METHODS OF TREATING A SUBJECT WITH mRNA ENCODING COAGULATION FACTOR VIII (F8)

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 haemophilia A comprising administering an effective amount of a pharmaceutical composition comprising a purified mRNA encoding coagulation factor VIII (F8) to the subject, wherein the mRNA comprises a 5′ cap structure; wherein the mRNA comprises a poly-A tail; wherein the mRNA is comprised in a liposomal system. 
     
     
         2 . The method of  claim 1 , wherein the mRNA has been purified by chromatography. 
     
     
         3 . The method of  claim 2 , wherein the mRNA has been purified by affinity chromatography. 
     
     
         4 . The method of  claim 3 , wherein the mRNA has been purified by HPLC using a porous reverse phase as the stationary phase. 
     
     
         5 . The method of  claim 1 , wherein the subject is a human subject. 
     
     
         6 . The method of  claim 1 , wherein the pharmaceutical composition is administered by injection. 
     
     
         7 . The method of  claim 6 , wherein the pharmaceutical composition is administered by intravenous injection. 
     
     
         8 . The method of  claim 1 , wherein the poly-A tail comprises 10 to 200 adenosine nucleotides. 
     
     
         9 . The method of  claim 1 , wherein the mRNA additionally comprises a poly-C tail of 10 to 200 cytosine nucleotides. 
     
     
         10 . The method of  claim 1 , wherein the mRNA further comprises a 5′ and/or a 3′ untranslated region (UTR). 
     
     
         11 . 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. 
     
     
         12 . The method of  claim 1 , wherein the mRNA comprises a G/C content in the F8 coding region which is greater than the G/C content of a coding region of the native mRNA sequence encoding F8. 
     
     
         13 . The method of  claim 1 , wherein the mRNA comprises an F8 coding sequence that is modified, compared with a native mRNA encoding F8, such that at least one codon of the native mRNA which codes for a tRNA which is relatively rare in the subject is exchanged for a codon which codes for a tRNA which is relatively frequent in the subject. 
     
     
         14 . The method of  claim 11 , wherein the mRNA comprises a chemical modification relative to a naturally occurring mRNA. 
     
     
         15 . The method of  claim 11 , 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-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. 
     
     
         16 . The method of  claim 11 , 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, puromycin-5′-triphosphate, and xanthosine-5′-triphosphate. 
     
     
         17 . The method of  claim 16 , wherein the nucleotide analog is chosen from the group consisting of: 5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate. 
     
     
         18 . The method of  claim 1 , wherein the liposomal system comprises a cationic lipid. 
     
     
         19 . The method of  claim 18 , wherein the mRNA is provided in complex with the cationic lipid. 
     
     
         20 . A method of inducing or enhancing an innate immune response in a subject in need thereof, comprising administering to the subject a complexed ribonucleic acid (RNA) comprising at least one RNA molecule complexed with one or more oligopeptides, wherein the at least one RNA molecule is not covalently bound to the one or more oligopeptides, wherein the nitrogen/phosphate ratio (N/P-ratio) of the RNA to the one or more oligopeptides is in the range of 0.75-25, and wherein the oligopeptide is 8 to 15 amino acids in length and comprises the following 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 is 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 is any amino acid selected from native or non-native amino acids except Arg, Lys, His or Orn; and   x is 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.   
     
     
         21 . A method of treating a subject having an ornithine transcarbamylase deficiency comprising administering an effective amount of a pharmaceutical composition comprising mRNA encoding human ornithine transcarbamylase (OTC) to the subject, wherein the mRNA comprises a 5′ cap structure; wherein the mRNA comprises a poly-A tail; wherein the mRNA is comprised in a liposomal system.