Methods of immunostimulation with complexes of rna and cationic carriers

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 stimulating an immune response in a subject comprising administering an effective amount of a pharmaceutical composition comprising a purified mRNA encoding a coronavirus antigen to the subject, wherein said mRNA is complexed or formulated with a cationic carrier molecule. 
     
     
         2 . The method of  claim 1 , wherein the coronavirus antigen is from a SARS-associated coronavirus. 
     
     
         3 . The method of  claim 1 , wherein the coronavirus antigen is from human coronavirus 229E or human coronavirus Oc43. 
     
     
         4 . The method of  claim 1 , further defined as a method for preventing disease from an infection with a SARS-associated coronavirus. 
     
     
         5 . The method of  claim 1 , wherein the administering is by injection. 
     
     
         6 . The method of  claim 5 , wherein the administering is by intramuscular injection. 
     
     
         7 . The method of  claim 1 , wherein the cationic carrier comprises a cationic polymer or cationic polypeptide. 
     
     
         8 . The method of  claim 1 , wherein the cationic carrier comprises a cationic lipid. 
     
     
         9 . The method of  claim 1 , wherein the mRNA is complexed with a cationic carrier and forms nanoplexes, lipoplexes or polyplexes. 
     
     
         10 . The method of  claim 1 , wherein the mRNA is complexed with a cationic carrier and forms lipoplexes. 
     
     
         11 . The method of  claim 1 , wherein the mRNA comprises a 5′ cap structure. 
     
     
         12 . The method of  claim 1 , wherein the mRNA comprises a poly-A tail of 10 to 200 adenosine nucleotides. 
     
     
         13 . The method of  claim 1 , wherein the mRNA comprises a poly-C tail of 10 to 200 cytosine nucleotides. 
     
     
         14 . The method of  claim 1 , wherein the mRNA comprises a 5′ and/or a 3′ untranslated region (UTR). 
     
     
         15 . The method of  claim 1 , wherein the mRNA comprises a G/C content in the antigen coding sequence which is greater than the G/C content of a coding region of the native mRNA sequence encoding the antigen. 
     
     
         16 . The method of  claim 1 , wherein the mRNA comprises an antigen coding sequence that is modified, compared with a native RNA encoding the antigen, 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. 
     
     
         17 . 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. 
     
     
         18 . The method of  claim 17 , wherein the mRNA comprises a chemical modification relative to a naturally occurring mRNA. 
     
     
         19 . The method of  claim 17 , 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, 06-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, xanthosine-5′-triphosphate. 
     
     
         20 . The method of  claim 1 , wherein the mRNA has been purified by chromatography. 
     
     
         21 . The method of  claim 20 , wherein the mRNA has been purified by HPLC. 
     
     
         22 . The method of  claim 21 , wherein the mRNA has been purified by reverse phase (RP) HPLC. 
     
     
         23 . The method of  claim 1 , wherein the composition comprises polyethylene glycol (PEG). 
     
     
         24 . The method of  claim 1 , further defined as a method of stimulating antibody production in the subject. 
     
     
         25 . The method of  claim 1 , further defined as a method for stimulating a CD4+ and/or a CD8+ T-cell response.