Zinc finger nuclease for the cftr gene and methods of use thereof

Abstract

The present invention provides new zinc finger proteins and zinc finger nuclease (ZFNs) that find particular using in repairing the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

1 . A zinc three-finger binding domain that targets a nucleotide sequence selected from the group consisting of GTGGAATTA (SEQ ID NO:1) and GAGTGGTTA (SEQ ID NO:2). 
     
     
         2 . The zinc three-finger binding domain of  claim 1 , where said zinc three-finger binding domain comprises a sequence selected from SEQ ID NOS: 3-38 for GTGGAATTA (SEQ ID NO:1) and selected from the group consisting of SEQ ID NOS: 39-110 for GAGTGGTTA (SEQ ID NO:2). 
     
     
         3 . The zinc three-finger binding domain of  claim 1 , wherein said zinc three-finger binding domain comprises the nucleotide sequence of SEQ ID NO:116 for GTGGAATTA (SEQ ID NO:1) and the nucleotide sequence of SEQ ID NO:117 for GAGTGGTTA (SEQ ID NO:2). 
     
     
         4 . The zinc three-finger binding domain of  claim 1 , wherein said zinc three-finger binding domain is linked to a non-specific nuclease. 
     
     
         5 . The zinc three-finger binding domain of  claim 4 , wherein said non-specific nuclease is FokI. 
     
     
         6 . A zinc three-finger binding domain dimer that targets a double-stranded nucleic acid comprising a first monomer that targets nucleotide sequence GTGGAATTA (SEQ ID NO:1) in one strand and a second monomer that targets nucleotide sequence GAGTGGTTA (SEQ ID NO:2) in the other strand. 
     
     
         7 . The zinc three-finger binding domain dimer of  claim 6 , wherein one monomer of said dimer comprises a sequence selected from SEQ ID NOS: 3-5, 6-8, 9-11, 12-14, 15-17, 18-20, 21-23, 24-26, 27-29, 30-32, 33-35 and 36-38 for GTGGAATTA (SEQ ID NO:1) and the other monomer comprises a sequence selected from the group consisting of SEQ ID NOS: 39-41, 42-44, 45-47, 48-50, 51-53, 54-56, 57-59, 60-62, 63-65, 66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86, 87-89, 90-92, 91-93, 94-96, 97-99, 100-102, 103-105, 106-108, and 109-111 for GAGTGGTTA (SEQ ID NO:2). 
     
     
         8 . The zinc three-finger binding domain dimer of  claim 6 , wherein one monomer comprises the nucleotide sequence of SEQ ID NO:116 for GTGGAATTA (SEQ ID NO:1) and the other monomer comprises the nucleotide sequence of SEQ ID NO:117 for GAGTGGTTA (SEQ ID NO:2). 
     
     
         9 . The zinc three-finger binding domain dimer of  claim 6 , wherein each monomer of said zinc three-finger binding domain dimer is linked to a non-specific nuclease monomer. 
     
     
         10 . The zinc three-finger binding domain dimer of  claim 9 , wherein said non-specific nuclease is FokI. 
     
     
         11 . The zinc three-finger binding domain dimer of  claim 10 , wherein said each FokI nuclease monomer is different. 
     
     
         12 . A vector comprising a nucleic acid segment encoding a zinc three-finger binding domain that targets a nucleotide sequence selected from the group consisting of GTGGAATTA (SEQ ID NO:1) and GAGTGGTTA (SEQ ID NO:2), said nucleic acid under the control of a promoter operable in a eukaryotic cell. 
     
     
         13 . The vector of  claim 12 , further comprising a selectable or screenable marker. 
     
     
         14 . The vector of  claim 12 , further comprising an origin of replication. 
     
     
         15 . The vector of  claim 12 , wherein said vector is a viral vector. 
     
     
         16 . The vector of  claim 15 , wherein said viral vector is an adenoviral vector, an adeno-associated viral vector, a pox viral vector, a herpes viral vector, a retroviral vector, a lentiviral vector. 
     
     
         17 . The vector of  claim 16 , wherein the lentiviral vector is an integrase-deficient vector. 
     
     
         18 . The vector of  claim 12 , wherein said vector comprises two nucleic acid segements, each encoding a zinc three-finger binding domain, one that targets GTGGAATTA (SEQ ID NO:1) and one that targets GAGTGGTTA (SEQ ID NO:2). 
     
