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 ;(Om) o ;(Xaa) x with the majority of residues being selected from Arg, Lys, His, Om. 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 . A method for treating a subject having hemophilia B comprising administering a pharmaceutical composition comprising an effective amount of a mRNA encoding PTC (Factor IX). 
     
     
         2 . The method of  claim 1 , wherein the pharmaceutical composition comprises a lipid. 
     
     
         3 . The method of  claim 2 , wherein the lipid comprises a cationic lipid. 
     
     
         4 . The method of  claim 3 , wherein the lipid comprises DOTMA, DOPE, DOSPA, DOTAP DC-Chol or EDMPC. 
     
     
         5 . The method of  claim 1 , the mRNA encoding PTC comprises a polypeptide coding sequence having a G/C content that is increased relative to the G/C content of the wild type sequence for PTC. 
     
     
         6 . The method of  claim 1 , wherein the mRNA comprises a 5′ cap structure. 
     
     
         7 . The method of  claim 1 , wherein the mRNA additionally comprises a poly-A tail of 10 to 200 adenosine nucleotides. 
     
     
         8 . The method of  claim 1 , wherein the mRNA further comprises a 5′ and/or a 3′ untranslated region (UTR). 
     
     
         9 . The method of  claim 1 , wherein the mRNA comprises a further chemical modification relative to a naturally occurring mRNA. 
     
     
         10 . The method  claim 9 , wherein the mRNA comprises at least one further nucleotide that is substituted with a nucleotide analog selected from the group consisting of: 2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate), 2′-deoxy-2′-deamine oligoribonucleotide (2′-amino-2′-deoxycytidine-5′-triphosphate, 2′-amino-2′-deoxyuridine-5′-triphosphate), 2′-O-alkyl oligoribonucleotide, 2′-deoxy-2′-C-alkyl oligoribonucleotide (2′-O-methylcytidine-5′-triphosphate, 2′-methyluridine-5′-triphosphate), 2′-C-alkyl oligoribonucleotide, and isomers thereof (2′-aracytidine-5′-triphosphate, 2′-arauridine-5′-triphosphate), or azidotriphosphate (2′-azido-2′-deoxycytidine-5′-triphosphate, 2′-azido-2′-deoxyuridine-5′-triphosphate)4-thio-uridine-5′-(mono)phosphate, 2-Aminopurine-riboside-5′-(mono)phosphate, 5-Aminoallylcytidine-5′-(mono)phosphate, 5-Aminoallyluridine-5′-(mono)phosphate, 5-Bromocytidine-5′-(mono)phosphate, 5-Bromo-2′-deoxycytidine-5′-(mono)phosphate, 5-Bromouridine-5′-(mono)phosphate, 5-Bromo-2′-deoxyuridine-5′-(mono)phosphate, 5-Iodocytidine-5′-(mono)phosphate, 5-Iodo-2′-deoxycytidine-5′-(mono)phosphate, 5-Iodouridine-5′-(mono)phosphate, 5-Iodo-2′-deoxyuridine-5′-(mono)phosphate, 5-Propynyl-2′-deoxycytidine-5′-(mono)phosphate, 5-Propynyl-2′-deoxyuridine-5′-(mono)phosphate, 5-formylcytidine-5′-(mono)phosphate, 5,2′-O-dimethylcytidine-5′-(mono)phosphate, 5-hydroxymethylcytidine-5′-(mono)phosphate, 5-formyl-2′-O-methylcytidine-5′-(mono)phosphate, 5,2′-O-dimethyluridine-5′-(mono)phosphate, 5-methyl-2-thiouridine-5′-(mono)phosphate, 5-hydroxyuridine-5′-(mono)phosphate, 5-methoxyuridine-5′-(mono)phosphate, uridine 5-oxyacetic acid-5′-(mono)phosphate, uridine 5-oxyacetic acid methyl ester-5′-(mono)phosphate, 5-(carboxyhydroxymethyl)uridine-5′-(mono)phosphate, 5-(carboxyhydroxymethyl)uridine methyl ester-5′-(mono)phosphate, 5-methoxycarbonylmethyluridine-5′-(mono)phosphate, 5-methoxycarbonylmethyl-2′-O-methyluridine-5′-(mono)phosphate, 5-methoxycarbonylmethyl-2-thiouridine-5′-(mono)phosphate, 5-aminomethyl-2-thiouridine-5′-(mono)phosphate, 5-methylaminomethyluridine-5′-(mono)phosphate, 5-methylaminomethyl-2-thiouridine-5′-(mono)phosphate, 5-methylaminomethyl-2-selenouridine-5′-(mono)phosphate, 5-carbamoylmethyluridine-5′-(mono)phosphate, 5-carbamoylmethyl-2′-O-methyluridine-5′-(mono)phosphate, 5-carboxymethylaminomethyluridine-5′-(mono)phosphate, 5-carboxymethylaminomethyl-2′-O-methyluridine-5′-(mono)phosphate, 5-carboxymethylaminomethyl-2-thiouridine-5′-(mono)phosphate, 5-carboxymethyluridine-5′-(mono)phosphate, 5-methyldihydrouridine-5′-(mono)phosphate, 5-taurinomethyluridine-5′-(mono)phosphate, 5-taurinomethyl-2-thiouridine-5′-(mono)phosphate, 5-(isopentenylaminomethyl)uridine-5′-(mono)phosphate, 5-(isopentenylaminomethyl)-2-thiouridine-5′-(mono)phosphate, 5-(isopentenylaminomethyl)-2′-O-methyluridine-5′-(mono)phosphate, 6-Azacytidine-5′-(mono)phosphate, 7-Deazaadenosine-5′-(mono)phosphate, 7-Deazaguanosine-5′-(mono)phosphate, 8-Azaadenosine-5′-(mono)phosphate, 8-Azidoadenosine-5′-(mono)phosphate, Pseudouridine-5′-(mono)phosphate, 2′-Amino-2′-deoxycytidine-(mono)phosphate, 2′-Fluorothymidine-5′-(mono)phosphate, inosine-5′-(mono)phosphate, and 2′-O-Methyl-inosine-5′-(mono)phosphate. 
     
     
         11 . The method of  claim 10 , wherein the mRNA comprises at least one nucleotide that is substituted with a nucleotide analog selected from the group consisting of: 2-amino-6-chloropurineriboside-5′-triphosphate, 2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate, 7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, xanthosine-5′-triphosphate. 
     
     
         12 . The method of  claim 11 , wherein the nucleotide analog is chosen from the group consisting of: 5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate. 
     
     
         13 . The method of  claim 12 , wherein the nucleotide analog is pseudouridine 5′-triphosphate. 
     
     
         14 . The method of  claim 1 , wherein the pharmaceutical composition comprises water, a saline solution or Ringer-Lactat solution. 
     
     
         15 . The method of  claim 1 , wherein the pharmaceutical composition comprises a pH of 7.0 to 7.6. 
     
     
         16 . The method of  claim 1 , wherein the pharmaceutical composition is administered by injection. 
     
     
         17 . The method of  claim 16 , wherein the pharmaceutical composition is administer by intramuscular or intravenous injection.