Methods and compositions for treating hemophilia

• US 9,771,403 B2
• Filed: 12/09/2014
• Issued: 09/26/2017
• Est. Priority Date: 12/09/2013
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Miller, Jeffrey C., Paschon, David, Rebar, Edward J.
• Primary Examiner(s): Kelly, Robert M
• Application Number:
  US14/564,722
• Publication Number:
  US 20150166618A1
• Time in Patent Office: 1,022 Days
• Field of Search: None
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14133 Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
• CPC Class Codes: C12N 2750/14142 virus or viral particle as vehicle, e.g. encapsulating small organic molecule
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/644 Coagulation factor IXa (3.4.21.22)

Abstract

Disclosed herein are methods and compositions for insertion of transgene sequences encoding proteins involved in clotting into the genome of a cell for treating conditions including hemophilias.

Claims

1. A zinc finger protein comprising five or six zinc finger domains designated and ordered F1 to F5 or F1 to F6, wherein F1 to F5 or F1 to F6 comprise the following recognition helix region sequences:

(i) F1;

QSGNLAR (SEQ ID NO;

4);

F2;

LKQNLCM (SEQ ID NO;

5);

F3;

WADNLQN (SEQ ID NO;

6);

F4;

TSGNLTR (SEQ ID NO;

7); and

F5;

RQSHLCL (SEQ ID NO;

8);

or

(ii) F1;

TPQLLDR (SEQ ID NO;

9);

F2;

LKWNLRT_(SEQ ID NO;

10);

F3;

DQSNLRA_(SEQ ID NO;

11);

F4;

RNFSLTM_(SEQ ID NO;

12);

F5;

LRHDLDR (SEQ ID NO;

13); and

F6;

HRSNLNK_(SEQ ID NO;

14).

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

3. A polynucleotide encoding one or more zinc finger proteins comprising five or six zinc finger domains designated and ordered F1 to F5 or F1 to F6, wherein F1 to F5 or F1 to F6 comprise the following recognition helix sequences:

(i) F1;

QSGNLAR (SEQ ID NO;

4);

F2;

LKQNLCM (SEQ ID NO;

5);

F3;

WADNLQN (SEQ ID NO;

6);

F4;

TSGNLTR (SEQ ID NO;

7); and

F5;

RQSHLCL (SEQ ID NO;

8);

or

(ii) F1;

TPQLLDR (SEQ ID NO;

9);

F2;

LKWNLRT (SEQ ID NO;

10);

F3;

DQSNLRA (SEQ ID NO;

11);

F4;

RNFSLTM (SEQ ID NO;

12);

F5;

LRHDLDR (SEQ ID NO;

13); and

F6;

HRSNLNK (SEQ ID NO;

14).

4. An expression vector comprising a polynucleotide according to claim 3.

5. The expression vector of claim 4, wherein the vector is an AAV vector.

6. The expression vector of claim 5, wherein the AAV vector is an AAV2/6 vector.

7. A pharmaceutical composition comprising one or more expression vectors encoding one or more fusion proteins according to claim 4 and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition of claim 7, comprising

a pharmaceutically acceptable excipient;

a first expression vector encoding a first zinc finger nuclease comprising a wild-type or engineered cleavage half-domain and a zinc finger protein comprising five zinc finger domains designated and ordered F1 to F5, wherein F1 to F5 comprise the following recognition helix region sequences;

(i) F1;

QSGNLAR (SEQ ID NO;

4);

F2;

LKQNLCM (SEQ ID NO;

5);

F3;

WADNLQN (SEQ ID NO;

6);

F4;

TSGNLTR (SEQ ID NO;

7); and

F5;

RQSHLCL (SEQ ID NO;

8); and

a second expression vector encoding a second zinc finger nuclease comprising a wild-type or engineered cleavage half-domain and a zinc finger protein comprising six zinc finger domains designated and ordered F1 to F6, wherein F1 to F6 comprise the following recognition helix regions sequences;

(ii) F1;

TPQLLDR (SEQ ID NO;

9);

F2;

LKWNLRT_(SEQ ID NO;

10);

F3;

DQSNLRA_(SEQ ID NO;

11);

F4;

RNFSLTM_(SEQ ID NO;

12);

F5;

LRHDLDR (SEQ ID NO;

13); and

F6;

HRSNLNKASEQ ID NO;

14).

