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 nuclease comprising a DNA-binding domain that binds to a target site in an endogenous CCR5 gene, wherein the target site comprises a sequence as shown in any of SEQ ID NO:1, 9, 19 and 24.
2 . A polynucleotide encoding at least one nuclease according to claim 1 .
3 . A cell comprising the nuclease according to claim 1 .
4 . A cell comprising a polynucleotide according to claim 2 .
5 . A method for targeted cleavage of an endogenous CCR5 gene in a cell, the method comprising:
expressing a nuclease according to claim 1 in the cell under conditions such that the CCR5 gene is cleaved.
6 . The method of claim 5 , wherein a pair of nucleases cleaves the endogenous CCR5 gene.
7 . The method of claim 5 , wherein the cleaved CCR5 gene is partially or fully inactivated.
8 . The method of claim 5 , further comprising the step of introducing a donor polynucleotide into the cell, wherein all or part of the donor polynucleotide is incorporated into the CCR5 following cleavage.
9 . A cell produced by the method of claim 7 .
10 . A method of treating a subject with HIV, the method comprising administering a cell according to claim 9 .
11 . A kit for producing a nuclease, the kit comprising a nuclease according to claim 1 contained in one or more containers, optional hardware, and instructions for use of the kit.
12 . A kit for producing a nuclease, the kit comprising a polynucleotide according to claim 2 contained in one or more containers, optional hardware, and instructions for use of the kit.
13 . The kit of claim 12 , further comprising a donor polynucleotide.