Alternative nucleic acid molecules and uses thereof

Abstract

The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them.

1 - 159 . (canceled) 
     
     
         160 . An mRNA comprising:
 (i) a 5′ cap structure;   (ii) a 5′-untranslated region (UTR);   (iii) an open reading frame encoding a polypeptide; and   (iv) a 3′-UTR,   wherein alpha-thio-adenosine and/or alpha-thio-guanosine represent about 2%-100% of the adenosines and/or guanosines in the mRNA.   
     
     
         161 . The mRNA of  claim 160 , wherein alpha-thio-adenosine and/or alpha-thio-guanosine represent about 2%-10% of the adenosines and/or guanosines in the mRNA. 
     
     
         162 . The mRNA of  claim 161 , wherein alpha-thio-adenosine and/or alpha-thio-guanosine represent about 2% of the adenosines and/or guanosines in the mRNA. 
     
     
         163 . The mRNA of  claim 160 , wherein alpha-thio-adenosine represents about 2%-10% of the adenosines in the mRNA. 
     
     
         164 . The mRNA of  claim 160 , wherein alpha-thio-guanosine represents about 2%-10% of the guanosines in the mRNA. 
     
     
         165 . The mRNA of  claim 160 , wherein the 5′ cap structure is Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine. 
     
     
         166 . The mRNA of  claim 160 , wherein the mRNA further comprises a poly-A tail. 
     
     
         167 . The mRNA of  claim 160 , wherein the mRNA is codon optimized. 
     
     
         168 . The mRNA of  claim 167 , wherein the mRNA is codon optimized to minimize base runs that impair gene expression. 
     
     
         169 . The mRNA of  claim 160 , wherein the open reading frame consists of nucleosides selected from 1-methyl-pseudouridine, cytidine, adenosine or alpha-thio-adenosine, and guanosine or alpha-thio-guanosine. 
     
     
         170 . A pharmaceutical composition comprising the mRNA of  claim 160  and a pharmaceutically acceptable excipient. 
     
     
         171 . A method of expressing a polypeptide of interest in a mammalian cell, said method comprising the steps of:
 (i) providing an mRNA of  claim 160 ; and   (ii) introducing said mRNA to a mammalian cell under conditions that permit the expression of the polypeptide of interest by the mammalian cell.   
     
     
         172 . The method of  claim 171 , wherein the innate immune response associated with the mRNA is reduced relative to the innate immune response induced by a corresponding mRNA that does not comprise alpha-thio-adenosine and/or alpha-thio-guanosine. 
     
     
         173 . The method of  claim 169 , wherein the expression of the polypeptide by the mRNA is increased relative to the expression of a corresponding mRNA that does not comprise alpha-thio-adenosine and/or alpha-thio-guanosine.