Process for preparing purified nucleic acid and the use thereof

Abstract

The invention relates to a nucleic acid preparation with a content of below 1% protein, preferably below 0.1% protein, free of ethidium bromide, phenol, cesium chloride and detergents based on octyl phenol poly(ethylene glycol ether) n and with a content of below 1EU/mg DNA of endotoxins. Said preparation is suitable as a drug particularly in gene therapy.

1 . DNA preparation comprising a DNA, proteins in an amount of less than about 0.1% DNA and endotoxins in an amount of less than about 1 EU/mg DNA. 
     
     
         2 . The DNA preparation of  claim 1 , comprising endotoxins of less than about 0.06 EU/mg DNA. 
     
     
         3 . The DNA preparation of  claim 1 , comprising endotoxins of about 0.01 to 0.1 EU/mg DNA. 
     
     
         4 . The DNA preparation of  claim 1 , wherein the DNA preparation is free of ethidium bromide, phenol, cesium chloride, octylphenolpoly(ethylene glycol ether) n  detergents and MOPS buffer. 
     
     
         5 . The DNA preparation of  claim 1 , wherein the DNA can be replicated in gram-negative bacteria. 
     
     
         6 . The DNA preparation of  claim 15 , wherein the gram-negative bacteria is  Escherichia coli.    
     
     
         7 . The DNA preparation of  claim 1 , wherein the DNA is plasmid DNA. 
     
     
         8 . The DNA preparation of  claim 7 , wherein the plasmid DNA is capable of replication. 
     
     
         9 . A pharmaceutical composition suitable for gene therapy comprising a therapeutically effective amount of the DNA preparation of  claim 5  and a pharmaceutically acceptable carrier. 
     
     
         10 . The composition of  claim 9 , wherein the DNA preparation contains endotoxins in an amount of less than about 0.06 EU/mg DNA. 
     
     
         11 . The composition of  claim 9 , wherein the DNA preparation contains endotoxins in an amount of about 0.01 to 0.1 EU/mg DNA. 
     
     
         12 . The composition of  claim 9 , wherein the DNA is a plasmid DNA. 
     
     
         13 . The composition of  claim 12 , wherein the plasmid DNA is encapsulated in liposomes. 
     
     
         14 . A method for gene therapy of cystic fibrosis caused by the absence of a normal first gene or the presence of a defective second gene, comprising administering an effective amount of the DNA preparation of  claim 18  to a patient in need thereof, wherein the plasmid DNA contains said normal first gene or a normal second gene corresponding to the defective second gene. 
     
     
         15 . A process for making the DNA preparation of  claim 1 , comprising the following steps
 (a) providing gram-negative bacteria containing said DNA;   (b) lysing said bacteria to obtain a lysate, wherein the lysate is a DNA-containing fraction and thereafter   (c) chromatographing said DNA-containing fraction on hydroxylapatite in order to obtain said DNA preparation.   
     
     
         16 . The method of  claim 15 , further comprising replicating said DNA in gram-negative bacteria after step (a) and before step (b). 
     
     
         17 . The method of  claim 16 , further comprising transfecting the gram-negative bacteria with a cloning vector containing said DNA before step (a). 
     
     
         18 . The method of  claim 16 , further comprising eluting said hydroxylapatite in step (c) with a solution of phosphate, citrate, sulfate or divalent metal ions to obtain said DNA preparation. 
     
     
         19 . The method of  claim 15 , further comprising, after step (b) and before step (c), filtering the lysate to obtain a filtrate and fractionating said filtrate by gel filtration to obtain said DNA-containing fraction. 
     
     
         20 . The method of  claim 16 , further comprising, after step (b) and before step (c), filtering the lysate to obtain a filtrate and fractionating said filtrate by gel filtration to obtain said DNA-containing fraction. 
     
     
         21 . The method of  claim 17 , further comprising, after step (b) and before step (c), filtering the lysate to obtain a filtrate and fractionating said filtrate by gel filtration to obtain said DNA-containing fraction. 
     
     
         22 . The method of  claim 18 , further comprising, after step (b) and before step (c), filtering the lysate to obtain a filtrate and fractionating said filtrate by gel filtration to obtain said DNA-containing fraction.