Modified polynucleotides for the production of secreted proteins

Abstract

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

1 . A pharmaceutical composition comprising: 
       a plurality of lipid nanoparticles comprising a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, 
       wherein the plurality of lipid nanoparticles has a mean particle size of between 80 nm and 160 nm; and 
       wherein the lipid nanoparticles comprise an mRNA encoding a secreted protein, wherein the mRNA comprises:
 (i) at least one 5′-cap structure; 
 (ii) a 5′-UTR; 
 (iii) an open reading frame encoding the secreted protein and consisting of nucleotides including N1-methyl-pseudouridine, cytosine, adenine, and guanine; 
 (iv) a 3′-UTR; and 
 (v) a poly-A region of least 100 nucleotides in length. 
 
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the cationic lipid is a biodegradable cationic lipid. 
     
     
         3 . The pharmaceutical composition of  claim 2 , wherein the biodegradable cationic lipid comprises an ester linkage. 
     
     
         4 . The pharmaceutical composition of  claim 3 , wherein the biodegradable cationic lipid comprises DLin-DMA with an internal ester, DLin-DMA with a terminal ester, DLin-MC3-DMA with an internal ester, or DLin-MC3-DMA with a terminal ester. 
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the at least one 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. 
     
     
         6 . The pharmaceutical composition of  claim 5 , wherein the at least one 5′-cap structure is cap0, cap1, or ARCA. 
     
     
         7 . The pharmaceutical composition of  claim 1 , wherein the 3′-UTR is an alpha-globin 3′-UTR. 
     
     
         8 . The pharmaceutical composition of  claim 1 , wherein the poly-A tail is at least 160 nucleotides in length. 
     
     
         9 . The pharmaceutical composition of  claim 1 , wherein the 5′-UTR comprises a Kozak sequence. 
     
     
         10 . The pharmaceutical composition of  claim 1 , wherein the plurality of lipid nanoparticles has a mean PDI of between 0.02 and 0.2. 
     
     
         11 . The pharmaceutical composition of  claim 1 , wherein the plurality of lipid nanoparticles has a mean lipid to polynucleotide ratio (wt/wt) of between 10 and 20. 
     
     
         12 . The pharmaceutical composition of  claim 1 , wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded secreted protein relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         13 . The pharmaceutical composition of  claim 1 , wherein, upon administration to a mammalian cell, the mRNA has a longer half-life or greater area under the curve of secreted protein expression relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         14 . The pharmaceutical composition of  claim 1 , wherein, upon administration to peripheral blood mononuclear cells, the mRNA induces detectably lower levels of IFN-α or TNF-α relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine.