Modified polynucleotides for the production of secreted proteins
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 polypeptide, wherein the mRNA comprises:
(i) at least one 5′-cap structure;
(ii) a 5′-UTR;
(iii) an open reading frame encoding a plasma membrane protein and consisting of nucleotides including a modified uracil, 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 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 16 nucleotides iia 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 claim 1 , wherein the modified uracil is N1-methyl-pseudouridine.
13. The pharmaceutical composition of claim 1 , wherein the modified uracil is 5-methoxy-uracil.
14. The pharmaceutical composition of claim 1 , wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded polypeptide relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine.
15. The pharmaceutical composition of claim 1 , where upon administration to a mammalian cell, the mRNA has a longer half-life or greater area under the curve of protein expression relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine.
16. 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.