Alternative nucleic acid molecules containing reduced uracil content and uses thereof

Abstract

The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them. In some aspects, the disclosure provides mRNA wherein the uracil content has been modified and which may be particularly effective for use in therapeutic compositions, because they may benefit from both high expression levels and limited induction of the innate immune response. In some aspects, the disclosure provides methods for the production of pharmaceutical compositions including mRNA without reverse phase chromatography.

1 . An mRNA encoding a polypeptide comprising:
 (i) at least one 5′-cap structure;   (ii) a 5′-UTR;   (iii) an open reading frame encoding the polypeptide and consisting of nucleotides that contain 5-methoxy-uracil, cytosine, adenine, or guanine, wherein the open reading frame has uracil content between the theoretical minimum and 200% of the theoretical minimum and at least one codon is a low frequency codon;   (iv) a 3′-UTR; and   (v) a poly-A region,   wherein, upon administration to a mammalian cell, the mRNA has increased expression of the encoded polypeptide relative to a corresponding mRNA wherein all of the codons are high frequency codons.   
     
     
         2 . The mRNA of  claim 1 , wherein the mRNA does not contain more than four consecutive uracils. 
     
     
         3 . The mRNA of  claim 1 , wherein the open reading frame has the theoretical minimum uracil content. 
     
     
         4 . The mRNA of  claim 1 , wherein the uracil content of the open reading frame is less than 20% of the total nucleotide content in the open reading frame. 
     
     
         5 . The mRNA of  claim 1 , wherein the mRNA contains fewer than ten uracil pairs. 
     
     
         6 . The mRNA of  claim 1 , wherein the uracil content within any 20 nucleotide window within the open reading frame does not exceed 50%. 
     
     
         7 . The mRNA of  claim 1 , wherein the open reading frame contains at least one of the following codons: GCG, GGG, CCG, AGG, ACG, CUC, CGC, UCC, and GUC. 
     
     
         8 . The mRNA of  claim 1 , wherein the uracil content of the 5′-UTR and/or the 3′-UTR is between the theoretical minimum and 200% of the theoretical minimum. 
     
     
         9 . The mRNA of  claim 8 , wherein the 5′-UTR and/or the 3′-UTR has the theoretical minimum uracil content. 
     
     
         10 . The mRNA of  claim 1 , wherein the at least one 5′-cap structure is cap0, cap1, or ARCA. 
     
     
         11 . The mRNA of  claim 1 , wherein the 3′-UTR is an alpha-globin 3′-UTR. 
     
     
         12 . The mRNA of  claim 1 , wherein the poly-A region is at least 160 nucleotides in length. 
     
     
         13 . The mRNA of  claim 1 , wherein, upon administration to a mammalian cell, the mRNA induces a detectably lower level of IFN-β relative to a corresponding mRNA having uracil content of greater than 200% of the theoretical minimum. 
     
     
         14 . 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 having uracil content of greater than 200% of the theoretical minimum. 
     
     
         15 . A method of producing a codon-modified mRNA comprising an open reading frame encoding a polypeptide, the method comprising:
 (a) providing a parent sequence of an open reading frame encoding the polypeptide;   (b) modifying the parent sequence to produce a second sequence of an open reading frame having uracil content between the theoretical minimum and 200% of the theoretical minimum, by replacing codons containing uracils with codons having the lowest number of uracils, wherein at least one replacement codon is a low frequency codon; and   (c) producing the codon-modified mRNA having the second sequence consisting of nucleotides including 5-methoxy-uracil as the uracil source, cytosine, adenine, and guanine, wherein the open reading frame has uracil content between the theoretical minimum and 200% of the theoretical minimum and at least one codon is a low frequency codon.   
     
     
         16 - 27 . (canceled)