Methods and compositions for treatment of a beta thalessemia

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 .