MODIFIED NUCLEOSIDES, NUCLEOTIDES, AND NUCLEIC ACIDS, AND USES THEREOF
The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using them.
1 . An isolated polynucleotide encoding a polypeptide of interest, said isolated polynucleotide comprising:
(a) a sequence of n number of linked nucleosides or nucleotides comprising at least one modified nucleoside or nucleotide as compared to the chemical structure of an A, G, U or C nucleoside or nucleotide,
(b) a 5′ UTR comprising at least one Kozak sequence,
(c) a 3′ UTR, and
(d) at least one 5′ cap structure.
2 . The isolated polynucleotide of claim 1 , further comprising a poly-A tail.
3 . The isolated polynucleotide of claim 2 which is purified.
4 . The isolated polynucleotide of claim 3 , 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.
5 . The isolated polynucleotide of claim 4 , wherein the modification of the nucleoside is located in the nucleoside base and/or sugar portion of the nucleoside.
6 - 10 . (canceled)
11 . A pharmaceutical composition comprising the isolated polynucleotide of claim 1 and a pharmaceutically acceptable excipient.
12 . The pharmaceutical composition of claim 11 , 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, lipoplexe peptide, protein, cell, hyaluronidase, and mixtures thereof.
13 . A method of increasing the level of a polypeptide of interest in a mammalian subject comprising administering to said subject the isolated polynucleotide of claim 1 .
14 . The method of claim 13 , wherein the polynucleotide is formulated.
15 . The method of claim 13 , wherein isolated polynucleotide has a Protein:Cytokine Ratio of greater than 100 for either TNF-alpha or IFN-alpha.
16 . The method of claim 13 , wherein the isolated polynucleotide is administered at a total daily dose of between 1 ug and 150 ug.
17 . The method of claim 16 , wherein administration is by injection.
18 . The method of claim 16 , wherein administration is intradermal or subcutaneous or intramuscular.
19 . The method of claim 13 , wherein levels of the polypeptide of interest in the serum of the mammal are at least 50 pg/mL at least two hours after administration.
20 . The method of claim 19 , wherein the levels of the polypeptide of interest in the serum of the mammal remain above 50 pg/mL for at least 72 hours after administration.
21 . The method of claim 20 , wherein the levels of the polypeptide of interest in the serum of the mammal remain above 60 pg/mL for at least 72 hours after administration.
22 . The method of claim 13 , wherein administration is in two or more equal or unequal split doses.
23 . The method of claim 22 , wherein the level of the polypeptide produced by the subject by administering split doses of the polynucleotide is greater than the levels produced by administering the same total daily dose of polynucleotide as a single administration.
24 . The method of claim 13 , wherein the mammalian subject is a human patient in need of an increased level of the polypeptide of interest.
25 . The method of claim 24 , wherein the increased level of the polypeptide of interest is detectable in a bodily fluid of said patient.
26 - 72 . (canceled)