Pharmaceutical composition containing a stabilised mRNA optimised for translation in its coding regions
The present invention relates to a pharmaceutical composition comprising a modified mRNA that is stabilised by sequence modifications and optimised for translation. The pharmaceutical composition according to the invention is particularly well suited for use as an inoculating agent, as well as a therapeutic agent for tissue regeneration. In addition, a process is described for determining sequence modifications that promote stabilisation and translational efficiency of modified mRNA of the invention.
1. A method for stimulating an immune response to an ebola virus antigen in a subject comprising administering an effective amount of a pharmaceutical composition comprising a modified mRNA that encodes an ebola virus glycoprotein to the subject, wherein said mRNA lacks any promoter sequences, and wherein the mRNA comprises an increased G/C content of at least 7% percentage points relative to a wild-type mRNA encoding the ebola virus glycoprotein.
2. The method of claim 1 , wherein the pharmaceutical composition is administered by injection.
3. The method of claim 1 , wherein the pharmaceutical composition is administered intravenously, intradermally, subcutaneously, intramuscularly, topically or orally.
4. The method of claim 3 , wherein the pharmaceutical composition is administered intradermally or intramuscularly.
5. The method of claim 1 , wherein the mRNA encoding the ebola virus glycoprotein comprises a sequence wherein at least one codon of a wild-type mRNA recognized by a rare cellular tRNA is replaced with a codon recognized by an abundant cellular tRNA, and wherein said rare cellular tRNA and said abundant cellular tRNA recognize the same amino acid.
6. The method of claim 1 , wherein the mRNA encoding the ebola virus glycoprotein comprises a stabilizing 5′ untranslated region (UTR) or 3′ UTR.
7. The method of claim 1 , wherein the mRNA comprises a 5′ cap structure and/or a poly-A tail of at least 50 nucleotides.
8. The method of claim 1 , wherein the mRNA encoding the ebola glycoprotein comprises at least one chemical modification of the mRNA.
9. The method of claim 1 , wherein the mRNA encoding the ebola virus glycoprotein comprises at least one nucleotide of the wild-type mRNA substituted with an analog of the naturally occurring nucleotide.
10. The method of claim 1 , wherein the mRNA encoding the ebola virus glycoprotein comprises at least one nucleotide position of the wild-type mRNA replaced with a nucleotide analogue selected from the group consisting of phosphorus amidates, phosphorus thioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.
11. The method of claim 1 , wherein the mRNA further encodes a secretion signal.
12. The method of claim 1 , wherein the mRNA is dissolved in the aqueous carrier.
13. The method of claim 12 , wherein the aqueous carrier is water for injection (WFI), a buffered solution or a salt solution.
14. The method of claim 13 , wherein the salt solution comprises sodium chloride or potassium chloride solution.
15. The method of claim 1 , wherein the pharmaceutical composition comprises a component selected from the group consisting of human serum albumin, a polycationic protein, polysorbate 80, a sugar and an amino acid.
16. The method of claim 15 , wherein the pharmaceutical composition comprises a polycationic protein.
17. The method of claim 16 , wherein the polycationic protein comprises protamine.
18. The method of claim 17 , wherein the mRNA is in complex with protamine.
19. The method of claim 1 , wherein the mRNA is provided in a liposome complex.
20. The method of claim 1 , wherein the pharmaceutical composition further comprises an adjuvant.
21. The method of claim 1 , further comprising administering the pharmaceutical composition to the subject two or more times.
22. The method of claim 1 , further comprising administering a cytokine to the subject.
23. The method of claim 1 , wherein the composition is administered intradermally and wherein the mRNA is provided in complex with protamine and encodes an ebola virus glycoprotein.