In vivo production of proteins

Abstract

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 plasma membrane 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 plasma membrane 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 plasma membrane 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 tail 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).