MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF CYTOPLASMIC AND CYTOSKELETAL 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 - 92 . (canceled)
93 . A method of expressing a secreted protein in a mammalian subject, the method comprising administering to the subject a pharmaceutical composition comprising a plurality of lipid nanoparticles encapsulating a polynucleotide, wherein the lipid nanoparticles comprise a cationic lipid, a neutral lipid, cholesterol, and a PEGylated lipid, wherein the plurality of lipid nanoparticles has a mean particle size of from 80 nm to 160 nm, and
wherein the polynucleotide comprises:
(a) an open reading frame encoding the secreted protein and consisting of nucleosides selected from uridine, cytidine, adenosine, and guanosine;
(b) a 5′-UTR;
(c) a 5′ cap structure;
(d) a 3′-UTR; and
(e) a 3′ tailing sequence of linked nucleosides.
94 . The method of claim 93 , wherein the cationic lipid is a biodegradable cationic lipid.
95 . The method of claim 94 , wherein the biodegradable cationic lipid comprises an ester linkage.
96 . The method of claim 93 , wherein the method comprises administering about 0.05 to about 0.5 mg/kg of polynucleotide.
97 . The method of claim 93 , wherein the administration is intramuscular administration.
98 . The method of claim 93 , wherein the administration is intravenous administration.
99 . The method of claim 93 , wherein upon administration, expression of the secreted protein is maximal at 8-24 hours.
100 . The method of claim 93 , wherein the 3′-tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail and a polyA-G quartet.
101 . The method of claim 100 , wherein the poly-A tail comprises at least 100 nucleosides.
102 . The method of claim 93 , wherein the 5′ cap structure is Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine.
103 . The method of claim 102 , wherein the 5′-cap structure is cap0, cap1, or ARCA.
104 . The method of claim 93 , wherein the plurality of lipid nanoparticles has a mean PDI of between 0.02 and 0.2.
105 . The method of claim 93 , wherein the plurality of lipid nanoparticles has a mean lipid to polynucleotide ratio (wt/wt) of between 10 and 20.
106 . The method of claim 93 , wherein the 3′-UTR comprises a miR binding site.
107 . The method of claim 93 , wherein the 5′-UTR comprises a Kozak sequence.
108 . The method of claim 93 , wherein the neutral lipid is a phospholipid.
109 . The method of claim 93 , wherein the open reading frame is codon optimized to bias GC content.
110 . The method of claim 93 , wherein the lipid nanoparticles comprise about 50 mol % biodegradable cationic lipid, about 38.5% cholesterol, about 10% neutral lipid, and about 1.5% PEGylated lipid.
111 . The method of claim 93 , wherein the polynucleotide includes at least two stop codons before the 3′-UTR.