Process for the preparation of endotoxin-free or endotoxin-depleted nucleic acids and/or oligonucleotides for gene therapy

Abstract

A process for the isolation and purification of nucleic acids and/or oligonucleotides for use in gene therapy wherein said nucleic acids and/or oligonucleotides are isolated or purified from an essentially biological source, characterized in that said essentially biological sources are lysed, the fractions obtained are optionally freed or depleted from the remainder of said biological sources by per se known mechanical methods, such as centrifugation, filtration; the fractions thus treated are subsequently treated with affinity chromatographic material or with inorganic chromatographic material for the removal of endotoxins; followed by isolation of said nucleic acids and/or oligonucleotides on an anion exchanger which is designed such that DNA begins to desorb from the anion exchanger only at an ionic strength corresponding to a sodium chloride solution of a concentration higher by at least 100 mM than one corresponding to the ionic strength at which RNA begins to desorb from the anion exchanger material.

1 . A process for the isolation and purification of nucleic acids and/or oligonucleotides for use in gene therapy wherein said nucleic acids and/or oligonucleotides are isolated or purified from an essentially biological source, characterized in that 
 said essentially biological sources are lysed, the fractions obtained are optionally freed or depleted from the remainder of said biological sources by per se known mechanical methods, such as centrifugation, filtration;    the fractions thus treated are subsequently treated with affinity chromatographic material or with inorganic chromatographic material for the removal of endotoxins; followed by    isolation of said nucleic acids and/or oligonucleotides on an anion exchanger which is designed such that DNA begins to desorb from the anion exchanger only at an ionic strength corresponding to a sodium chloride solution of a concentration higher by at least 100 mM than one corresponding to the ionic strength at which RNA begins to desorb from the anion exchanger material.    
     
     
         2 . The process according to  claim 1 , characterized in that porous or non-porous inorganic and/or organic support materials modified with anion exchanging groups are used as the support material of the inorganic chromatographic material.  
     
     
         3 . The process according to any of claims  1  and/or  2 , characterized in that silica gel, diatomaceous earth, glass, aluminium oxides, titanium oxides, zirconium oxides, hydroxyapatite are used as inorganic support materials, and dextrane, agarose, acrylic amide, polystyrene resins, or copolymers of the materials mentioned are used as organic support materials.  
     
     
         4 . The process according to at least one of  claims 1  to  3 , wherein said modified support material is obtained by reacting one of the support materials mentioned in  claim 3  in a first step with a silanizing reagent of the general formula I,  
       R 1 R 2 R 3 SiR 4   (I)  
       wherein R 1  is an alkoxy residue of from 1 to 10 carbon atoms, especially —OCH 3 , —OC 2 H 5  or —OC 3 H 7 , or a halogen atom, especially —Cl, or a dialkylamino group with identical or different alkyl residues of from 1 to 6 carbon atoms; 
 R 2  and R 3  are independently a hydrocarbon residue of from 1 to 10 carbon atoms, especially —CH 3 , —C 2 H 5  or —C 3 H 7 , or an alkoxy residue of from 1 to 10 carbon atoms, especially —OCH 3 , —OC 2 H 5  or —OC 3 H 7 , or a halogen atom, or an alkyl residue of from 4 to 20 carbon atoms which is interrupted by at least one oxa or amino group wherein said residue may also be substituted with one or more of halogen, cyano, nitro, amino, monoalkylamino, dialkylamino, hydroxy or aryl;  
 R 4  is a hydrocarbon chain of from 1 to 20 carbon atoms, or an alkyl residue which is interrupted by at least one oxa or amino group wherein said residue may also be substituted with one or more of halogen, cyano, nitro, amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, aryl, and/or epoxy, especially  
                     
 followed by a second step wherein the support which has been modified in the first step is reacted with a reagent of the general formula II:  
 X-R-Y  (II)  
 wherein X is an amino, hydroxy, epoxy group or a halogen atom;  
 R is a hydrocarbon chain of from 2 to 20 carbon atoms, or an alkyl residue which is interrupted by at least one oxa or amino group wherein said residue may also be substituted with one or more of halogen, cyano, nitro, amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, aryl, and/or epoxy;  
 Y is a hydrocarbon residue having functional groups which form an anion exchange material and having from 1 to 10 carbon atoms which may be substituted with one or more of amino, monoalkylamino, dialkylamino, quarternary alkylamino.  
 
     
     
         5 . The process according to at least one of  claims 1  to  4 , wherein diethylaminoethyl (DEAE) groups or dimethylaminoethyl (DMAE) groups are arranged on the surface of the support either directly or through so-called spacers.  
     
     
         6 . The process according to at least one of  claims 1  to  5 , wherein DNA, such as plasmids, cosmids, DNA isolated from viruses, also in enzymatically or chemically modified form, and/or RNA in any form and of any origin, or ribozymes are isolated as said nucleic acids.  
     
     
         7 . The process according to any of  claims 1  to  6 , wherein a treatment of said fractions containing nucleic acids with non-ionic detergents, such as Triton X 100 or a treatment with affinity chromatographic supports, such as nickel/NTA, nickel/IDA, polymyxin, DNA ETOX, is performed for the removal of endotoxins from the nucleic acid fractions.  
     
     
         8 . The process according to any of  claims 1  to  7 , wherein the salts, which are necessary for eluting the nucleic acids under conditions of high ionic strength, are removed by treating with a mineral support material, such as one essentially consisting of glass, the fractions containing nucleic acids and having the high salt concentrations, wherein the nucleic acid adsorbs to the surface of said mineral supports, and subsequent desorption of the nucleic acids with water or buffer solutions of low ionic strength.  
     
     
         9 . The process according to any of  claims 1  to  7 , wherein cell debris of the cell lysate are separated off by filtration, in particular using a filter the pore size of which decreases in the direction of flow of the sample to be filtrated and/or a filter having a filter layer consisting of glass, silica gel, alumina or packed diatomaceous earth or interlaced or bonded non-wovens made of fiber glass and silica gel as well as cellulose, paper, pressed paper, non-wovens made of paper.  
     
     
         10 . The process according to at least one of  claims 1  to  9 , wherein a preliminary purification of a sample of a cell lysate containing nucleic acids is performed on a layer of unmodified diatomaceous earth.  
     
     
         11 . Use of the anion exchange materials mentioned in  claims 1  to  6  for the separation, purification and isolation of nucleic acids for the preparation of an agent containing nucleic acids for gene therapy.  
     
     
         12 . The use according to  claim 11  for in vivo and ex vivo gene therapy.  
     
     
         13 . The use according to any of claims  11  or  12  for the preparation of an agent for the treatment of genetically caused diseases, such as cystic fibrosis, muscular dystrophy.  
     
     
         14 . Use of the anion exchange materials mentioned in  claims 1  to  6  for the purification of oligonucleotides for in vivo/ex vivo gene therapy by the antisense/sense strategy.  
     
     
         15 . Use of the anion exchange materials mentioned in  claims 1  to  6  for the purification of virus particles, also intact virus particles, for in vivo/ex vivo gene therapy.  
     
     
         16 . Use of isopropanol as a reagent for the purification of nucleic acids in a process according to any of  claims 1  to  10 .  
     
     
         17 . A kit containing the components necessary for performing the process according to any of  claims 1  to  10 , in particular, reagents, also in concentrated form for final mixing by the user, chromatographic materials for the separation of the nucleic acids, aqueous solutions (buffers, optionally also in concentrated form for final adjusting by the user), further auxiliaries, and substances for the removal of endotoxins.