Cationic reagents for transfection

Abstract

Cationic cytofectins and liposomes comprising the same are disclosed which are especially useful for delivering exogenous compounds into cells in vitro and in vivo. The liposomes may comprise (a) a neutral lipid such as dioleoylphosphatidyl-ethanolamine (DOPE) or similar lipid-like compounds such as 1,2-dioleoyl-oxiphosphatidylethanolamine or other lipid-like structures and (b) one or more of the cationic cytofectins provided herein. The invention provides transfection kits and methods for delivery of exogenous compounds into cells.

1 . Use of a compound according to Formula (I),  
       
         
           
           
               
               
           
         
       
       wherein 
 A denotes an anion selected from the group of chloride, bromide, iodide, hydrogenphosphate(HPO 4   2− ), dihydrogenphosphate (H 2 PO 4   - ), sulphate, thiosulphate, hydroxy and/or oxalate.  
 k denotes an integer 1, 2, 3, 4 or 5;  
 B denotes an alkandiyl bridge (CH 2 ) n ; wherein  
 n denotes an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;  
 R 1 , R 3  and R 4 , which may be identical to one another or different, denote hydrogen, straight chained or branched C 1 -C 6 -alkyl, C 1 -C 6 -alkenyl, C 1 -C 6 -alkynyl;  
 R 2  denotes straight-chained or branched C 8 -C 20 -alkyl, C 8 -C 20 -alkenyl, C 8 -C 20 -alkynyl;  
 R 5  denotes for k=1 
 straight-chained or branched C 8 -C 20 -alkyl, C 8 -C 20 -alkenyl, C 8 -C 20 -alkynyl;  
 denotes for k>1  
 hydrogen, straight-chained or branched C 1 -C 6 -alkyl, C 1 -C 6 -alkenyl, C 1 -C 6 -alkynyl;  
 
 R 6  denotes for k=1 
 hydrogen, straight-chained or branched C 1 -C 6 -alkyl, c 1 -C 6 -alkenyl, C 1 -C 6 -alkynyl;  
 denotes for k>1  
 a straight-chained or branched C 8 -C 20 -alkyl, C 8 -C 20 -alkenyl, C 8 -C 20 -alkynyl  
 and the repeating unit —B—NR 4 R 6  may be identical to one another or different;  
 for transfection:  
 
 
     
     
         2 . Use of a compound according to  claim 1 , wherein 
 A denotes an anion selected from the group of chloride, bromide, iodide, hydrogenphosphate(HPO 4   2− ), dihydrogenphosphate (H 2 PO 4   - ), sulphate, thiosulphate, hydroxy and/or oxalate.    k denotes an integer 1, 2 or 3;    B denotes an alkandiyl bridge (—CH 2 ) n ; and    n denotes an integer 1, 2, 3, 4, 5 or 6;    R 1 , R 3  and R 4 , which may be identical to one another or different, denote hydrogen or straight-chained or branched C 1 -C 6 -alkyl;    R 2  denotes straight-chained or branched C 8 -C 20 -alkyl, C 8 -C 20 -alkenyl, C 8 -C 20 -alkynyl;    R 5  denotes for k=1 
 a straight-chained or branched C 8 -C 20 -alkyl, C 8 -C 20 -alkenyl, C 8 -C 20 -alkynyl;  
 denotes for k>1  
 hydrogen, straight-chained or branched C 1 -C 6 -alkyl;  
   R 6  denotes for k=1 
 hydrogen, straight-chained or branched C 1 -C 6 -alkyl, C 1 -C 6 -alkenyl, C 1 -C 6 -alkynyl;  
 denotes for k>1  
 a straight-chained or branched C 8 -C 20 -alkyl, C 8 -C 20 -alkenyl, C 8 -C 20 -alkynyl  
 and the repeating unit —B—NR 4 R 6  is preferably identical to one another.  
   
     
     
         3 . Use of a compound according to  claim 1  or 2, wherein 
 A denotes an anion selected from the group of bromide, iodide, dihydrogenphosphate (H 2 PO 4   - ) and/or thiosulphate;  
 k denotes an integer 1 or 2;  
 B denotes for k=1 
 an alkandiyl bridge —(CH 2 ) n  wherein  
 n represents an integer 2, 3 or 4;  
 
 B denotes for k=2 
 an ethylenebridge —(CH 2 —CH 2 )—;  
 R 1 , R 3  and R 4 , which are identical to one another, denote CH 3 ;  
 
 R 2  denotes straight-chained C 10 -C 20 -alkyl;  
 R 5  denotes for k=1 
 straight-chained C 10 -C 20 -alkyl and is identical to R 2 ;  
 denotes for k=2  
 CH 3 ;  
 
 R 6  denotes for k=1 
 CH 3    
 denotes for k=2  
 straight-chained C 10 -C 20 -alkyl and is identical to R 2 .  
 
 
     
     
         4 . Use of a compound according to any one of  claims 1  to  3 , wherein said compound is part of a liposome further comprising a neutral lipid or lipid like compound.  
     
     
         5 . Use of a compound according to  claim 4 , wherein said neutral lipid or lipid like compound is dioleoylphosphatidylethanolamine (DOPE) and/or 1,2-dioleoyloxiphosphatidylethanolamine and/or Cholesterole and/or Dioleyl-phosphatidylcholin (DOPC).  
     
     
         6 . Use of a compound according to any one of  claims 1  to  5 , wherein said compound comprises a cell targeting component.  
     
     
         7 . Use of a compound according to  claim 6 , wherein said cell targeting compound is a ligand or ligand-like component for a specific cell surface receptor or nuclear receptor.  
     
     
         8 . Use of a compound according to any one of  claims 1  to  7  for in vitro transfection of cell cultures, wherein the DNA/liposome ratio is 0.01 μg to 10 μg DNA/μg liposome.  
     
     
         9 . Use of a compound according to  claim 8 , wherein the DNA/liposome ratio is 0.1 μg to 1 μg DNA/μg liposome.  
     
     
         10 . Use of a compound according to any one of  claims 1  to  7  for in vivo transfection, wherein the DNA/liposome ratio is in the range of DNA/liposome (w/w) 2:1 to 1:3/1 μg to 100 mg per kg body weight.  
     
     
         11 . Kit for transfection, characterized in that it comprises a compound as defined in any one of  claims 1  to  10 .  
     
     
         12 . Kit according to  claim 11 , characterized in that it further comprises at least one suitable buffer.  
     
     
         13 . Use of a compound according to any one of  claims 1  to  12  for the delivery of a nucleic acid, or derivative thereof, into a target cell.  
     
     
         14 . Use of a compound according to  claim 13 , characterized in that the nucleic acid is single stranded and/or double stranded DNA and/or RNA and/or a DNA/RNA-Hybrid, or derivatives thereof.  
     
     
         15 . Use of a compound according to  claim 13  or  14 , characterized in that the DNA is selected form the group of plasmids, vectors, cDNA, CpG-motifs, and/or oligonucleotides, and the RNA is selected from the group of mRNA, oligonucleotides or ribozymes.  
     
     
         16 . Use of a compound according to any one of  claims 1  to  15  as a pharmaceutical substance.  
     
     
         17 . Use of a compound according to any one of  claims 1  to  15  as a prophylactic and/or therapeutic vaccine.