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 lipid nanoparticle comprising an mRNA, wherein the mRNA comprises
(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;
and wherein each uridine in the mRNA is a modified uridine nucleoside.
2. The lipid nanoparticle of claim 1 , wherein each uridine in the mRNA is a pseudouridine analog.
3. The lipid nanoparticle of claim 2 , wherein the pseudouridine analog is 1-methyl pseudouridine.
4. The lipid nanoparticle of claim 3 , wherein each cytidine in the mRNA is a 5-methyl cytidine.
5. The lipid nanoparticle of claim 3 , wherein the microRNA binding site is for an immune cell specific microRNA.
6. The lipid nanoparticle of claim 5 , wherein the microRNA binding site is for a microRNA selected from miR-142-3p, miR-142-5p, miR-146a and miR-146b.
7. The lipid nanoparticle of claim 6 , wherein the microRNA binding site is miR-142-3p.
8. The lipid nanoparticle of claim 7 , comprising a cationic or ionizable lipid.
9. The lipid nanoparticle of claim 8 , wherein the cationic lipid is DLin-MC3-DMA, DLin-DMA, C12-200, or DLin-KC2-DMA.
10. The lipid nanoparticle of claim 8 , comprising a PEG lipid.
11. The lipid nanoparticle of claim 10 , comprising 1-5% PEG lipid.
12. The lipid nanoparticle of claim 1 , wherein the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody or a fusion protein.
13. The lipid nanoparticle of claim 12 , wherein the region encoding the polypeptide of interest is codon optimized.
14. The lipid nanoparticle of claim 1 , wherein the mRNA further comprises a 5′ cap structure.
15. The lipid nanoparticle of claim 14 , wherein the 5′ cap structure is Cap 1.
16. The lipid nanoparticle of claim 1 , wherein the 5′ UTR comprises a translation initiation sequence selected from a group consisting of a Kozak sequence and an internal ribosome entry site (IRES).
17. The lipid nanoparticle of claim 1 , wherein the 3′ tailing region comprises a poly A tail of at least 100 nucleosides.
18. The lipid nanoparticle of claim 1 , wherein the microRNA binding site is for a microRNA selected from miR-122, miR-133, miR-206, miR-208, miR-17-92, miR-126, miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27, let-7, miR-30c, miR-1d, miR-149, miR-192, miR-194, and miR-204.
19. The lipid nanoparticle of claim 3 , wherein the microRNA binding site is for a microRNA selected from miR-122, miR-133, miR-206, miR-208, miR-17-92, miR-126, miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27, let-7, miR-30c, miR-1d, miR-149, miR-192, miR-194, and miR-204.
20. The lipid nanoparticle of claim 1 , wherein the microRNA binding site is for miR-142-3p.
21. A lipid nanoparticle comprising an mRNA, wherein the mRNA comprises
(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 miR-142-3p binding site; and
(d) a 3′ tailing region of linked nucleosides;
and wherein each uridine in the mRNA is a modified uridine nucleoside.
22. The lipid nanoparticle of claim 21 , comprising a cationic or ionizable lipid.
23. The lipid nanoparticle of claim 22 , comprising a PEG lipid.
24. The lipid nanoparticle of claim 23 , comprising 1-5% PEG lipid.
25. The lipid nanoparticle of claim 21 , wherein the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody or a fusion protein.
26. A lipid nanoparticle comprising an mRNA, wherein the mRNA comprises
(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 miR-126-3p binding site; and
(d) a 3′ tailing region of linked nucleosides;
and wherein each uridine in the mRNA is a modified uridine nucleoside.
27. The lipid nanoparticle of claim 26 , comprising a cationic or ionizable lipid.
28. The lipid nanoparticle of claim 27 , comprising a PEG lipid.
29. The lipid nanoparticle of claim 28 , comprising 1-5% PEG lipid.
30. The lipid nanoparticle of claim 26 , wherein the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody or a fusion protein.
31. A lipid nanoparticle comprising an mRNA, wherein the mRNA comprises
(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 miR-122 binding site; and
(d) a 3′ tailing region of linked nucleosides;
and wherein each uridine in the mRNA is a modified uridine nucleoside.
32. The lipid nanoparticle of claim 31 , comprising a cationic or ionizable lipid.
33. The lipid nanoparticle of claim 32 , comprising a PEG lipid.
34. The lipid nanoparticle of claim 33 , comprising 1-5% PEG lipid.
35. The lipid nanoparticle of claim 31 , wherein the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody or a fusion protein.
36. The lipid nanoparticle of claim 31 , wherein the miR-122 binding site is a miR-122-3p binding site.
37. The lipid nanoparticle of claim 31 , wherein the miR-122 binding site is a miR-122-5p binding site.