Long-lived polynucleotide molecules
The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of long-lived polynucleotides, primary transcripts and mmRNA molecules.
1. A method of expressing a fusion polypeptide in a human subject, comprising administering to the human subject a lipid nanoparticle (LNP) comprising a codon optimized mRNA, wherein the mRNA encodes the fusion polypeptide, wherein the fusion polypeptide comprises a secreted polypeptide operably linked at the carboxy terminus to an albumin or a functional fragment thereof, and wherein the mRNA is fully modified at each uridine with 1-methylpseudouridine, and optionally modified at each cytidine with 5-methylcytosine, thereby expressing the fusion polypeptide in the human subject.
2. The method of claim 1 , wherein the mRNA comprises a poly-A tail and wherein the poly-A tail is at least 140 nucleotides in length.
3. The method of claim 1 , wherein the mRNA comprises a 5′ cap structure and wherein the 5′ cap structure is selected from the group consisting of: Cap0, Cap1, anti-reverse cap analog (ARCA), inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid (LNA)-guanosine, and 2-azido-guanosine.
4. The method of claim 1 , wherein the fusion polypeptide comprises a signal peptide sequence.
5. The method of claim 1 , wherein the albumin is human serum albumin.
6. An LNP comprising a codon optimized mRNA encoding a fusion polypeptide, wherein the fusion polypeptide comprises a secreted polypeptide operably linked at the carboxy terminus to an albumin or a functional fragment thereof, and wherein the mRNA is fully modified at each uridine with 1-methylpseudouridine, and optionally modified at each cytidine with 5-methylcytosine.
7. A pharmaceutical composition comprising the LNP of claim 6 and a pharmaceutically acceptable excipient.
8. The LNP of claim 6 , wherein the fusion polypeptide comprises a signal peptide sequence.
9. The LNP of claim 6 , wherein the albumin is human serum albumin.
10. A method of increasing a level of a fusion polypeptide in a mammal, comprising administering to the mammal an LNP comprising a codon optimized mRNA, wherein the mRNA encodes the fusion polypeptide, wherein the fusion polypeptide comprises a secreted polypeptide operably linked at the carboxy terminus to an albumin or a functional fragment thereof, and wherein the mRNA is fully modified at each uridine with 1-methylpseudouridine, and optionally modified at each cytidine with 5-methylcytosine, thereby increasing the level of the fusion polypeptide in the mammal.
11. The method of claim 10 , wherein the level of the fusion polypeptide is increased relative to the secreted polypeptide not fused at the carboxy terminus to the albumin or a functional fragment thereof.
12. The method of claim 10 , wherein the level of the fusion polypeptide is increased in serum or plasma of the mammal.
13. The method of claim 12 , wherein the level of the fusion polypeptide is determined by measuring the area under the curve (AUC) or the maximum serum or plasma concentration (C max ) of the fusion polypeptide following administration to the mammal.
14. The method of claim 10 , wherein the fusion polypeptide comprises a signal peptide sequence.
15. The method of claim 10 , wherein the albumin is human serum albumin.