Engineered target specific nucleases

Abstract

Described herein are engineered nucleases comprising mutations in the cleavage domain (e.g., FokI or homologue thereof) and/or DNA binding domain (zinc finger protein, TALE, single guide RNA) such that on-target specificity is increased.

What is claimed is: 
     
         1 . A zinc finger nuclease that cleaves a T cell receptor constant region gene (TRAC) gene, the zinc finger nuclease comprising a first zinc finger nuclease comprising an ELD engineered FokI cleavage domain and a first zinc finger DNA-binding domain that binds to a target site in the TRAC gene and a second zinc finger nuclease comprising a KKR FokI cleavage domain and a second zinc finger DNA-binding domain, the first and second zinc finger DNA-binding domains comprising wherein
 (i) at least one of the ELD or KKR FokI cleavage domains further comprises a mutation in the FokI cleavage domain selected from the group consisting of: K393S, K394S, R398S, K400S, K402S, R416S, R422S, K427S, K434S, R439S, K441S, R447S, K448S, K469S, R487S, R495S, K497S, K506S, K516S, K525S, K529S, R534S, K559S, R569S, and R570S, numbered relative to wild-type FokI; and/or   (ii) at least one of the first and second zinc finger DNA-binding domains comprises one or more mutations in amino acid residues (−5), (−9) and/or (−14), numbered relative to the start of the alpha helix region.   
     
     
         2 . The zinc finger nuclease of  claim 1 , wherein the mutation in the ELD and/or KKR FokI cleavage domain is a R416S, R422S, R4475, K448S or K525S mutation. 
     
     
         3 . The zinc finger nuclease of  claim 1 , wherein the amino acid at residue at position −5 is mutated in one, two or three fingers. 
     
     
         4 . One or more polynucleotides encoding the zinc finger nuclease according to  claim 3 . 
     
     
         5 . An isolated cell comprising the one or more polynucleotides of  claim 4 . 
     
     
         6 . A method for cleaving a TCR alpha (TRAC) gene in a mammalian cell, the method comprising:
 expressing the one or more polynucleotides according to  claim 4  in a cell, wherein the zinc finger nuclease is expressed in the cell and site-specifically cleaves to a nucleotide sequence in the TRAC gene.   
     
     
         7 . The method of  claim 6 , further comprising contacting the cell with a donor polynucleotide; wherein cleavage of the cellular chromatin facilitates homologous recombination between the donor polynucleotide and the cellular chromatin. 
     
     
         8 . An isolated cell comprising at least one site-specific genomic modification in a TRAC gene made by a zinc finger nuclease according to  claim 1 . 
     
     
         9 . A composition comprising first and second polynucleotides of  claim 4 , wherein the zinc finger nuclease comprises first and second zinc finger nucleases, wherein the first polynucleotide encodes the first zinc finger nuclease and a second polynucleotide encodes the second zinc finger nuclease and the ratio of the first and second zinc finger nucleases is not one to one. 
     
     
         10 . The composition of  claim 9 , further comprising one or more donor polynucleotides. 
     
     
         11 . The composition of  claim 10 , wherein the donor polynucleotide comprises a sequence encoding one or more chimeric antigen receptors (CARs) or antibody-coupled T-cell receptor (ACTRs). 
     
     
         12 . An isolated cell comprising the composition of  claim 10 , wherein the zinc finger nuclease cleaves the TRAC gene and the donor polynucleotide is integrated into the TRAC gene. 
     
     
         13 . A method of providing a therapeutic protein to a subject in need thereof, the method comprising infusing an isolated cell according to  claim 12  into the subject, wherein the donor polynucleotide integrated into the TRAC gene expresses the therapeutic protein.