Methods and compositions for gene inactivation

Abstract

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.

What is claimed is: 
     
         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.