Modified nucleosides, nucleotides, and nucleic acids, and uses thereof

Abstract

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.