9. The pharmaceutical composition of claim 8, wherein the first and second expression vectors are AAV vectors and further wherein the pharmaceutical composition further comprises an AAV vector comprising a donor encoding a Factor IX protein.

10. The pharmaceutical composition of claim 9, wherein the AAV expression vectors and the AAV vector comprising the donor are provided in a ratio about 1:

1;

1, about 1;

1;

2, about 1;

1;

3, about 1;

1;

4, about 1;

1;

5, about 1;

1;

6, about 1;

1;

7, about 1;

1;

8, about 1;

1;

9, about 1;

1;

10, about 1;

1;

11, about 1;

1;

12, about 1;

1;

13, about 1;

1;

14, about 1;

1;

15, about 1;

1;

16, about 1;

1;

17, about 1;

1;

18, about 1;

1;

19, or about 1;

1;

20.

11. The pharmaceutical composition of claim 8, wherein the first polynucleotide encodes a polypeptide comprising or consisting of a sequence as in SEQ ID NO. 15 and the second polynucleotide encodes a polypeptide comprising or consisting of a sequence as in SEQ ID NO. 16.

12. The pharmaceutical composition of claim 11, further comprising a donor sequence.

13. The pharmaceutical composition of claim 12, wherein an expression vector comprises the donor sequence.

14. The pharmaceutical composition of claim 12, wherein the donor sequence comprises SEQ ID NO. 17.

15. The pharmaceutical composition of claim 14, wherein an AAV vector comprises the donor sequence.

16. The pharmaceutical composition of claim 15, wherein the AAV vector is an AAV2/6 vector.

17. The pharmaceutical composition of claim 8, wherein the first and second zinc finger nucleases and a donor sequence are provided in the composition in a ratio about 1:

1;

1, about 1;

1;

2, about 1;

1;

3, about 1;

1;

4, about 1;

1;

5, about 1;

1;

6, about 1;

1;

7, about 1;

1;

8, about 1;

1;

9, about 1;

1;

10, about 1;

1;

11, about 1;

1;

12, about 1;

1;

13, about 1;

1;

14, about 1;

1;

15, about 1;

1;

16, about 1;

1;

17, about 1;

1;

18, about 1;

1;

19, or about 1;

1;

20.

18. A method of cleaving an albumin gene in a cell, the method comprising:

introducing, into the cell, one or more expression vectors according to claim 4, under conditions such that the one or more fusion proteins are expressed and the albumin gene is cleaved.

19. The method of claim 18, further comprising integrating a donor sequence into the cleaved albumin gene.

20. The method of claim 19, wherein an AAV vector comprises the donor sequence.

21. The method of claim 20, wherein the AAV vector is an AAV2/6 vector.

22. The method of claim 19, wherein the donor sequence encodes a Factor IX (F.IX) protein.

23. The method of claim 18, wherein the cell is a liver cell.

24. A method of treating a patient with hemophilia B, the method comprising administering to the patient an expression vector according to claim 4 that mediates targeted integration of a transgene encoding a functional Factor IX protein into an endogenous albumin gene, wherein the Factor IX protein is expressed in the patient, thereby treating the patient.

25. The method of claim 24, wherein a system is administered to the patient, the system comprising:

(i) an AAV vector comprising a polynucleotide encoding a zinc finger nuclease, the zinc finger nuclease comprising a Fokl cleavage domain and a zinc finger protein comprising 5 zinc finger domains ordered F1 to F5, wherein each zinc finger domain comprises a recognition helix region and wherein the recognition helix regions of the zinc finger protein are shown in the first row of Table 1 (SEQ ID NOs;

4-8);

(ii) an AAV vector comprising a polynucleotide encoding a zinc finger nuclease, the zinc finger nuclease comprising a Fokl cleavage domain and a zinc finger protein comprising 6 zinc finger domains ordered F1 to F6, wherein each zinc finger domain comprises a recognition helix region and wherein the recognition helix regions of the zinc finger protein are shown in the second row of Table 1 (SEQ ID NOs;

9-14); and

(iii) an AAV vector comprising a donor encoding a Factor IX protein.

26. The method of claim 24, wherein the expression vector(s) or pharmaceutical composition and transgene are administered intravenously.