Complexes of rna and cationic peptides for transfection and for immunostimulation

Abstract

The present invention relates to a complexed RNA, comprising at least one RNA complexed with one or more oligopeptides, wherein the oligopeptide, which has the function of cell-penetrating peptide (CPP), has a length of 8 to 15 amino acids and has the empirical formula (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; (Xaa) x with the majority of residues being selected from Arg, Lys, His, Orn. The invention further relates to a method for transfecting a cell or an organism, thereby applying the inventive complexed RNA. Additionally, pharmaceutical compositions and kits comprising the inventive complexed RNA, as well as the use of the inventive complexed RNA for transfecting a cell, tissue or an organism and/or for modulating, preferably inducing or enhancing, an immune response are disclosed herein.

1 . Complexed RNA, comprising at least one RNA (molecule) complexed with one or more oligopeptides, wherein the oligopeptide has a length of 8 to 15 amino acids and has the following empirical formula:
   (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x   (formula I)   
       wherein
 l+m+n+o+x=8-15, and 
 l, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and 
 Xaa may be any amino acid selected from native or non-native amino acids except of Arg, Lys, His or Orn; and 
 x may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8, provided, that the overall content of Xaa does not exceed 50% of all amino acids of the oligopeptide. 
 
     
     
         2 . Complexed RNA according to  claim 1 , wherein the at least one RNA (molecule) is an mRNA. 
     
     
         3 . Complexed RNA according to  claim 1 , wherein the oligopeptide has a length of 8 to 14, 8 to 13, 8 to 12, or 9 to 12 or 9 to 11 amino acids. 
     
     
         4 . Complexed RNA according to  claim 1 , wherein the overall content of Arg, Lys, His and Orn represents at least 60%, 70%, 80%, 90%, or 95% of all amino acids of the oligopeptide of the complexed RNA. 
     
     
         5 . Complexed RNA according to  claim 1 , wherein Xaa in empirical formula (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; (Xaa) x  is selected from amino acids having a neutral side chain including amino acids having a neutral hydrophobic side chain and amino acids having a neutral polar side chain. 
     
     
         6 . Complexed RNA according to  claim 1 , wherein the oligopeptide according to empirical formula (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; (Xaa) x  does not comprise amino acids at one or both terminal ends, which comprise an acidic side chain. 
     
     
         7 . Complexed RNA according to  claim 1 , wherein the oligopeptide according to empirical formula (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; (Xaa) x  comprises neutral or basic amino acids at one or both terminal ends, or basic amino acids at one or both terminal ends or at least one, preferably at least two, more preferably at least three, basic amino acids at both terminal ends. 
     
     
         8 . Complexed RNA according to  claim 1 , wherein the oligopeptide according to empirical formula (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; (Xaa) x  comprises a stretch of at least 3 contiguous basic amino acids within its sequence. 
     
     
         9 . Complexed RNA according to  claim 1 , wherein the oligopeptide according to empirical formula (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; (Xaa) x  comprises at least 1, 2, or 3 non-cationic amino acid(s) at one or both terminal ends, the non-cationic amino acid preferably being selected from histidine. 
     
     
         10 . Complexed RNA according to  claim 1 , wherein the at least one RNA (molecule) of the complexed RNA is an mRNA, wherein the mRNA codes for a therapeutically active protein or peptide, an immunostimulating protein or peptide, a tumor antigen or an antibody. 
     
     
         11 . Complexed RNA according to  claim 1 , wherein the RNA is a modified RNA, in particular a stabilized RNA, as compared to the wt RNA. 
     
     
         12 . Complexed RNA according to  claim 11 , wherein the G/C content of the coding region of the modified RNA is increased compared with the G/C content of the coding region of the wild-type RNA, and wherein the coded amino acid sequence of the modified RNA is preferably not modified compared with the coded amino acid sequence of the wild-type RNA. 
     
