Multimeric coding nucleic acid and uses thereof
The present invention provides, among other things, multimeric coding nucleic acids that exhibit superior stability for in vivo and in vitro use. In some embodiments, a multimeric coding nucleic acid (MCNA) comprises two or more encoding polynucleotides linked via 3′ ends such that the multimeric coding nucleic acid compound comprises two or more 5′ ends.
1. A composition comprising a multimeric coding nucleic acid (MCNA) encapsulated or complexed with a liposomal delivery vehicle,
wherein the MCNA comprises two messenger RNAs (mRNAs) linked at 3′ ends, via stable linkage, such that the multimeric coding nucleic acid has two 5′ ends, and wherein the stable linkage is an oligonucleotide bridge comprising an internal 3′-to- 3′ inverted phosphodiester linkage.
2. The composition of claim 1 , wherein the delivery vehicle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEGylated lipids.
3. A method of delivering MCNA for in vivo protein production, comprising administering the composition of claim 1 to a subject in need of delivery.
4. The method of claim 3 , wherein the composition is administered via a route of delivery selected from the group consisting of intravenous delivery, subcutaneous delivery, oral delivery, subdermal delivery, ocular delivery, pulmonary delivery, intramuscular delivery, intrathecal delivery, and intraarticular delivery.
5. The method of claim 3 , wherein the composition comprises human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) mRNA.
6. The method of claim 3 , wherein the composition comprises human phenylalanine hydroxylase (hPAH) mRNA.
7. The composition of claim 1 , wherein each of the mRNAs encodes a protein of interest.
8. The composition of claim 7 , wherein each of the mRNAs encodes a same protein.
9. The composition of claim 7 , wherein each of the mRNAs encodes a distinct protein.
10. The composition of claim 1 , wherein the mRNAs comprise a 5′ untranslated region (5′ UTR) and/or a 3′ untranslated region (3′ UTR).
11. The composition of claim 1 , wherein the mRNAs comprise a 3′ UTR.
12. The composition of claim 11 , wherein the 3′ UTR comprises a plurality of multi-A segments with spacers in between.
13. The composition of claim 1 , wherein the oligonucleotide bridge comprises nucleosides selected from the group consisting of 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ψU), and 1-methyl-pseudouridine.
14. The composition of claim 1 , wherein the mRNAs comprise one or more modified nucleosides.
15. The composition of claim 14 , wherein the modified nucleosides are selected from the group consisting of 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ψU), and 1-methyl-pseudouridine.
16. The composition of claim 1 , wherein each of the mRNAs encodes an enzyme, a receptor, a ligand, a light chain or heavy chain of an antibody, a nuclease, or a DNA-binding protein.
17. The composition of claim 11 , wherein the 3′ UTR does not include a polyA tail.
18. The composition of claim 1 , wherein each of the mRNAs is unmodified.
19. The composition of claim 1 , wherein the oligonucleotide bridge is unmodified.
20. The composition of claim 1 , wherein each of the mRNAs has a 5′ cap.