     
         19 . The vector of  claim 18 , wherein each of said nucleic acid segments is under the control of a separate promoter active in said eukaryotic cell. 
     
     
         20 . The vector of  claim 18 , wherein both of said nucleic acid segments are under the control of a the same promoter. 
     
     
         21 . The vector of  claim 19 , wherein said nucleic acid segments are separated by a transcription termination signal and/or a picornavirus T2A sequence. 
     
     
         22 . The vector of  claim 20 , wherein said nucleic acid segments are separated by an internal ribosome entry site. 
     
     
         23 . A method of promoting recombination within a CTFR gene in a human cell comprising contacting said cell with a first zinc three-finger binding domain that targets a nucleotide sequence GTGGAATTA (SEQ ID NO:1) and a second zinc three-finger binding domain that targets a nucleotide sequence GAGTGGTTA (SEQ ID NO:2), wherein each of said first and second zinc three-finger binding domains are linked to a non-specific nuclease. 
     
     
         24 . The method of  claim 23 , wherein said first zinc three-finger binding domain comprises a sequence selected from SEQ ID NOS: 3-5, 6-8, 9-11, 12-14, 15-17, 18-20, 21-23, 24-26, 27-29, 30-32, 33-35 and 36-38 for GTGGAATTA (SEQ ID NO:1) and said second zinc three-finger binding domain comprises a sequence selected from the group consisting of SEQ ID NOS: 39-41, 42-44, 45-47, 48-50, 51-53, 54-56, 57-59, 60-62, 63-65, 66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86, 87-89, 90-92, 91-93, 94-96, 97-99, 100-102, 103-105, 106-108, and 109-111 for GAGTGGTTA (SEQ ID NO:2) 
     
     
         25 . The method of  claim 23 , wherein said first zinc three-finger binding domain comprises the nucleotide sequence of SEQ ID NO:116 for GTGGAATTA (SEQ ID NO:1) and said second zinc three-finger binding domain comprises the nucleotide sequence of SEQ ID NO:117 for GAGTGGTTA (SEQ ID NO:2). 
     
     
         26 . The method of  claim 23 , wherein said human cell is a lung epithelial cell, and intestinal epithelial cell, a biliary duct epithelial cell, a gall bladder epithelial cell or pancreatic epithelial cell. 
     
     
         27 . The method of  claim 26 , wherein said epithelial lung cell or pancreatic cell comprises a CFTR gene with a ΔF508 mutation. 
     
     
         28 . The method of  claim 27 , wherein said epithelial lung cell or pancreatic epithelial cell is located in a living human subject. 
     
     
         29 . The method of  claim 28 , wherein contacting comprises administering said first and second zinc three-finger binding domains to lung or pancreatic tissue of said subject. 
     
     
         30 . The method of  claim 29 , wherein administration to lung tissue comprises inhalation or topical instillation. 
     
     
         31 . The method of  claim 29 , wherein administration to pancreatic tissue comprises injection. 
     
     
         32 . The method of  claim 23 , wherein contacting comprises administering to said subject an expression vector comprising a first nucleic acid segment encoding a first zinc three-finger binding domain that targets GTGGAATTA (SEQ ID NO:1) and a second nucleic acid segment encoding a second zinc three-finger binding domain that targets GAGTGGTTA (SEQ ID NO:2), said nucleic acids under the control of one or more promoters operable in a eukaryotic cell. 
     
     
         33 . The method of  claim 32 , wherein said vector is a viral vector. 
     
     
         34 . The method of  claim 33 , wherein said viral vector is an adenoviral vector, an adeno-associated viral vector, a pox viral vector, a herpes viral vector, a retroviral vector, a lentiviral vector. 
     
     
         35 . The method of  claim 34 , wherein the lentiviral vector is an integrase-deficient vector. 
     
     
         36 . The method of claim of  claim 32 , wherein each of said nucleic acid segments is under the control of a separate promoter active in said eukaryotic cell. 
     
     
         37 . The method of  claim 32 , wherein both of said nucleic acid segments are under the control of a the same promoter. 
     
     
         38 . The method of  claim 36 , wherein said nucleic acid segments are separated by a transcription termination signal. 
     
     
         39 . The method of  claim 37 , wherein said nucleic acid segments are separated by an internal ribosome entry site and/or a picornavirus T2A sequence.