     
         13 . Complexed RNA according to  claim 1 , wherein the mass ratio of the at least one RNA (molecule) of the complexed RNA to the one or more oligopeptides is in a range of about 1:100 to about 1:0.5, or has a value of about 1:50 to about 1:1, about 1:100, about 1:90, about 1:80, about 1:70, about 1:60, about 1:50, about 1:45, about 1:40, about 1:35, about 1:30, about 1:25, about 1:20, about 1:15, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1 or about 1:0.5. 
     
     
         14 . Complexed RNA according to  claim 1 , wherein the molar ratio of the at least one RNA (molecule) of the complexed RNA to the one or more oligopeptides is in a range of about 1:20000 to about 1:500 or 1:250, or in a range of about 1:10000 to about 1:1000, or has a value of about 1:9500, about 1:9000, about 1:8500, about 1:8000, about 1:7500, about 1:7000, about 1:6500, about 1:6000, about 1:5500, about 1:5000, about 1:4500, about 1:4000, about 1:3500, about 1:3000, about 1:2500, about 1:2000, about 1:1500, about 1:1000, about 1:500, about 1:450, about 1:400, about 1:350, about 1:300, or about 1:250. 
     
     
         15 . Complexed RNA according to  claim 1 , wherein the nitrogen/phosphate ratio (N/P-ratio) of the at least one RNA (molecule) of the complexed RNA to the one or more oligopeptides is in a range of about 0, 2-50, in a range of about 0.5-50 or in a range of about 0.75-25. 
     
     
         16 . Complexed RNA according to  claim 1 , wherein the oligopeptide is selected from the group consisting of:
 Arg 9 His 3 , His 3 Arg 9 His 3 , TyrSerSerArg 9 SerSerTyr, His 3 Arg 9 SerSerTyr, (ArgLysHis) 4 , Tyr(ArgLysHis) 2 Arg;   or from   Arg 8 , Arg 9 , Arg 10 , Arg 11 , Arg 12 , Arg 13 , Arg 14 , Arg 15 , (SEQ ID NOs: 1-8);   Lys 8 , Lys 9 , Lys 10 , Lys 11 , Lys 12 , Lys 13 , Lys 14 , Lys 15 , (SEQ ID NOs: 9-16);   His 8 , His 9 , His 10 , His 11 , His 12 , His 13 , His 14 , His 15 , (SEQ ID NOs: 17-24); and   Orn 8 , Orn 9 , Orn 10 , Orn 11 , Orn 12 , Orn 13 , Orn 14 , Orn 15 , (SEQ ID NOs: 25-32).   
     
     
         17 . Pharmaceutical composition comprising the complexed RNA according to  claim 1  and optionally a pharmaceutically suitable carrier. 
     
     
         18 . A method for transfection of a cell or an organism, comprising the following steps:
 a. optionally preparing and/or providing a complexed RNA according to  claim 1 , comprising at least one RNA complexed with one or more oligopeptides having the empirical formula (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; (Xaa) x ;   b. transfecting a cell, a tissue or an organism using the complexed RNA prepared and/or provided according to step a).   
     
     
         19 . Method according to  claim 18 , wherein the organism is a mammal, selected from the group comprising humans, goat, cattle, swine, dog, cat, donkey, monkey, ape or rodents, including mouse, hamster and rabbit. 
     
     
         20 . Use of a complexed RNA according to any of  claim 1  for transfecting a cell or an organism. 
     
     
         21 . Use of a complexed RNA according to any of  claim 1  for preparing an agent for treating a disease selected from a tumour or cancer disease, a cardiovascular disease, an infectious disease, an (infectious) virus disease, an autoimmune disease, a (mono-) genetic disease and/or an allergy. 
     
     
         22 . Use according to  claim 21 , wherein transfection of the complexed RNA elicits an immune response in the organism. 
     
     
         23 . Kit according to  claim 22  comprising a cytokine or a composition containing a cytokine and/or at least one additional adjuvant or a composition containing at least one additional adjuvant.