or 5.

9. The genetically modified cell of claim 8, wherein the donor sequence comprises a novel restriction enzyme cleavage site with respect to the endogenous Hbb gene.

10. A genetically modified differentiated cell descended from the stem cell of claim 1.

11. The genetically modified cell of claim 10, wherein the cell is a red blood cell (RBC).

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