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 method for inactivating a CCR-5 gene in an isolated CD34+ cell, the method comprising administering to the cell a polynucleotide encoding a first fusion protein comprising a first cleavage domain and a first 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:
(SEQ ID NO: 2)
DRSNLSR
F3:
(SEQ ID NO: 4)
RSDNLAR
F4:
(SEQ ID NO: 8)
TSGNLTR.
2. The method of claim 1 , wherein F2 comprises the amino acid sequence ISSNLNS (SEQ ID NO:5).
3. The method according to claim 1 , wherein F2 comprises the amino acid sequence VSSNLTS (SEQ ID NO:6).
4. The method of claim 1 , wherein the first cleavage domain comprises a FokI cleavage domain.
5. The method of claim 1 , further comprising the step of administering a viral vector encoding a second fusion protein comprising a second cleavage domain and a second engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition helices as follows:
F1:
(SEQ ID NO: 10)
RSDNLSV
F2:
(SEQ ID NO: 17)
QKINLQV
F3:
(SEQ ID NO: 12)
RSDVLSE
F4:
(SEQ ID NO: 13)
QRNHRTT.
6. The method of claim 5 , wherein the second cleavage domain comprises a FokI cleavage domain.
7. The method of claim 1 , wherein the polynucleotide comprises a viral vector.
8. The method of claim 7 , where the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated vector.
9. The method of claim 7 , wherein the viral vector encodes the first and second fusion proteins.
10. The method of claim 1 wherein the polynucleotide comprises a plasmid or RNA.
11. The method of claim 1 , wherein the CD34+ cell is a human cell.
12. A method of treating or preventing HIV infection in a subject, the method comprising:
inactivating a CCR5 gene in an isolated CD34+ cell according to the method of claim 1 ;
introducing the isolated CD34+ cell into the subject.
13. The method of claim 12 , wherein the subject is a human.