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 has a length of 8 to 15 amino acids and has the empirical formula (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; (Xaa) x . 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 of treating a subject having a ornithine transcarbamylase deficiency comprising administering an effective amount of a pharmaceutical composition comprising mRNA encoding ornithine transcarbamylase (OTC) to the subject. 
     
     
         2 . The method of  claim 1 , wherein the subject is a human subject. 
     
     
         3 . The method of  claim 1 , wherein the pharmaceutical composition is administered by intravenous injection or intramuscular injection. 
     
     
         4 . The method of  claim 1 , wherein the mRNA comprises a 5′ cap structure. 
     
     
         5 . The method of  claim 1 , wherein the mRNA additionally comprises a poly-A tail of 10 to 200 adenosine nucleotides. 
     
     
         6 . The method of  claim 1 , wherein the mRNA additionally comprises a poly-C tail of 10 to 200 cytosine nucleotides. 
     
     
         7 . The method of  claim 1 , wherein the mRNA further comprises a 5′ and/or a 3′ untranslated region (UTR). 
     
     
         8 . The method of  claim 1 , wherein the mRNA has been modified by introduction of a non-native nucleotide compared with a corresponding native mRNA nucleotide and/or by covalent coupling of the mRNA with a further chemical moiety. 
     
     
         9 . The method of  claim 1 , wherein the mRNA comprises a G/C content in the OTC coding region which is greater than the G/C content of a coding region of the native mRNA sequence encoding OTC. 
     
     
         10 . The method of  claim 1 , wherein the mRNA comprises an OTC coding sequence that is modified, compared with a native mRNA encoding OTC, such that at least one codon of the native mRNA which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is relatively frequent in the cell. 
     
     
         11 . The method of  claim 8 , wherein the mRNA comprises a chemical modification relative to a naturally occurring mRNA. 
     
     
         12 . The method of  claim 8 , wherein the mRNA comprises at least one 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-formyl cytidine-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. 
     
     
         13 . The method of  claim 8 , 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′-tri phosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, puromycin-5′-triphosphate, and xanthosine-5′-triphosphate. 
     
     
         14 . The method of  claim 13 , wherein the nucleotide analog is chosen from the group consisting of: 5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate. 
     
     
         15 . The method of  claim 1 , wherein the pharmaceutical composition further comprises a cationic polymer. 
     
     
         16 . The method  claim 1 , wherein the pharmaceutical composition further comprises a cationic lipid. 
     
     
         17 . The method  claim 15 , wherein the cationic polymer is a cationic polypeptide. 
     
     
         18 . The method  claim 15 , wherein the mRNA is provided in complex with the cationic polymer or cationic lipid. 
     
     
         19 . A pharmaceutical composition comprising an isolated mRNA comprising a sequence encoding OTC, wherein:
 the sequence encoding OTC is linked to a heterologous 5′ and/or 3′ untranslated region (UTR); and/or   (ii) the pharmaceutical composition further comprises a cationic polymer or cationic lipid.   
     
     
         20 . The pharmaceutical composition of  claim 19 , wherein the mRNA is modified by introduction of a non-native nucleotide compared with a native mRNA sequence and/or by covalent coupling of the mRNA with a further chemical moiety.