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 . The isolated polynucleotide of claim 5 , wherein the modification is located in the nucleotide base and the nucleoside base has the formula:
wherein:
denotes a single or double bond;
X is O or S;
U and W are each independently C or N;
V is O, S, C or N;
wherein when V is C then R 1 is H, C 1-6 alkyl, 01_6 alkenyl, C 1-6 alkynyl, halo, or —OR c , wherein C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl are each optionally substituted with —OH, —NR a R b , —SH, —C(O)R c , —C(O)OR c , —NHC(O)R c , or —NHC(O)OR c ;
and wherein when V is O, S, or N then R 1 is absent;
R 2 is H, —OR c , —SR c , —NR a R b , or halo;
or when V is C then R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, —OH, —SH, —NR a R b , C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 1-20 alkoxy, or C 1-20 thioalkyl;
R 3 is H or C 1-20 alkyl;
R 4 is H or C 1-20 alkyl; wherein when denotes a double bond then R 4 is absent, or N—R 4 , taken together, forms a positively charged N substituted with C 1-20 alkyl;
R a and R b are each independently H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, or C 6-20 aryl; and
R c is H, C 1-20 alkyl, C 2-20 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
7 . The isolated polynucleotide of claim 6 , wherein the modification is located in the nucleoside base and the nucleoside base has the formula:
wherein:
R 3 is C 1-20 alkyl.
8 . The isolated polynucleotide of claim 7 , wherein R 3 is C 1-4 alkyl.
9 . The isolated polynucleotide of claim 7 , wherein R 3 is CH 3 .
10 . The isolated polynucleotide of claim 1 , wherein the modified nucleoside is not pseudouridine (ψ) or 5-methyl-cytidine (m 5 C).
11 . A pharmaceutical composition comprising the isolated polynucleotide of claim 6 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 6 .
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.