Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (CFTR) gene

• US 9,161,995 B2
• Filed: 07/25/2012
• Issued: 10/20/2015
• Est. Priority Date: 07/25/2011
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc., Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Guschin, Dmitry M., Holmes, Michael C., Tam, Phillip, Paschon, David
• Primary Examiner(s): Leavitt, Maria
• Assistant Examiner(s): Aron, Kimberly A
• Application Number:
  US13/558,101
• Publication Number:
  US 20130145485A1
• Time in Patent Office: 1,182 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2015/8536 Animal models for genetic diseases
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 5/0662 Stem cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Nucleases and methods of using these nucleases for alteration of a CFTR gene and generation of cells and animal models.

Claims

1. A protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four, five or six zinc finger recognition regions ordered F1 to F4, F1 to F5 or F1 to F6 from N-terminus to C-terminus, and wherein the protein is selected from the group consisting of:

(i) a 5-finger zinc finger protein comprising the following recognition regions;

F1;

WPSCLYA (SEQ ID NO;

8);

F2;

NGVLLKR (SEQ ID NO;

9);

F3;

QSGNLAR (SEQ ID NO;

10);

F4;

RSDNLSE (SEQ ID NO;

11); and

F5;

NPRNRFT (SEQ ID NO;

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

37;

(ii) a 5-finger zinc finger protein comprising the following recognition regions;

F1;

RSDVLSE (SEQ ID NO;

13);

F2;

QSGNLAR (SEQ ID NO;

10);

F3;

QSGHLSR (SEQ ID NO;

14);

F4;

RSDVLSE (SEQ ID NO;

15); and

F5;

WSASLSK (SEQ ID NO;

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

38;

(iii) a 4-finger zinc finger protein comprising the following recognition regions;

F1;

QNATRIN (SEQ ID NO;

17);

F2;

QSGNLAR (SEQ ID NO;

10);

F3;

RSDNLST (SEQ ID NO;

18); and

F4;

QSADRKK (SEQ ID NO;

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

39;

(iv) a 6-finger zinc finger protein comprising the following recognition regions;

F1;

TNQNRIT (SEQ ID NO;

20);

F2;

RNQTRIT (SEQ ID NO;

21);

F3;

QSGNLAR (SEQ ID NO;

10);

F4;

QSNTRIM (SEQ ID NO;

22);

F5;

TSGNLTR (SEQ ID NO;

23); and

F6;

QSNALHQ (SEQ ID NO;

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

40;

(v) a 5-finger zinc finger protein comprising the following recognition regions;

F1;

TSSDRKK (SEQ ID NO;

25);

F2;

QSSDLSR (SEQ ID NO;

26);

F3;

DRSNLTR (SEQ ID NO;

27);

F4;

TSGNLTR (SEQ ID NO;

23); and

F5;

WRLSLQV (SEQ ID NO;

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

41;

(vi) a 6-finger zinc finger protein comprising the following recognition regions;

F1;

QSGNLAR (SEQ ID NO;

10);

F2;

QGANLIK (SEQ ID NO;

29);

F3;

RSDHLSA (SEQ ID NO;

30);

F4;

ESRYLMV (SEQ ID NO;

31);

F5;

RSDNLST (SEQ ID NO;

18); and

F6;

DRSNRKT (SEQ ID NO;

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

42;

(vii) a 5-finger zinc finger protein comprising the following recognition regions;

F1;

TSGNLTR (SEQ ID NO;

23);

F2;

QSNALHQ (SEQ ID NO;

24);

F3;

QSGNLAR (SEQ ID NO;

10);

F4;

TSGNLTR (SEQ ID NO;

23); and

F5;

WWTSRAL (SEQ ID NO;

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

43; and

(viii) a 4-finger zinc finger protein comprising the following recognition regions;

F1;

HSNARKT (SEQ ID NO;

34);

F2;

TSGNLTR (SEQ ID NO;

23);

F3;

TLQNRMS (SEQ ID NO;

35); and

F4;

DQSTLRN (SEQ ID NO;

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

44.

2. A fusion protein comprising a protein according to claim 1 and a cleavage domain.

3. The fusion protein of claim 2, wherein the cleavage domain is a cleavage half-domain.

4. The fusion protein of claim 3, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

5. The fusion protein of claim 3, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

6. A polynucleotide encoding the protein of claim 1.

7. An isolated cell comprising the protein of claim 1.

8. An isolated cell comprising the polynucleotide of claim 6.

9. The cell of claim 7, wherein the cell is selected from the group consisting of an embryonic stem cell (ESC), a hematopoietic stem cell, a nerve stem cell, a skin stem cell, a muscle stem cell, a lung stem cell, an induced pluripotent stem cell (iPSC) and a fibroblast cell.

10. The cell of claim 8, wherein the cell is selected from the group consisting of an embryonic stem cell (ESC), a hematopoietic stem cell, a nerve stem cell, a skin stem cell, a muscle stem cell, a lung stem cell, an induced pluripotent stem cell (iPSC) and a fibroblast cell.

11. A method of modifying a cystic fibrosis transmembrane conductance regulator (CFTR) gene in an isolated cell, the method comprising;

cleaving the CFTR gene with one or more fusion proteins according to claim 2.

12. The method of claim 11, wherein the modification is selected from the group consisting of an insertion, a deletion, a substitution and combinations thereof.

13. The method of claim 11, further comprising introducing an exogenous sequence into the CFTR gene.

14. The method of claim 11, wherein the modification corrects a mutation in the CFTR gene.

15. The method of claim 14, wherein the mutation is selected from the group consisting of Δ

F508, Δ

I507 and combinations thereof.

16. A method of generating a model system for the study of cystic fibrosis (CF), the method comprising modifying cells according to the method of claim 11.

17. The method of claim 16, wherein the model system comprises a cell line.

18. The method of claim 16, wherein the model system comprises a non-human animal.

19. A kit comprising the protein according to claim 1.