Gene repair involving in vivo excision of targeting DNA

Abstract

Methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell are disclosed. Also disclosed are methods of treating or prophylaxis of a genetic disease in an individual in need thereof.

What is claimed is:  
     
         1 . A method of attenuating an endogenous gene of interest in a cell comprising the steps of: 
 a) introducing into the cell a first vector comprising targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to a targeting site of the endogenous gene of interest and (2) DNA which attenuates the gene of interest upon recombination between said targeting DNA and the gene of interest; and    b) introducing into the cell a second vector comprising a nucleic acid encoding a restriction endonuclease which cleaves the restriction endonuclease site present in the first vector.    
     
     
         2 . The method of  claim 1  wherein the first vector is a viral vector.  
     
     
         3 . The method of  claim 1  wherein the second vector is a viral vector.  
     
     
         4 . The method of  claim 1  wherein the first vector is a plasmid.  
     
     
         5 . The method of claims  1  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         6 . A method of attenuating an endogenous gene of interest in a cell comprising the steps of: 
 a) introducing into the cell a vector comprising targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to a targeting site of the endogenous gene of interest and (2) DNA which attenuates the gene of interest upon recombination between said targeting DNA and the gene of interest; and    b) introducing into the cell a restriction endonuclease which cleaves the restriction endonuclease site present in the vector.    
     
     
         7 . The method of  claim 6  wherein the vector is a viral vector.  
     
     
         8 . The method of  claim 6  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         9 . A method of attenuating an endogenous gene of interest in a cell comprising introducing into the cell a vector comprising (a) targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to a targeting site of the endogenous gene of interest and (2) DNA which attenuates the gene of interest upon recombination between said targeting DNA and the gene of interest; and (b) a nucleic acid encoding a restriction endonuclease which cleaves the restriction endonuclease site.  
     
     
         10 . The method of  claim 9  wherein the vector is a viral vector.  
     
     
         11 . The method of claims  9  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         12 . A method of introducing a mutation into a targeting site of chromosomal DNA of a cell comprising the steps of: 
 a) introducing into the cell a first vector comprising targeting DNA, wherein said is flanked by a restriction endonuclease site and comprises (1) DNA homologous to said target site and (2) the mutation to be introduced into the chromosomal DNA; and    b) introducing into the cell a second vector comprising a nucleic acid encoding a restriction endonuclease which cleaves the restriction endonuclease site present in the first vector.    
     
     
         13 . The method of  claim 12  wherein the first vector is a viral vector.  
     
     
         14 . The method of  claim 12  wherein the second vector is a viral vector.  
     
     
         15 . The method of  claim 14  wherein the first vector is a plasmid.  
     
     
         16 . The method of claims  12  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         17 . A method of introducing a mutation into a target site of chromosomal DNA of a cell comprising the steps of: 
 a) introducing into the cell a vector comprising targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to the target site and (2) the mutation to be introduced into the chromosomal DNA; and    b) introducing into the cell a restriction endonuclease which cleaves the restriction endonuclease site present in the vector.    
     
     
         18 . The method of  claim 17  wherein the vector is a viral vector.  
     
     
         19 . The method of  claim 17  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         20 . A method of introducing a mutation into a target site of chromosomal DNA of a cell comprising introducing into the cell a vector comprising (a) targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to the target site and (2) the mutation to be introduced into the chromosomal DNA; and (b) a nucleic acid encoding a restriction endonuclease which cleaves the restriction endonuclease site.  
     
     
         21 . The method of  claim 20  wherein the vector is a viral vector.  
     
     
         22 . The method of claims  20  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         23 . A method of treating or prophylaxis of a genetic disease in an individual in need thereof comprising the steps of: 
 a) introducing into the individual cells which comprise a first vector comprising targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to chromosomal DNA adjacent to a specific sequence of interest and (2) DNA which repairs the specific sequence of interest upon recombination between said targeting DNA and the chromosomal DNA; and    b) introducing into the individual a second vector comprising a nucleic acid encoding a restriction endonuclease which cleaves the restriction endonuclease site present in the first vector.    
     
     
         24 . The method of  claim 23  wherein the first vector is a viral vector.  
     
     
         25 . The method of  claim 24  wherein the second vector is a viral vector.  
     
     
         26 . The method of  claim 24  wherein the first vector is a plasmid.  
     
     
         27 . The method of claims  23  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         28 . A method for treating or prophylaxis of a genetic disease in an individual in need thereof comprising the steps of: 
 a) introducing into the individual cells which comprise a vector comprising targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to chromosomal DNA adjacent to a specific sequence of interest and (2) DNA which repairs the specific sequence of interest upon recombination between said targeting DNA and the chromosomal DNA; and    b) introducing into the cell a restriction endonuclease which cleaves the restriction endonuclease site present in the vector.    
     
     
         29 . The method of  claim 28  wherein the vector is a viral vector.  
     
     
         30 . The method of  claim 28  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         31 . A method of modifying a specific sequence in chromosomal DNA of a cell comprising the steps of: 
 a) introducing into the cell a first vector comprising targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to the specific sequence to be modified and (2) DNA which modifies the specific sequence upon recombination between said targeting DNA and the chromosomal DNA; and    b) introducing into the cell a second vector comprising a nucleic acid encoding a restriction endonuclease which cleaves the restriction endonuclease site present in the first vector.    
     
     
         32 . The method of  claim 31  wherein the first vector is a viral vector.  
     
     
         33 . The method of  claim 31  wherein the second vector is a viral vector.  
     
     
         34 . The method of  claim 33  wherein the first vector is a plasmid.  
     
     
         35 . The method of claims  31  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         36 . A method of modifying a specific sequence in chromosomal DNA of a cell comprising the steps of: 
 a) introducing into the cell a vector comprising targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to the specific sequence to be modified and (2) DNA modifies the specific sequence upon recombination between said targeting DNA and the chromosomal DNA; and    b) introducing into the cell a restriction endonuclease which cleaves the restriction endonuclease site present in the vector, under conditions appropriate for the restriction endonuclease to cleave the restriction endonuclease site in the vector of step a).    
     
     
         37 . The method of  claim 36  wherein the vector is a viral vector.  
     
     
         38 . The method of  claim 36  wherein said targeting DNA is flanked by two restriction endonuclease sites.  
     
     
         39 . A method of modifying a specific sequence in chromosomal DNA of a cell comprising introducing into the cell a vector comprising (a) targeting DNA, wherein said targeting DNA is flanked by a restriction endonuclease site and comprises (1) DNA homologous to the specific sequence to be modified and (2) DNA which results in modification of the specific sequence upon recombination between said targeting DNA and the chromosomal DNA; and (b) a nucleic acid encoding a restriction endonuclease which cleaves the restriction endonuclease site.  
     
     
         40 . The method of  claim 39  wherein the vector is a viral vector.  
     
     
         41 . The method of claims  39  wherein said targeting DNA is flanked by two restriction endonuclease sites.