Modified RNA with decreased immunostimulatory properties
The present invention provides a method for providing modified mRNAs of reduced immunogenicity and/or immunostimulatory capacity for use in protein replacement therapy. The invention further provides modified mRNAs and pharmaceutical compositions comprising the modified mRNAs according to the invention for use in protein replacement therapy.
1. A method for synthesis of a modified mRNA that provides decreased immunogenicity or immune stimulating activity comprising the steps of:
(a) identifying a target mRNA wild type sequence coding for a biologically active polypeptide or protein;
(b) modifying at least 70% of the codons of the wild type sequence which are cytosine-content optimizable by replacing at least 70% of said cytosine-content optimizable codons of the wild type sequence with a codon coding for the same amino add but having a higher cytosine-content than the cytosine-content optimizable codon being replaced, thereby increasing the cytosine-content of the mRNA such that the cytosine-content of the coding region of the modified mRNA is greater than the cytosine-content of the coding region of the wild type sequence, whereby the amino add sequence encoded by the modified mRNA is unchanged compared to the wild type sequence, and wherein the codons replacing the cytosine-content optimizable codons do not have a higher G-content than the wild type sequence; and
(c) synthesizing the modified mRNA.
2. The method according to claim 1 , wherein the cytosine-content of the coding region of the modified mRNA is at least 10% greater than the cytosine-content of the coding region of the wild type sequence.
3. The method according to claim 2 , wherein the cytosine-content of the coding region of the modified mRNA is at least 12.5% greater than the cytosine-content of the coding region of the wild type mRNA.
4. The method according to claim 3 , wherein the cytosine-content of the coding region of the modified mRNA is at least 15% greater than the cytosine-content of the coding region of the wild type mRNA.
5. The method according to claim 1 , wherein all codons of the wild type sequence that are not cytosine-content optimizable and that code for a rare tRNA codons in the cell are replaced by codons that code for a more frequent tRNA in the cell, which carries the same amino acid as the rare tRNA.
6. The method according to claim 1 , wherein the codon adaptation index (CAI) of the coding region of the modified mRNA is at least 0.05 greater than the CAI of the coding region of the wild type mRNA coding for the polypeptide or protein.
7. The method according to claim 6 , wherein the codon adaptation index (CAI) of the coding region of the modified mRNA is at least 0.1 greater than the CAI of the coding region of the wild type mRNA coding for the polypeptide or protein.
8. The method according to claim 7 , wherein the codon adaptation index (CAI) of the coding region of the modified mRNA is at least 0.15 greater than the CAI of the coding region of the wild type mRNA coding for the polypeptide or protein.
9. The method according to claim 1 , wherein the method further comprises the step of determining the immunogenicity and/or immunostimulatory capacity of the modified mRNA.
10. The method according to claim 9 , wherein determining the immunogenicity and/or immunostimulatory capacity of the modified mRNA comprises the sub-steps of:
(i) transfecting peripheral blood mononuclear cells (PBMCs) with the modified mRNA,
(ii) cultivating the cells for at least 8 hours,
(iii) measuring the amount of pro-inflammatory cytokines in a cell supernatant from step (ii).
11. The method according to claim 10 , wherein the modified mRNA coding for the polypeptide or protein that has lower immunogenicity and/or immunostimulatory capacity than the wild type mRNA is selected and steps (a), (b) and (c) are repeated to further decrease the immunogenicity and/or immunostimulatory capacity of the modified mRNA.
12. The method according to claim 10 , wherein the cultivating of step (ii) lasts for at least 12 hours.
13. The method according to claim 12 , wherein the cultivating of step (ii) lasts for at least 20 hours.
14. The method according to claim 1 , wherein the method is carried out by executing at least one algorithm on a computer with the aid of software.
15. The method according to claim 1 , wherein the modified mRNA is obtained by in vitro transcription.
16. The method according to claim 15 , wherein the in vitro transcription is bacteriophage polymerase-mediated in vitro transcription, Sp6 polymerase in vitro transcription, T3 polymerase-mediated in vitro transcription, or T7 polymerase-mediated in vitro transcription.
17. The method according to claim 1 , wherein step (b) further comprises modifying at least 70% of codons for amino acids not eligible for cytosine-content optimization, but eligible for guanosine-content optimization, thereby increasing the guanosine-content of the modified mRNA.