Modified polynucleotides for the production of biologics and proteins associated with human disease
View Patent ↗The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.
1. An isolated mRNA comprising;
(a) a first region comprising an open reading frame having at least 95% identity to SEQ ID NO: 1871 encoding a polypeptide having the sequence of SEQ ID NO: 967 consisting of nucleotides selected from 1-methyl-pseudouridine, cytidine, adenosine, and guanosine;
(b) a first flanking region located at the 5′ terminus of said first region comprising;
(i) a 5′-UTR including a Kozak sequence; and
(ii) at least one 5′ terminal cap;
(c) a second flanking region located at the 3′ terminus of said first region comprising;
(i′) a 3′-UTR; and
(ii′) a 3′ tailing sequence of linked nucleosides.
2. The isolated mRNA of claim 1 wherein the open reading frame comprises the sequence of SEQ ID NO: 1871.
3. The isolated mRNA of claim 1 , wherein the 3′ tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail of approximately 160 nucleotides and a polyA-G quartet.
4. The isolated mRNA of claim 1 which is purified.
5. The isolated mRNA of claim 1 , wherein the at least one 5′ terminal cap 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.
6. The isolated mRNA of claim 1 , further comprising a targeting moiety, wherein said targeting moiety is covalently bound to said polynucleotide.
7. The isolated mRNA of claim 6 , wherein said targeting moiety is an antibody, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic, or an aptamer.
8. A pharmaceutical composition comprising the isolated mRNA of claim 1 .
9. A pharmaceutical composition comprising a plurality of lipid nanoparticles, wherein the plurality of lipid nanoparticles has a mean particle size of between 80 nm and 160 nm, a mean PDI of between 0.02 and 0.2, and a mean lipid to polynucleotide ratio (wt/wt) of between 10 and 20; and wherein the lipid nanoparticles comprise the isolated mRNA of claim 1 and a pharmaceutically acceptable excipient.
10. The pharmaceutical composition of claim 9 , wherein the excipient is selected from a solvent, aqueous solvent, non-aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplex, peptide, protein, cell, hyaluronidase, and mixtures thereof.
11. The pharmaceutical composition of claim 10 , where the pharmaceutical composition comprises a lipid and wherein said lipid is selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA and PEGylated lipids and mixtures thereof.
12. A method of producing a polypeptide of interest in a mammalian cell, tissue or organism comprising administering to said cell, tissue or organism the the pharmaceutical composition of claim 9 .