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 mRNA encoding a polypeptide, wherein the mRNA comprises:
 (i) at least one 5′-cap structure;   (ii) a 5′-UTR including a Kozak sequence;   (iii) an open reading frame encoding the polypeptide and consisting of nucleotides including 5-methoxy-uracil, cytosine, adenine, and guanine; and   (iv) a 3′-UTR.   
     
     
         2 . The mRNA of  claim 1 , wherein, upon administration to peripheral blood mononuclear cells, the mRNA induces detectably lower levels of IFN-α or TNF-α relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         3 . The mRNA of  claim 1 , wherein, upon administration to peripheral blood mononuclear cells, the mRNA induces detectably lower levels of IFN-α or TNF-α relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including pseudouracil or N1-methyl-pseudouracil, cytosine, adenine, and guanine. 
     
     
         4 . The mRNA of  claim 1 , wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded polypeptide relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         5 . The mRNA of  claim 1 , wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded polypeptide relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including pseudouracil, cytosine, adenine, and guanine. 
     
     
         6 . The mRNA of  claim 1 , wherein, upon administration to a mammalian cell, the mRNA has a longer half-life or greater area under the curve of protein expression relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         7 . The mRNA of  claim 1 , wherein the at least one 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. 
     
     
         8 . The mRNA of  claim 7 , wherein the at least one 5′-cap structure is cap0, cap1, or ARCA. 
     
     
         9 . The mRNA of  claim 1 , wherein the 3′-UTR is an alpha-globin 3′-UTR. 
     
     
         10 . The mRNA of  claim 1 , wherein the poly-A region is 160 nucleotides in length. 
     
     
         11 . An mRNA encoding a polypeptide, wherein the mRNA comprises:
 (i) at least one 5′-cap structure;   (ii) a 5′-UTR including a Kozak sequence;   (iii) an open reading frame encoding the polypeptide and consisting of nucleotides including 5-methoxy-uracil, cytosine, adenine, and guanine;   (iv) a 3′-UTR; and   (v) a poly-A region of 150 to 165 nucleotides in length.   
     
     
         12 . The mRNA of  claim 11 , wherein, upon administration to peripheral blood mononuclear cells, the mRNA induces detectably lower levels of IFN-α or TNF-α relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         13 . The mRNA of  claim 11 , wherein, upon administration to peripheral blood mononuclear cells, the mRNA induces detectably lower levels of IFN-α or TNF-α relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including pseudouracil or N1-methyl-pseudouracil, cytosine, adenine, and guanine. 
     
     
         14 . The mRNA of  claim 11 , wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded polypeptide relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         15 . The mRNA of  claim 11 , wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded polypeptide relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including pseudouracil, cytosine, adenine, and guanine. 
     
     
         16 . The mRNA of  claim 11 , wherein, upon administration to a mammalian cell, the mRNA has a longer half-life or greater area under the curve of protein expression relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         17 . The mRNA of  claim 11 , wherein the at least one 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. 
     
     
         18 . The mRNA of  claim 17 , wherein the at least one 5′-cap structure is cap0, cap1, or ARCA. 
     
     
         19 . The mRNA of  claim 11 , wherein the 3′-UTR is an alpha-globin 3′-UTR. 
     
     
         20 . The mRNA of  claim 11 , wherein the poly-A region is 160 nucleotides in length. 
     
     
         21 . An mRNA encoding a polypeptide, wherein the mRNA comprises:
 (i) at least one 5′-cap structure;   (ii) a 5′-UTR including a Kozak sequence;   (iii) an open reading frame encoding the polypeptide and consisting of nucleotides including 5-methoxy-uracil, cytosine, adenine, and guanine;   (iv) a 3′-UTR; and   (v) a poly-A region of 150 to 165 nucleotides in length,   
       wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded polypeptide relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including pseudouracil, cytosine, adenine, and guanine. 
     
     
         22 . The mRNA of  claim 21 , wherein, upon administration to peripheral blood mononuclear cells, the mRNA induces detectably lower levels of IFN-α or TNF-α relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         23 . The mRNA of  claim 21 , wherein, upon administration to peripheral blood mononuclear cells, the mRNA induces detectably lower levels of IFN-α or TNF-α relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including pseudouracil or N1-methyl-pseudouracil, cytosine, adenine, and guanine. 
     
     
         24 . The mRNA of  claim 21 , wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded polypeptide relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         25 . The mRNA of  claim 21 , wherein, upon administration to a mammalian cell, the mRNA has a longer half-life or greater area under the curve of protein expression relative to a corresponding mRNA comprising an open reading frame consisting of nucleotides including uracil, cytosine, adenine, and guanine. 
     
     
         26 . The mRNA of  claim 21 , wherein the at least one 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. 
     
     
         27 . The mRNA of  claim 26 , wherein the at least one 5′-cap structure is cap0, cap1, or ARCA. 
     
     
         28 . The mRNA of  claim 21 , wherein the 3′-UTR is an alpha-globin 3′-UTR. 
     
     
         29 . The mRNA of  claim 21 , wherein the poly-A region is 160 nucleotides in length.