Modification of CXCR4 using engineered zinc finger proteins
Disclosed herein are methods and compositions for modulating activity of CXCR4 genes, for example using zinc finger transcription factors (ZF-TFs) or zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding ZF-TFs or ZFNs, vectors comprising polynucleotides encoding ZF-TFs or ZFNs and cells comprising polynucleotides encoding ZF-TFs or ZFNs and/or cells comprising ZF-TF or ZFNs are also provided.
1. A zinc finger protein comprising 4 zinc finger DNA-binding domains, wherein
(i) each zinc finger DNA-binding domain binds to a 3 base pair target subsite;
(ii) the zinc finger protein binds to a target site in a CXCR4 gene; and
(iii) the target site is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21 and SEQ ID NO:25.
2. A fusion protein comprising a zinc finger DNA-binding domain according to claim 1 and at least one cleavage domain or at least one cleavage half-domain.
3. The fusion protein of claim 2 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.
4. The fusion protein of claim 2 , wherein the cleavage half-domain is an engineered FokI cleavage half-domain.
5. A polynucleotide encoding the zinc finger DNA-binding domain according to claim 1 .
6. A gene delivery vector comprising the polynucleotide of claim 5 .
7. The gene delivery vector of claim 6 , wherein the vector comprises an adenovirus vector.
8. An isolated cell comprising a protein according to claim 1 .
9. A method of inactivating an endogenous cellular CXCR4 gene in a cell, the method comprising:
(a) introducing, into a cell, a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises:
(i) a zinc finger DNA-binding domain according to claim 1 ; and
(ii) a cleavage domain; such that the polypeptide is expressed in the cell, whereby the polypeptide binds to the target site and cleaves the CXCR4 gene.
10. The method of claim 9 , further comprising introducing a nucleic acid encoding a second polypeptide, wherein the second polypeptide comprises:
(i) a zinc finger DNA-binding domain that is engineered to bind to a second target site in the CXCR4 gene; and
(ii) a cleavage domain; such that the second polypeptide is expressed in the cell, whereby the first and second polypeptides bind to their respective target sites and cleave the CXCR4 gene.
11. The method of claim 9 , wherein the first and second polypeptides are encoded by the same nucleic acid.
12. The method of claim 9 , wherein the first and second polypeptides are encoded by different nucleic acids.
13. The method of claim 9 , further comprising introducing a polynucleotide into the cell, wherein the polynucleotide comprises a first region of homology to sequences upstream of the double-strand break and a second region of homology to sequences downstream of the double-strand breaks.
14. The method of claim 9 , wherein the nucleic acid is carried on a gene delivery vector.
15. The method of claim 14 , wherein the gene delivery vector is an adenovirus vector.
16. The method of claim 15 , wherein the adenovirus vector is an Ad5/F35 vector.
17. The method of claim 9 , 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. A method for treating or preventing HIV infection in a subject, the method comprising:
(a) inactivating an endogenous cellular CXCR4 gene in a cell according to the method of claim 9 , and
(b) introducing the cell into the subject.
19. The method of claim 18 , further comprising inactivating an endogenous CCR5 gene in the cell using a sequence encoding a nuclease prior to introducing the cell into the subject.