Methods and compositions for gene inactivation
Disclosed herein are methods and compositions for inactivating CCR-5 genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs, such as adenovirus (Ad) vectors, and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided.
1. A protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F3, and F4 comprise the following amino acid sequences:
F1: DRSNLSR
(SEQ ID NO: 2)
F3: RSDNLAR
(SEQ ID NO: 4)
F4: TSGNLTR.
(SEQ ID NO: 8)
2. The protein according to claim 1 , wherein F2 comprises the amino acid sequence ISSNLNS (SEQ ID NO:5).
3. The protein according to claim 1 , wherein F2 comprises the amino acid sequence VSSNLTS (SEQ ID NO:6).
4. A protein according to claim 1 , further comprising a cleavage domain.
5. The protein of claim 4 , wherein the cleavage domain is a cleavage half-domain.
6. The protein of claim 5 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.
7. The protein of claim 5 , wherein the cleavage half-domain is an engineered FokI cleavage half-domain.
8. A polynucleotide encoding the protein of claim 1 .
9. A gene delivery vector comprising a polynucleotide according to claim 8 .
10. The gene delivery vector of claim 9 , wherein the vector is an adenovirus vector.
11. The gene delivery vector of claim 8 , wherein the adenovirus vector is an Ad5/35 vector.
12. An isolated cell comprising the protein of claim 1 .
13. An isolated cell comprising the polynucleotide of claim 8 .
14. The cell of claim 12 , wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.
15. The cell of claim 14 , wherein the T-cell is a CD4 + cell.
16. The cell of claim 13 , wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.
17. The cell of claim 16 , wherein the T-cell is a CD4 + cell.
18. A method for inactivating the CCR-5 gene in a human cell, the method comprising administering to the cell a polynucleotide according to claim 8 .
19. The method of claim 18 , wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.
20. The method of claim 19 , wherein the T-cell is a CD4 + cell.
21. The isolated cell of claim 12 further comprising a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition helices as follows:
F1: RSDNLSV
(SEQ ID NO: 10)
F2: QKINLQV
(SEQ ID NO: 17)
F3: RSDVLSE
(SEQ ID NO: 12)
F4: QRNHRTT.
(SEQ ID NO: 13)
22. The isolated cell of claim 13 , further comprising a polynucleotide encoding a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition helices as follows:
F1: RSDNLSV (SEQ ID NO: 10)
F2: QKINLQV (SEQ ID NO: 17)
F3: RSDVLSE (SEQ ID NO: 12)
F4: QRNHRTT (SEQ ID NO: 13).