Complexes of RNA and cationic peptides for transfection and for immunostimulation
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 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.
2. The method according to claim 1 , wherein the at least one RNA molecule is a messenger RNA (mRNA).
3. The method according to claim 1 , wherein the oligopeptide is 8 to 14 amino acids in length.
4. The method according to claim 1 , wherein the overall content of Arg, Lys, His and Orn represents at least 60% of all amino acids of the oligopeptide of the complexed RNA.
5. The method according to claim 1 , wherein Xaa in the formula (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x is selected from amino acids having a neutral side chain and amino acids having a neutral polar side chain.
6. The method according to claim 1 , wherein the oligopeptide according to the formula (Arg) l ; (Lys) m ; (His) n ;(Orn) o ;(Xaa) x does not comprise amino acids having an acidic side chain at one or both terminal ends.
7. The method according to claim 1 , wherein the oligopeptide according to the formula (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x comprises neutral or basic amino acids at one or both terminal ends.
8. The method according to claim 1 , wherein the oligopeptide according to the formula (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x comprises a stretch of at least 3 contiguous basic amino acids within its sequence.
9. The method according to claim 1 , wherein the oligopeptide according to the formula (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x comprises at least 1 non-cationic amino acid(s) at one or both terminal ends.
10. The method according to claim 2 , wherein the mRNA codes for a therapeutically active protein or peptide, an immunostimulating protein or peptide, a tumor antigen, or an antibody.
11. The method according to claim 10 , wherein the mRNA encodes an immunostimulating protein.
12. The method according to claim 1 , wherein the RNA is a modified RNA with increased stability, compared to the RNA without modification.
13. The method according to claim 12 , wherein the G/C content of the coding region of the modified RNA is increased compared with the G/C content of the coding region of the RNA without modification.
14. The method according to claim 1 , wherein the mass ratio of the at least one RNA molecule of the complexed RNA to the one or more oligopeptides is in a range of 1:100 to 1:0.5.
15. The method according to claim 1 , wherein the molar ratio of the at least one RNA molecule of the complexed RNA to the one or more oligopeptides is in a range of 1:20000 to 1:250.
16. The method according to claim 1 , wherein the oligopeptide comprises an oligopeptide selected from the group consisting of:
Arg 9 His 3 (SEQ ID NO: 39), His 3 Arg 9 His 3 (SEQ ID NO: 40), TyrSerSerArg 9 SerSerTyr (SEQ ID NO: 41), His 3 Arg 9 SerSerTyr (SEQ ID NO: 42), (ArgLysHis) 4 (SEQ ID NO: 43) Tyr(ArgLysHis) 2 Arg (SEQ ID NO: 44);
Arg 8 (SEQ ID NO: 1), Arg 9 (SEQ ID NO: 2), Arg 10 (SEQ ID NO: 3), Arg 11 (SEQ ID NO: 4), Arg 12 (SEQ ID NO: 5), Arg 13 (SEQ ID NO: 6), Arg 14 (SEQ ID NO: 7), Arg 15 (SEQ ID NO: 8);
Lys 8 (SEQ ID NO: 9), Lys 9 (SEQ ID NO: 10), Lys 10 (SEQ ID NO: 11), Lys 11 (SEQ ID NO: 12), Lys 12 (SEQ ID NO: 13), Lys 13 (SEQ ID NO: 14), Lys 14 (SEQ ID NO: 15), Lys 15 (SEQ ID NO: 16);
His 8 (SEQ ID NO: 17), His 9 (SEQ ID NO: 18), His 10 (SEQ ID NO: 19), His 11 (SEQ ID NO: 20), His 12 (SEQ ID NO: 21), His 13 (SEQ ID NO: 22), His 14 (SEQ ID NO: 23), His 15 (SEQ ID NO: 24); and
Orn 8 (SEQ ID NO: 25), Orn 9 (SEQ ID NO: 26), Orn 10 (SEQ ID NO: 27), Orn 11 (SEQ ID NO: 28), Orn 12 (SEQ ID NO: 29), Orn 13 (SEQ ID NO: 30), Orn 14 (SEQ ID NO: 31), Orn 15 (SEQ ID NO: 32).
17. The method according to claim 1 , wherein the subject is a mammal selected from the group consisting of humans, goat, cattle, swine, dog, cat, donkey, monkey, ape and rodents.
18. The method according to claim 1 , wherein the subject has a disease selected from a tumour or cancer disease, a cardiovascular disease, an infectious disease, an autoimmune disease, a genetic disease, or an allergy.
19. The method according to claim 1 , wherein the complexed RNA further induces or enhances an immune response to an antigen.
20. The method according to claim 19 , wherein the antigen is a tumour antigen, an autoimmune antigen, an allergy antigen, or an infectious disease antigen.
21. The method according to claim 1 , wherein the at least one RNA is a coding RNA.
22. The method according to claim 1 , wherein the at least one RNA is single stranded.
23. The method according to claim 1 , wherein the at least one RNA is double stranded.
24. The method according to claim 1 , wherein the at least one RNA is an immunostimulatory RNA (isRNA).
25. The method according to claim 1 , wherein the innate immune response comprises stimulation of at least one of toll-like receptor (TLR) 3, TLR7, TLR8, RIG-1, and Melanoma Differentiation-Associated protein 5 (MDA-5).