Talen-based gene correction

Abstract

The invention is directed to transcription activator-like effector nuclease (TALEN)-mediated DNA editing of disease-causing mutations in the context of the human genome and human cells to treat patients with compromised genetic disorders.

We claim: 
     
         1 . A composition comprising at least one nucleic acid comprising:
 (i) a first nucleic acid encoding a first transcription activator-like (TAL) effector endonuclease (TALEN) monomer,   (ii) a second nucleic acid encoding a second TALEN monomer, and   (iii) and a donor sequence,   wherein each of the first and second TALEN monomers comprises a plurality of TAL effector repeat sequences and a FokI endonuclease domain, wherein each of the plurality of TAL effector repeat sequences comprises a repeat-variable diresidue,   wherein the first TALEN monomer is capable of binding to a first half-site sequence of a target DNA within a cell, the target DNA having a genetic mutation, and the first TALEN monomer is capable of cleaving the target DNA when the second TALEN monomer is bound to a second half-site sequence of the target DNA,   wherein the target DNA comprises the first half-site sequence and the second half-site sequence separated by a spacer sequence, and wherein the first and second half-sites have the same nucleotide sequence or different nucleotide sequences, wherein the donor sequence comprises a region homologous to the target DNA at least at the 5′ and 3′ ends of the target DNA and is a template for DNA repair resulting in a correction of the genetic mutation in the target DNA.   
     
     
         2 . The composition of  claim 1 , wherein the cell is selected from the group consisting of a fibroblast, keratinocyte, inducible pluripotent stem cell, hematopoietic stem cell, mesenchymal stem cell, embryonic stem cell, hematopoietic progeny cell, T-cell, B-cell, glial cell, neural cell, neuroglial progenitor cell, neuroglial stem cell, muscle cell, lung cell, pancreatic cell, liver cell, and a cell of the reticular endothelial system. 
     
     
         3 . The composition of  claim 1 , wherein the first nucleic acid encoding the first TALEN protein is selected from the group consisting of SEQ ID Nos: 28, 29, 30, 31, and combinations thereof. 
     
     
         4 . The composition of  claim 1 , wherein the second nucleic acid encoding the second TALEN protein is selected from the group consisting of SEQ ID Nos: 28, 29, 30, 31, and combinations thereof. 
     
     
         5 . A vector comprising the first nucleic acid of  claim 1 . 
     
     
         6 . The vector of  claim 5  selected from the group consisting of a plasmid, adenovirus, adeno-associated virus (AAV), lentivirus, modified HIV-1, SIV or FIV, retrovirus, ASV, ALV, MoMLV), and transposons. 
     
     
         7 . A vector comprising the second nucleic acid of  claim 1 . 
     
     
         8 . The vector of  claim 7  selected from the group consisting of a plasmid, adenovirus, adeno-associated virus (AAV), lentivirus, modified HIV-1, SIV or FIV, retrovirus, ASV, ALV, MoMLV), and transposons. 
     
     
         9 . An isolated host cell comprising the first nucleic acid of  claim 1 . 
     
     
         10 . An isolated host cell comprising the second nucleic acid of  claim 1 . 
     
     
         11 . An isolated host cell comprising one or more of exogenous SEQ ID NOs: 22, 31, 28, 29 or 30 or the proteins expressed from such sequences. 
     
     
         12 . A protein coded for or expressed by the first nucleic acid of  claim 1 . 
     
     
         13 . A protein coded for or expressed by the second nucleic acid of  claim 1 . 
     
     
         14 . A protein coded for or expressed by a nucleic acid sequence selected from the group consisting of SEQ ID Nos: 28, 29, 30, 31, and combinations thereof. 
     
     
         15 . The composition of  claim 1 , wherein the nucleic acid comprising the donor sequence, is a template for site specific DNA repair resulting in a correction of a genetic mutation, wherein the donor sequence comprises homology to at least the 5′ and 3′ ends of the target sequence, wherein a portion of the donor sequence comprises a repair sequence to correct the target sequence for use in conjunction with a TALEN protein. 
     
     
         16 . The nucleic acid of  claim 15  wherein the target is COL7A1. 
     
     
         17 . The nucleic acid of  claim 15 , wherein the donor comprises SEQ ID NO: 22.