Methods and compositions for genome engineering

• US 10,081,661 B2
• Filed: 12/09/2014
• Issued: 09/25/2018
• 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., Wechsler, Thomas, Zhang, Lei
• Primary Examiner(s): Hutson, Richard G
• Application Number:
  US14/565,014
• Publication Number:
  US 20150159172A1
• Time in Patent Office: 1,386 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 that is aberrantly expressed in disease or disorder such as a lysosomal storage disease.

Claims

1. A zinc finger protein that binds to an endogenous human albumin gene, the zinc finger protein comprising five or six zinc finger domains designated and ordered F1 to F5 or F1 to F6, the zinc finger domains comprising the recognition helix regions ordered and shown as follows:

(i) F1;

QSGNLSR (SEQ ID NO;

23);

F2;

LKQNLCM (SEQ ID NO;

18);

F3;

WADNLQN (SEQ ID NO;

24);

F4;

TSGNLTR (SEQ ID NO;

20); and

F5;

RQSHLCL (SEQ ID NO;

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

17;

or

(ii) F1;

TPQLLDR (SEQ ID NO;

14);

F2;

LKWNLRT (SEQ ID NO;

9);

F3;

DQSNLNA (SEQ ID NO;

25);

F4;

RNFSLTM (SEQ ID NO;

15);

F5;

LRHDLDR (SEQ ID NO;

16); and

F6;

HRSNLNK (SEQ ID NO;

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

7;

or

(iii) F1;

QSGNLAR (SEQ ID NO;

2);

F2;

LIQYLQS (SEQ ID NO;

26);

F3;

WADNLQN (SEQ ID NO;

24);

F4;

TSGNLTR (SEQ ID NO;

20); and

F5;

RQSHLSL (SEQ ID NO;

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

17;

or

(iv) F1;

TPQLLDR (SEQ ID NO;

14);

F2;

LKWNLRT (SEQ ID NO;

9);

F3;

DQSNLRA (SEQ ID NO;

10);

F4;

RNFSLTM (SEQ ID NO;

15);

F5;

LRHDLDR (SEQ ID NO;

16); and

F6;

HRSNLNK (SEQ ID NO;

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

7;

or

(v) F1;

QSGNLAR (SEQ ID NO;

2);

F2;

LIQYLQS (SEQ ID NO;

26);

F3;

WADNLQN (SEQ ID NO;

24);

F4;

TSGNLTR (SEQ ID NO;

20); and

F5;

RQSHLCL (SEQ ID NO;

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

17;

or

(vi) F1;

TPQLLDR (SEQ ID NO;

14);

F2;

LKHNLLT (SEQ ID NO;

28);

F3;

DQSNLNA (SEQ ID NO;

25);

F4;

RNFSLTM (SEQ ID NO;

15);

F5;

LRHDLDR (SEQ ID NO;

16); and

F6;

HRSNLNK (SEQ ID NO;

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

7;

or

(vii) F1;

TPQLLDR (SEQ ID NO;

14);

F2;

LKWNLRT (SEQ ID NO;

9);

F3;

DQSNLRA (SEQ ID NO;

10);

F4;

RNFSLTM (SEQ ID NO;

15);

F5;

LRHDLDR (SEQ ID NO;

16); and

F6;

HRSNLNK (SEQ ID NO;

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

7;

or

(viii) F1;

QSGNLAR (SEQ ID NO;

2);

F2;

LIQYLQS (SEQ ID NO;

26);

F3;

WQSNLQN (SEQ ID NO;

19);

F4;

TSGNLTR (SEQ ID NO;

20); and

F5;

RQSHLCL (SEQ ID NO;

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

17;

or

(ix) F1;

QSSDLSR (SEQ ID NO;

8);

F2;

LKHNLLT (SEQ ID NO;

28);

F3;

LKHNLLT (SEQ ID NO;

28);

F4;

RPYTLRL (SEQ ID NO;

11);

F5;

LRPDLER (SEQ ID NO;

41);

or

F6;

HRSNLNK (SEQ ID NO;

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

7.

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

3. A polynucleotide encoding one or more proteins of claim 1.

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 AAV2/8 vector.

7. A pharmaceutical composition comprising an expression vector according to claim 4.

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

introducing, into the cell, one or more expression vectors comprising a polynucleotide which encodes a fusion protein of claim 2, under conditions such that the one or more proteins are expressed and the albumin gene is cleaved.

9. The method of claim 8, further comprising introducing a donor sequence into the cell such that the donor sequence is integrated into the cleaved albumin gene.

10. The method of claim 9, wherein the donor sequence is introduced using an AAV vector.

11. The method of claim 10, wherein the AAV vector is an AAV2/8 vector.

12. The method of claim 9, wherein the donor sequence encodes a protein lacking or deficient in a lysosomal storage disease (LSD).

13. The method of claim 8, wherein the cell is a liver cell.

14. A kit comprising an expression vector according to claim 4.