Methods and compositions for treatment of a genetic condition

• US 9,963,715 B2
• Filed: 08/29/2013
• Issued: 05/08/2018
• Est. Priority Date: 08/29/2012
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Cost, Gregory J., Gregory, Philip D., Guschin, Dmitry, Holmes, Michael C., Miller, Jeffrey C., Paschon, David, Rebar, Edward J., Reik, Andreas, Urnov, Fyodor, Zhang, Lei
• Primary Examiner(s): SINGH, ANOOP KUMAR
• Application Number:
  US14/013,236
• Publication Number:
  US 20140093913A1
• Time in Patent Office: 1,713 Days
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are nucleases and methods of using these nucleases for alteration of a globin gene and generation of cells.

Claims

1. A zinc finger protein comprising five or six zinc finger domains designated and ordered F1 to F5 or F1 to F6 from N-terminus to C-terminus, wherein F1 to F5 or F1 to F6 comprises any one of the following recognition helix regions:

(i) F1;

DRSNLSR (SEQ ID NO;

9);

F2;

LRQNLIM (SEQ ID NO;

161);

F3;

TSANLTV (SEQ ID NO;

162);

F4;

RSDHLSR (SEQ ID NO;

94);

F5;

QSGNLAR (SEQ ID NO;

39); and

F6;

QRNDRKS (SEQ ID NO;

163), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

160;

(ii) F1;

DRSNLSR (SEQ ID NO;

9);

F2;

LRQNLIM (SEQ ID NO;

161);

F3;

LQSQLNR (SEQ ID NO;

164);

F4;

RSDHLSR (SEQ ID NO;

94);

F5;

QSGNLAR (SEQ ID NO;

39); and

F6;

QRNDRKS (SEQ ID NO;

163), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

160;

(iii) F1;

DRANLSR (SEQ ID NO;

165);

F2;

LRQNLIM (SEQ ID NO;

161);

F3;

LQSQLNR (SEQ ID NO;

164);

F4;

RSDHLSR (SEQ ID NO;

94);

F5;

QSGNLAR (SEQ ID NO;

39); and

F6;

QRNDRKS (SEQ ID NO;

163), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

160;

(iv) F1;

DRSNLSR (SEQ ID NO;

9);

F2;

TSSNRNH (SEQ ID NO;

166);

F3;

HSGNLTK (SEQ ID NO;

167);

F4;

RSDHLSR (SEQ ID NO;

94);

F5;

QSGNLAR (SEQ ID NO;

39); and

F6;

QKVDLSR (SEQ ID NO;

168), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

160;

(v) F1;

DRSNLSR (SEQ ID NO;

9);

F2;

TSSNRNH (SEQ ID NO;

166);

F3;

QANNLKV (SEQ ID NO;

169);

F4;

RSDHLSR (SEQ ID NO;

94);

F5;

QSGNLAR (SEQ ID NO;

39); and

F6;

QKVDLSR (SEQ ID NO;

168), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

160;

(vi) F1;

RSDHLSA (SEQ ID NO;

59);

F2;

DRSALAR (SEQ ID NO;

30);

F3;

RSDSLSR (SEQ ID NO;

171);

F4;

DRSVRTK (SEQ ID NO;

172);

F5;

RSDHLSA (SEQ ID NO;

59); and

F6;

QRSNLKV (SEQ ID NO;

173), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

170;

(vii) F1;

RSDHLTQ (SEQ ID NO;

35);

F2;

DRSALAR (SEQ ID NO;

30);

F3;

RSDSLSR (SEQ ID NO;

171);

F4;

DRSVRTK (SEQ ID NO;

172);

F5;

RSDHLSA (SEQ ID NO;

59); and

F6;

QRSNLKV (SEQ ID NO;

173), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

170;

(viii) F1;

RSDHLTT (SEQ ID NO;

174);

F2;

DRSALAR (SEQ ID NO;

30);

F3;

RSDSLSR (SEQ ID NO;

171);

F4;

DRSVRTK (SEQ ID NO;

172);

F5;

RSDHLSA (SEQ ID NO;

59); and

F6;

QRSNLKV (SEQ ID NO;

173), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

170;

(ix) F1;

RSDHLSA (SEQ ID NO;

59);

F2;

WATARDR (SEQ ID NO;

175);

F3;

RSDSLSR (SEQ ID NO;

171);

F4;

HTKSLSR (SEQ ID NO;

176);

F5;

RSDHLSA (SEQ ID NO;

59);

F6;

QRSNLKV (SEQ ID NO;

173);

wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

170;

(x) F1;

RSAHLTQ (SEQ ID NO;

177);

F2;

DRSVLRR (SEQ ID NO;

178)

F3;

RSDSLSR (SEQ ID NO;

171);

F4;

DRSVRTK (SEQ ID NO;

172);

F5;

RSDHLSA (SEQ ID NO;

59); and

F6;

QRSNLKV (SEQ ID NO;

173), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

170;

(xi) F1;

RSDVLSE (SEQ ID NO;

57);

F2;

RNQHRKT (SEQ ID NO;

58);

F3;

RSDHLSA (SEQ ID NO;

59);

F4;

RSANLTR (SEQ ID NO;

60;

F5;

RSDVLSN (SEQ ID NO;

61); and

F6;

DRSTRIT (SEQ ID NO;

62), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

56;

(xii) F1;

DRSNLSR (SEQ ID NO;

9);

F2;

HRQHLVT (SEQ ID NO;

64);

F3;

DRSNLTR (SEQ ID NO;

65);

F4;

QSGDLTR (SEQ ID NO;

13); and

F5;

HRSSLLN (SEQ ID NO;

66), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

63;

(xiii) F1;

QSGHLSR (SEQ ID NO;

32);

F2;

RSDHLST (SEQ ID NO;

67);

F3;

RSADLSR (SEQ ID NO;

68);

F4;

RSDNLSQ (SEQ ID NO;

69); and

F5;

ASNDRKK (SEQ ID NO;

70), wherein the zinc finger protein binds to a target site as shown in SEQ ID NO;

66;

or

(xiv) F1;

RSDNLSA (SEQ ID NO;

72);

F2;

RNNDRKT (SEQ ID NO;

73);

F3;

DRSDLSR (SEQ ID NO;

29);

F4;

TSSNRTK (SEQ ID NO;

74);

F5;

QSGNLAR (SEQ ID NO;

39); and

F6;

QSGDLTR (SEQ ID NO;

13), wherein the zinc finger protein binds to a target site as set forth in SEQ ID NO;

71.

2. A fusion protein comprising the zinc finger protein of claim 1 and a cleavage domain or cleavage half-domain.

3. An isolated polynucleotide encoding the zinc finger proteins of claim 1.

4. An isolated cell comprising the zinc finger proteins of claim 1.

5. An isolated cell comprising the polynucleotides of claim 3.

6. The cell of claim 5, wherein the cell is a red blood cell precursor cell.

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

8. The cell of claim 6, wherein the precursor cell is a CD34+ hematopoietic stem cell.

9. A kit comprising the protein of claim 1.

10. A kit comprising the polynucleotide of claim 3.

11. An isolated cell comprising the fusion protein of claim 2.