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 of expressing a polypeptide in a subject comprising administering an effective amount of a pharmaceutical composition comprising a mRNA encoding the polypeptide to the subject, wherein the mRNA encoding polypeptide comprises at least one nucleotide of the mRNA that is substituted with an analog of a naturally occurring nucleotide, wherein the polypeptide encoded by the mRNA is a therapeutic polypeptide, which is not an infectious disease antigen or a tumor antigen, and wherein the mRNA that encodes the polypeptide comprises a coding sequence encoding the polypeptide with an increased G/C content of at least 7 percentage points relative to the coding sequence of an original RNA sequence encoding the polypeptide.
2. The method of claim 1 , wherein the mRNA encoding the polypeptide comprises at least one nucleotide position replaced with a nucleotide analogue selected from the group consisting of phosphorus amidates, phosphorus thioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.
3. The method of claim 1 , wherein the pharmaceutical composition is administered by injection.
4. The method of claim 1 , wherein the pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, topically or orally.
5. The method of claim 1 , wherein the mRNA encoding the polypeptide comprises a stabilizing 5′ untranslated region (UTR) or 3′ UTR.
6. The method of claim 1 , wherein the mRNA comprises a 5′ cap structure.
7. The method of claim 1 , wherein the mRNA comprises a poly-A tail of at least 50 nucleotides.
8. The method of claim 1 , wherein the therapeutic polypeptide comprises erythropoietin (EPO).
9. The method of claim 1 , wherein the mRNA is dissolved in an aqueous carrier.
10. The method of claim 9 , wherein the aqueous carrier is water for injection (WFI), a buffered solution or a salt solution.
11. The method of claim 10 , wherein the salt solution comprises sodium chloride or potassium chloride solution.
12. 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.
13. The method of claim 12 , wherein the pharmaceutical composition comprises a polycationic protein.
14. The method of claim 1 , wherein the mRNA is provided in a liposome complex.
15. The method of claim 1 , further comprising administering the pharmaceutical composition to the subject two or more times.
16. The method of claim 1 , wherein the composition is administered by injection and wherein the mRNA is provided in a liposome complex.
17. The method of claim 1 , wherein the subject has a disease.
18. The method of claim 1 , wherein the mRNA that encodes the polypeptide comprises a coding sequence encoding the polypeptide with an increased G/C content of at least 15 percentage points relative to the coding sequence of an original RNA sequence encoding the polypeptide.
19. The method of claim 18 , wherein the mRNA that encodes the polypeptide comprises a coding sequence encoding the polypeptide with an increased G/C content of at least 20 percentage points relative to the coding sequence of an original RNA sequence encoding the polypeptide.