Method for producing therapeutic DNA

Abstract

The invention discloses the preparation of DNA, in particular plasmid DNA. More particularly it concerns the production of bacterial plasmid DNA to be used in gene therapy, in the form of plasmid, minicircle supercoiled, loose or linear.

1 . Method for the production of DNA which can be used in gene therapy, characterized in that the said DNA is produced in a cell containing a cassette for the expression of a DNA methyltransferase that enables the cytosine residues of the dinucleotides 5′-CG-3′ to be methylated.  
     
     
         2 . Method according to  claim 1 , characterized in that the cell is a prokaryotic cell.  
     
     
         3 . Method according to  claim 2 , characterized in that the cell is a bacterium.  
     
     
         4 . Method according to  claims 1  to  3 , characterized in that the expression cassette is carried by a replicative vector.  
     
     
         5 . Method according to  claims 1  to  3 , characterized in that the expression cassette is integrated in the genome of the cell.  
     
     
         6 . Method according to one of the preceding claims, characterized in that the expression cassette comprises a nucleic acid coding for a DNA methyltransferase that enables the cytosine residues of the dinucleotides 5′-CG-3′ to be methylated under the control of a promoter.  
     
     
         7 . Method according to  claim 6 , characterized in that the promoter is an inducible promoter.  
     
     
         8 . Method according to  claim 1 , characterized in that the DNA methyltransferase preferentially methylates the cytosine residues of the dinucleotides 5′-CG-3′.  
     
     
         9 . Method according to  claim 8 , characterized in that the DNA methyltransferase is chosen from methylase M.SssI, mouse methylase and human methylase.  
     
     
         10 . Method according to  claim 1 , characterized in that more than 50% of the cytosine residues of the dinucleotides 5′-CG-3′ of the plasmid DNA are methylated.  
     
     
         11 . Method according to  claim 1 , characterized in that more than 80% of the cytosine residues of the dinucleotides 5′-CG-3′ of the plasmid DNA are methylated.  
     
     
         12 . Method according to  claim 1 , characterized in that more than 90% of the cytosine residues of the dinucleotides 5′-CG-3′ of the plasmid DNA are methylated.  
     
     
         13 . Use of a plasmid DNA for the preparation of a pharmaceutical composition intended for the therapeutic or diagnostic treatment of the human or animal body, characterized in that more than 50% of the cytosine residues of the dinucleotides 5′-CG-3′ of the plasmid DNA are methylated.  
     
     
         14 . Use of a plasmid DNA for the preparation of a pharmaceutical composition intended for the therapeutic or diagnostic treatment of the human or animal body, characterized in that more than 80% of the cytosine residues of the dinucleotides 5′-CG-3′ of the plasmid DNA are methylated.  
     
     
         15 . Use of a plasmid DNA for the preparation of a pharmaceutical composition intended for the therapeutic or diagnostic treatment of the human or animal body, characterized in that more than 90% of the cytosine residues of the dinucleotides 5′-CG-3′ of the plasmid DNA are methylated.  
     
     
         16 . Method for the preparation of a pharmaceutical composition intended for the therapeutic or diagnostic treatment of the human or animal body, comprising the following steps: 
 production of DNA by culturing a cell comprising the said DNA and a cassette for the expression of a DNA methyltransferase that enables the cytosine residues of the dinucleotides 5′-CG-3′ to be methylated,    recovery of the said DNA, and    packaging of the said DNA with a pharmaceutically acceptable vehicle.    
     
     
         17 . Method according to  claim 16 , characterized in that the DNA is a plasmid DNA comprising a nucleic acid of therapeutic importance.  
     
     
         18 . Method according to  claim 16 , characterized in that the DNA is a minicircle comprising a nucleic acid of therapeutic importance.  
     
     
         19 . Composition comprising a bacterial DNA in which at least 50% of the cytosine residues of the dinucleotides 5′-CG-3′ are methylated.