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 producing a stabilized mRNA comprising synthesizing a stabilized mRNA molecule encoding a polypeptide, wherein the stabilized mRNA molecule encoding the polypeptide comprises a coding sequence that has an increased Guanine/Cytosine (G/C) content relative to the original coding sequence encoding the polypeptide, said relative G/C content being increased by at least 7 percentage points compared to the original coding sequence encoding the polypeptide, to thereby produce a stabilized mRNA molecule, wherein said increase in relative G/C content results in the elimination of at least one destabilizing sequence element (DSE), wherein the stabilized mRNA molecule exhibits enhanced expression of the polypeptide compared to mRNA having the original coding sequence encoding the polypeptide.
2. The method of claim 1 , wherein the sequence encoding the polypeptide has a G/C content increased by at least 15 percentage points compared to the original nucleic acid sequence encoding the polypeptide.
3. The method of claim 2 , wherein the sequence encoding the polypeptide has a G/C content increased by at least 20 percentage points compared to the original nucleic acid sequence encoding the polypeptide.
4. The method of claim 1 , wherein the sequence encoding the polypeptide has a G/C content increased sufficiently to reduce the susceptibility of the mRNA to exonuclease digestion compared to the original nucleic acid sequence encoding the polypeptide.
5. The method of claim 1 , wherein the stabilized mRNA comprises a nucleic acid sequence encoding the polypeptide that has at least one codon recognized by a rare cellular tRNA replaced with a codon recognized by an abundant tRNA relative to the original nucleic acid sequence encoding the polypeptide.
6. The method of claim 1 , wherein the stabilized mRNA comprises a 5′ cap.
7. The method of claim 1 , wherein synthesizing the stabilized mRNA comprises producing a DNA molecule encoding the stabilized mRNA.
8. The method of claim 7 , wherein synthesizing the stabilized mRNA further comprises transcribing the stabilized mRNA from the DNA molecule.
9. The method of claim 8 , wherein the transcription is in vitro transcription.
10. The method of claim 1 , wherein the polypeptide sequence is the polypeptide sequence of a virus, bacterium, protozoan or tumour antigen.
11. The method of claim 1 , wherein the polypeptide sequence is the polypeptide sequence of a biologically active polypeptide.
12. The method according to claim 1 , wherein synthesizing a stabilized mRNA comprises using a computer to determine the nucleic acid sequence encoding the polypeptide that has an increased Guanine/Cytosine (G/C) content and producing a DNA molecule encoding the stabilized mRNA.
13. The method of claim 12 , wherein using the computer comprises using a software program comprising a source code of Appendix I.
14. The method of claim 1 , further comprising the formulating the stabilized mRNA into a pharmaceutically acceptable carrier.
15. The method of claim 1 , further comprising synthesizing a stabilized mRNA comprising at least one nucleotide position replaced with a nucleotide analogue.
16. The method of claim 15 , wherein the nucleotide analogue is selected from the group consisting of phosphorus amidates, phosphorus thioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.