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

Abstract

The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using thereof.

1 .- 20 . (canceled) 
     
     
         21 . A method of inducing in vivo translation of a recombinant polypeptide in a mammalian subject in need thereof, comprising administering to the mammalian subject a synthetic messenger RNA (mRNA) in an effective amount such that the synthetic mRNA is localized into a cell population of the mammalian subject and the recombinant polypeptide is translated in the cell population from the synthetic mRNA,
 wherein the synthetic mRNA is prepared by a synthetic method comprising the steps of:   a) providing a complementary deoxyribonucleic acid (cDNA) encoding a pharmaceutical protein of interest;   b) selecting a nucleotide that disrupts a binding of a major groove binding partner with an mRNA, wherein the nucleotide has decreased binding affinity to the major groove binding partner selected from the group consisting of toll-like receptor (TLR) 3, TLR7, TLR8, retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), and wherein the nucleotide comprises a modification on the major groove face of the nucleobase where an atom of the major groove face of the nucleobase is replaced or substituted with an alkyl group; and   c) contacting the provided cDNA and the selected nucleotide with an RNA polymerase under conditions such that an RNA transcript encoding the recombinant polypeptide is synthesized.   
     
     
         22 . The method of  claim 21 , wherein the administration of the synthetic mRNA is repeated for one or more times such that the synthetic mRNA is localized into the cell population, or a tissue in which the cell population is present. 
     
     
         23 . The method of  claim 22 , wherein the administration of the synthetic mRNA is repeated for one or more times such that a predetermined efficiency of protein translation in the cell is achieved. 
     
     
         24 . The method of  claim 23 , wherein the administration of the synthetic mRNA is repeated for two, three, four, five, or more than five times. 
     
     
         25 . The method of  claim 21 , wherein the synthetic mRNA is at least 300 nucleotides in length. 
     
     
         26 . The method of  claim 21 , wherein the nucleotide comprises a modification on the major groove face of a pyrimidine nucleobase. 
     
     
         27 . The method of  claim 26 , wherein the pyrimidine nucleobase is selected from cytosine and uracil. 
     
     
         28 . The method of  claim 26 , wherein the pyrimidine nucleobase is uracil. 
     
     
         29 . The method of  claim 26 , wherein the pyrimidine nucleobase is cytosine. 
     
     
         30 . The method of  claim 28 , wherein the nucleotide comprises 1-methyl-pseudouridine or 5-methyl-uridine. 
     
     
         31 . The method of  claim 30 , wherein the nucleotide comprises 1-methyl-pseudouridine. 
     
     
         32 . The method of  claim 29 , wherein the nucleotide comprises 5-methyl-cytidine. 
     
     
         33 . The method of  claim 21 , wherein the major groove binding partner is TLR3, TLR7, or TLR8. 
     
     
         34 . The method of  claim 21 , wherein the major groove binding partner is RIG-I, MDA5, or LGP2. 
     
     
         35 . The method of  claim 21 , wherein the synthetic method further comprises after c), the step of:
 d) 5′-capping the mRNA transcript concomitantly or post-transcriptionally such that the synthetic mRNA is synthesized.   
     
     
         36 . The method of  claim 35 , wherein the 5′-capping is performed concomitantly. 
     
     
         37 . The method of  claim 21 , wherein the mammalian subject is a human. 
     
     
         38 . The method of  claim 21 , wherein the mammalian subject is a non-human mammal. 
     
     
         39 . The method of  claim 21 , wherein a pharmaceutical composition comprising the synthetic mRNA of  claim 21  and a pharmaceutically acceptable carrier is administered to the mammalian subject.