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 that binds to an endogenous CCR5 gene, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and further wherein the zinc finger DNA-binding domains comprise recognition regions selected from the group consisting of:
(i)
F1: DRSNLSR;
(SEQ ID NO: 2)
F2: ISSNLNS;
(SEQ ID NO: 5)
F3: RSDNLAR;
(SEQ ID NO: 4)
and
F4: TSANLSR
(SEQ ID NO: 3)
or
NRDNLSR
(SEQ ID NO: 7)
(ii)
F1: DRSNLSR;
(SEQ ID NO: 2)
F2: TSGNLTR;
(SEQ ID NO: 8)
F3: RSDNLAR;
(SEQ ID NO: 4)
and
F4: TSGNLTR.
(SEQ ID NO: 8)
2. A protein according to claim 1 , further comprising a cleavage domain.
3. The protein of claim 2 , wherein the cleavage domain is a cleavage half-domain.
4. The protein of claim 3 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.
5. The 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. A gene delivery vector comprising a polynucleotide according to claim 6 .
8. The gene delivery vector of claim 7 , wherein the vector is an adenovirus vector.
9. The gene delivery vector of claim 8 , wherein the adenovirus vector is an Ad5/35 vector.
10. An isolated cell comprising the protein of claim 1 .
11. The isolated cell of claim 10 , further comprising 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 zinc finger DNA-binding domains comprise recognition regions selected from the group consisting of:
(i)
F1: DRSNLSR
(SEQ ID NO: 2)
F3: RSDNLAR
(SEQ ID NO: 4)
F4: TSGNLTR;
(SEQ ID NO: 8)
and
(ii)
F2: QKINLQV
(SEQ ID NO: 17)
F3: RSDVLSE
(SEQ ID NO: 12)
F4: QRNHRTT.
(SEQ ID NO: 13)
12. An isolated cell comprising the polynucleotide of claim 6 .
13. The isolated cell of claim 12 , 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 regions ordered F1 to F4 from N-terminus to C-terminus, and wherein zinc finger DNA-binding domains comprise recognition regions selected from the group consisting of:
(i)
F1: DRSNLSR
(SEQ ID NO: 2)
F3: RSDNLAR
(SEQ ID NO: 4)
F4: TSGNLTR;
(SEQ ID NO: 8)
and
(ii)
F2: QKINLQV
(SEQ ID NO: 17)
F3: RSDVLSE
(SEQ ID NO: 12)
F4: QRNHRTT.
(SEQ ID NO: 13)
14. The cell of claim 10 , 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 14 , wherein the hematopoietic stem cell is a CD34+ cell.
17. 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.
18. The cell of claim 17 , wherein the T-cell is a CD4 + cell.
19. A method for inactivating the CCR-5 gene in a human cell, the method comprising administering to the cell a polynucleotide according to claim 6 .
20. The method of claim 19 , further comprising administering a polynucleotide encoding 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 zinc finger DNA-binding domains comprise recognition regions selected from the group consisting of:
(i)
F1: DRSNLSR
(SEQ ID NO: 2)
F3: RSDNLAR
(SEQ ID NO: 4)
F4: TSGNLTR;
(SEQ ID NO: 8)
and
(ii)
F2: QKINLQV
(SEQ ID NO: 17)
F3: RSDVLSE
(SEQ ID NO: 12)
F4: QRNHRTT.
(SEQ ID NO: 13)
21. The method of claim 19 , 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.
22. The method of claim 21 , wherein the T-cell is a CD4 + cell.
23. The cell of claim 21 , wherein the hematopoietic stem cell is a CD34+ cell.