RSV RNA molecules and compositions for vaccination
The present invention is directed to an artificial nucleic acid, particularly to an artificial RNA suitable for use in treatment and/or prophylaxis of an infection with Respiratory syncytial virus (RSV) or a disorder related to such an infection. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial RNA, compositions, and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial RNA, compositions and vaccines.
1. A purified mRNA molecule, said mRNA molecule comprising:
a) a 5′-cap structure comprising m7G;
b) a heterologous 5′ untranslated region (UTR);
c) at least one coding sequence encoding an antigenic RSV F protein having an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 4957, wherein said at least one coding sequence is at least 90% identical to the RNA sequence of SEQ ID NO: 4958; and
d) at least one poly(A) sequence comprising 30 to 150 consecutive adenosine nucleotides.
2. The purified mRNA molecule of claim 1 , further comprising a heterologous 3′ UTR.
3. The purified mRNA molecule of claim 1 , wherein the 5′-cap structure is a Cap1 or a modified Cap1.
4. The purified mRNA molecule of claim 2 , wherein the antigenic RSV F protein has an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 4957.
5. The purified mRNA molecule of claim 4 , wherein the antigenic RSV F protein has an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4957.
6. The purified mRNA molecule of claim 4 , wherein the mRNA molecule comprises at least one chemically modified nucleotide.
7. The purified mRNA molecule of claim 4 , wherein all uracil nucleotides of the mRNA molecule are replaced by N1-methylpseudouridine (m1ψ) nucleotides.
8. The purified mRNA molecule of claim 1 , wherein the at least one poly(A) sequence is located at the 3′ terminus of the mRNA molecule.
9. The purified mRNA molecule of claim 7 , wherein said at least one coding sequence is adapted to human codon usage with most frequently used codons such that the at least one coding sequence has a codon adaptation index (CAI) of at least 0.9.
10. The purified mRNA molecule of claim 9 , wherein said at least one poly(A) sequence is about 100 adenosine nucleotides.
11. The purified mRNA molecule of claim 5 , wherein the amino acid sequence of the antigenic RSV F protein comprises the following amino acid substitutions relative to the amino acid sequence of a native RSV F protein (SEQ ID NO: 68): S155C, S290C, S190F, V207L, A149C and Y458C.
12. The purified mRNA molecule of claim 11 , wherein the amino acid sequence of the antigenic RSV F protein comprises a GS linker in place of amino acids 104-144 of the amino acid sequence of the native RSV F protein (SEQ ID NO: 68).
13. The purified mRNA molecule of claim 12 , wherein the amino acid sequence of the antigenic RSV F protein lacks amino acids 554-574 of the amino acid sequence of the native RSV F protein (SEQ ID NO: 68).
14. The purified mRNA molecule of claim 13 , wherein the antigenic RSV F protein comprises the native RSV F protein transmembrane domain.
15. A pharmaceutical composition comprising purified mRNA molecules of claim 12 formulated in a pharmaceutically acceptable carrier.
16. The pharmaceutical composition of claim 15 , wherein the mRNA molecules are formulated with lipid nanoparticles (LNPs).
17. The pharmaceutical composition of claim 16 , wherein the LNPs comprise a cationic lipid.
18. The pharmaceutical composition of claim 17 , wherein the LNPs comprise a cationic lipid, a neutral lipid, a sterol, and a polymer conjugated lipid.
19. The pharmaceutical composition of claim 18 , wherein the LNPs comprise a cationic lipid, a neutral lipid, a sterol, and a PEG-lipid.
20. The pharmaceutical composition of claim 19 , wherein the LNPs comprise a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.
21. The pharmaceutical composition of claim 20 , wherein the neutral lipid is distearoylphosphatidylcholine (DSPC), the sterol is cholesterol and the PEG-lipid is 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG).
22. The pharmaceutical composition of claim 20 , wherein all uracil nucleotides of the mRNA molecules are replaced by N1-methylpseudouridine (mlv) nucleotides.
23. The pharmaceutical composition of claim 20 , wherein the antigenic RSV F protein comprises the native RSV F protein transmembrane domain.
24. The pharmaceutical composition of claim 20 , wherein the 5′-cap structure is a Cap1 or a modified Cap1.
25. A method of stimulating an anti-RSV immune response in a subject comprising administering to the subject the pharmaceutical composition of claim 20 , wherein the pharmaceutical composition is administered by intramuscular injection.
26. The method of claim 25 , wherein stimulating an anti-RSV immune response comprises stimulating production of RSV neutralizing antibodies in the subject.
27. The method of claim 25 , wherein stimulating an anti-RSV immune response comprises stimulating a RSV-specific CD8+ T-cell response in the subject.
28. The method of claim 26 , wherein all uracil nucleotides of the mRNA molecules are replaced by N1-methylpseudouridine (m1ψ) nucleotides.
29. A method of preventing nasal RSV production in a subject comprising administering to the subject the pharmaceutical composition of claim 20 , wherein the administration is by intramuscular injection.
30. The method of claim 29 , wherein the 5′-cap structure of the purified mRNA molecule is a Cap1 or a modified Cap1.
31. The pharmaceutical composition of claim 24 , wherein the amino acid sequence of the antigenic RSV F protein lacks amino acids 554-574 of the amino acid sequence of the native RSV F protein (SEQ ID NO: 68).
32. The pharmaceutical composition of claim 31 , wherein the antigenic RSV F protein comprises the native RSV F protein transmembrane domain.
33. The pharmaceutical composition of claim 32 , wherein the at least one coding sequence does not include a fibritin foldon domain.
34. The pharmaceutical composition of claim 33 , wherein, when expressed in a cell, the antigenic RSV F protein is expressed on the cell surface.
35. The method of claim 25 , wherein the neutral lipid is distearoylphosphatidylcholine (DSPC), the sterol is cholesterol, and the PEG-lipid is 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG).
36. The method of claim 35 , wherein all uracil nucleotides of the mRNA molecules are replaced by N1-methylpseudouridine (m1ψ) nucleotides.
37. The method of claim 36 , wherein the antigenic RSV F protein comprises the native RSV F protein transmembrane domain.
38. The method of claim 37 , wherein the 5′-cap structure is a Cap1 or a modified Cap1.
39. The method of claim 38 , wherein stimulating an anti-RSV immune response comprises stimulating production of RSV neutralizing antibodies in the subject.
40. The method of claim 39 , wherein stimulating an anti-RSV immune response comprises stimulating a RSV-specific CD8+ T-cell response in the subject.
41. The method of claim 39 , wherein the at least one coding sequence does not include a fibritin foldon domain.
42. The method of claim 41 , wherein, when expressed in a cell, the antigenic RSV F protein is expressed on the cell surface.