Method for increasing expression of RNA-encoded proteins
The invention relates to an RNA comprising at least one open reading frame (ORF) and comprising at least one modification, which increases the expression of the encoded peptide or protein. Furthermore, the invention relates to the medical use of such a modified RNA administered to a subject by jet injection. The invention relates further to a pharmaceutical composition and to a kit of parts comprising said modified RNA for administration by jet injection, preferably for use in the field of gene therapy and/or genetic vaccination. Additionally, the invention relates to a method for enhancing the (localized) expression of RNA-encoded peptides or proteins in the dermis or muscle (of a mammal) comprising administering the modified RNA by jet injection. And finally, the invention relates to a method of treatment comprising administering the modified RNA by jet injection to a subject in need thereof.
1. A method for enhancing the localized expression of an RNA-encoded polypeptide in the dermis or muscle of a mammal comprising administering by jet injection a modified RNA comprising an increased G/C content of a polypeptide coding region of the modified RNA as compared with the G/C content of the corresponding polypeptide coding region of the native RNA, wherein the polypeptide sequence encoded by the modified RNA is not altered compared with the polypeptide sequence encoded by the native RNA, the increased G/C content resulting in increased expression of the RNA-encoded polypeptide, wherein the RNA comprises at least one histone stem-loop.
2. The method of claim 1 , wherein the RNA is an mRNA and comprises a 5′ cap.
3. The method of claim 1 , the RNA further comprising at least one further modification selected from the group consisting of codon optimization, UTR modification, Poly(A) tail with more than 30 adenosine nucleotides, Poly(C) sequence, 5′-CAP structure except of m7GpppN, histone-stem-loop sequence, a base modification and a chemically modified nucleotide.
4. The method of claim 3 , wherein the at least one modification is a chemically modified ARCA-CAP.
5. The method of claim 4 , wherein the ARCA-CAP is modified with phosphorothioate.
6. The method of claim 1 , wherein the histone stem-loop comprises a nucleic acid sequence according to SEQ ID NO. 44.
7. The method according to claim 2 , wherein the mRNA sequence further comprises a stabilizing sequence from the alpha globin 3′ UTR, positioned 3′ relative to the coding region of the mRNA sequence.
8. The method of claim 2 , wherein the mRNA sequence comprises from a 5′ to 3′: a 5′-cap structure, a 5′ UTR sequence, the sequence encoding the polypeptide, a 3′ UTR, a poly(A) sequence, a poly(C) sequence and a histone stem-loop sequence.
9. The method of claim 1 , wherein the RNA is complexed with a cationic or polycationic compound.
10. The method of claim 9 , wherein the cationic or polycationic compound is protamine, poly-L-lysine (PLL), or poly-arginine.
11. The method of claim 3 , wherein the at least one modification comprises a base modification selected from the group consisting of 2-amino-6-chloropurineriboside-5′-triphosphate, 2-aminopurine-riboside-5′-triphosphate; 2-aminoadenosine-5′-triphosphate, 2′-amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 2′-fluorothymidine-5′-triphosphate, 2′-O-methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 5-bromo-2′-deoxycytidine-5′-triphosphate, 5-bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-iodo-2′-deoxyuridine-5′riphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 5-propynyl-2′-deoxycytidine-5′-triphosphate, 5-propynyl-2′-deoxyuridine-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, and puromycin-5′-triphosphate, xanthosine-5′-triphosphate.
12. The method of claim 1 , wherein the RNA is administered intradermally.
13. The method of claim 1 , wherein the RNA is administered intramuscularly.
14. The method of claim 1 , wherein the RNA is administered subcutaneously.
15. The method of claim 1 , wherein RNA-encoded polypeptide comprises a polypeptide antigen.
16. The method of claim 15 , wherein the polypeptide antigen is from Influenza virus, respiratory syncytial virus (RSV), Herpes simplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus , Dengue virus, Chlamydia trachomatis , Cytomegalovirus (CMV), Hepatitis B virus (HBV), Mycobacterium tuberculosis , Rabies virus or Yellow Fever Virus.
17. The method of claim 15 , further defined as a method of stimulating an immune response to the polypeptide antigen in the mammal.
18. The method of claim 1 , wherein the mammal is a human subject.
19. The method of claim 9 , wherein the RNA is complexed with a cationic or polycationic lipid.