HETEROLOGOUS UNTRANSLATED REGIONS FOR MRNA
The invention relates to compositions and methods for the manufacture and optimization of modified mRNA molecules via optimization of their terminal architecture.
1 . A synthetic isolated RNA comprising:
(a) a first region of linked nucleosides encoding a polypeptide of interest;
(b) a first flanking region located at the 5′ terminus of said first region, wherein said first flanking region comprises a heterologous 5′UTR relative to the said first region of linked nucleosides encoding a polypeptide of interest, with the proviso that said heterologous 5′UTR is not derived from the beta-globin gene;
(c) a second flanking region located at the 3′ terminus of said first region; and
(d) a 3′ tailing region of linked nucleosides.
2 . The synthetic isolated RNA of claim 1 wherein any of the regions (a)-(d) comprise at least one modified nucleoside.
3 . The synthetic isolated RNA of claim 1 , wherein the first flanking region comprises a heterologous 5′ untranslated region (UTR) selected from the group consisting of 5′UTR-005-5′UTR 68524.
4 . The synthetic isolated RNA of claim 3 , wherein the first flanking region comprises at least one 5′ cap structure.
5 . The synthetic isolated RNA of claim 4 , 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 and Cap4.
6 . The synthetic isolated RNA of claim 3 , wherein the first flanking region comprises a translation initiation sequence selected from the group consisting of Kozak sequence and an internal ribosome entry site (IRES).
7 . The synthetic isolated RNA of claim 1 , wherein the second flanking region comprises a 3′ UTR.
8 . The synthetic isolated RNA of claim 7 , wherein the 3′UTR is the native 3′UTR of the encoded polypeptide of interest.
9 . The synthetic isolated RNA of claim 1 , wherein the second flanking region comprises at least one sensor region.
10 . The synthetic isolated RNA of claim 9 , wherein the at least one sensor region is at least one miR binding site selected from the group consisting of SEQ ID NOs: 1188-2208 and 3230-4250.
11 . The synthetic isolated RNA of claim 9 , wherein the at least one sensor region is at least one miR binding site and wherein the at least one miR binding site lacks a miR seed.
12 . The synthetic isolated RNA of claim 11 , wherein the at least one miR binding site is one which binds miR-122.
13 . The synthetic isolated terminally optimized RNA of claim 9 , wherein the second flanking region comprises four sensor regions.
14 . The synthetic isolated RNA of claim 1 , wherein the 3′ tailing region is selected from the group consisting of a PolyA tail, PolyA-G quartet and a triple helix.
15 . The synthetic isolated RNA of claim 14 , wherein the 3′ tailing region is a PolyA tail.
16 . The synthetic isolated RNA of claim 1 , wherein the first flanking region comprises a structured untranslated region.
17 . A method of producing a protein of interest comprising contacting a mammalian cell, tissue or organ with the synthetic isolated RNA of claim 1 .
18 . A pharmaceutical composition comprising the synthetic isolated RNA of claim 1 and a pharmaceutically acceptable excipient.