Terminally modified RNA
The invention relates to compositions and methods for the manufacture and optimization of modified mRNA molecules via optimization of their terminal architecture.
1. A mRNA comprising
(a) a 5′ untranslated region (5′UTR);
(b) a region of linked nucleosides encoding a polypeptide of interest;
(c) a 3′ untranslated region (3′ UTR) comprising at least one microRNA binding site; and
(d) a 3′ tailing region of linked nucleosides,
wherein each uridine in the mRNA is a modified uridine nucleoside.
2. The mRNA of claim 1 , wherein the modified uridine nucleoside is a pseudouridine analog.
3. The mRNA of claim 2 , wherein the pseudouridine analog is a 1-methyl pseudouridine.
4. The mRNA of claim 3 , wherein each cytidine in the mRNA is 5-methyl cytidine.
5. The mRNA of claim 1 , wherein the 5′UTR comprises a translation initiation sequence selected from the group consisting of Kozak sequence and an internal ribosome entry site (IRES).
6. The synthetic isolated terminally optimized mRNA of claim 1 , comprising at least one 5′ cap structure.
7. The mRNA of claim 6 , wherein the at least one 5′ cap structure is selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, and CAP-003-CAP-225.
8. The mRNA of claim 1 , wherein the at least one microRNA binding site is for an immune cell specific microRNA.
9. The mRNA of claim 8 , wherein the immune cell specific microRNA is selected from the group consisting of miR-142-3p, miR-142-5p, miR-146a and miR-146b.
10. The mRNA of claim 1 , wherein the 3′ tailing region of linked nucleosides further comprises a chain terminating nucleoside.
11. The mRNA of claim 10 , wherein the chain terminating nucleoside is selected from the group consisting of 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine, 2′,3′-dideoxynucleosides, 2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, 2′,3′-dideoxythymine, a 2′-deoxynucleoside, and —O— methylnucleoside.
12. The mRNA of claim 1 , wherein the 3′ tailing region comprises a stem loop sequence.
13. The mRNA of claim 1 , wherein the modified uridine nucleoside is 1-methyl pseudouridine, and wherein each cytidine in the mRNA is 5-methyl cytidine.
14. The mRNA of claim 1 , wherein the 3′ tailing region of linked nucleosides comprises a poly A tail of at least 100, at least 120 or at least 140 nucleosides.
15. The mRNA of claim 1 , wherein the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody or fusion protein.