In vivo production of proteins
The invention relates to compositions including polynucleotides encoding polypeptides which have been chemically modified by replacing the uridines with 1-methyl-pseudouridine to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency, accessibility to circulation, protein half-life and/or modulation of a cell's status, function, and/or activity.
1. A method of expressing a protein in a mammalian subject, the method comprising administering a pharmaceutical composition comprising a plurality of lipid nanoparticles encapsulating a polynucleotide, wherein the lipid nanoparticle comprises a biodegradable cationic lipid, a neutral lipid, cholesterol, and a PEGylated lipid and the plurality of lipid nanoparticles has a mean particle size of between 80 nm to 160 nm, and
wherein the polynucleotide comprises:
(a) an open reading frame encoding the protein consisting of nucleotides selected from 1-methyl-pseudouridine, cytidine, adenosine, and guanosine;
(b) a 5′-UTR;
(c) at least one 5′ cap structure;
(d) a 3′-UTR; and
(e) a 3′ tailing sequence of linked nucleosides.
2. The method of claim 1 , wherein the biodegradable cationic lipid comprises an ester linkage.
3. The method of claim 1 , wherein the method comprises administering about 0.05 to about 0.5 mg/kg of polynucleotide.
4. The method of claim 1 , wherein the administration is intramuscular administration.
5. The method of claim 1 , wherein the administration is intravenous administration.
6. The method of claim 1 , wherein upon administration, expression of the protein is maximal at 8-24 hours.
7. The method of claim 1 , wherein the 3′-tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail and a polyA-G quartet.
8. The method of claim 7 , wherein the poly-A comprises approximately 160 nucleotides.
9. The method of claim 1 , 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, and 2-azido-guanosine.
10. The method of claim 9 , wherein the at least one 5′-cap structure is cap0, cap1, or ARCA.
11. The method of claim 1 , wherein the plurality of lipid nanoparticles has a mean PDI of between 0.02 and 0.2.
12. The method of claim 1 , wherein the plurality of lipid nanoparticles has a mean lipid to polynucleotide ratio (wt/wt) of between 10 and 20.
13. The method of claim 1 , wherein the 3′-UTR comprises a miR binding site.
14. The method of claim 1 , wherein the 5′-UTR comprises a Kozak sequence.
15. The method of claim 1 , wherein the neutral lipid is a phospholipid.
16. The method of claim 1 , wherein the open reading frame is codon optimized to bias GC content.
17. The method of claim 1 , wherein the plurality of lipid nanoparticles comprise about 50 mol % biodegradable cationic lipid, about 38.5% cholesterol, about 10% neutral lipid and about 1.5% PEGylated lipid.
18. The method of claim 1 , wherein the polynucleotide includes at least two stop codons before the 3′ untranslated region (UTR).