Reengineering mRNA Primary Structure for Enhanced Protein Production

Abstract

Described herein are rules to modify natural mRNAs or to engineer synthetic mRNAs to increase their translation efficiencies. These rules describe modifications to mRNA coding and 3′ UTR sequences intended to enhance protein synthesis by: 1) decreasing ribosomal diversion via AUG or non-canonical initiation codons in coding sequences, and/or 2) by evading miRNA-mediated down-regulation by eliminating one or more miRNA binding sites in coding sequences.

What is claimed is: 
     
         1 . A method of improving full-length protein expression efficiency comprising:
 a) providing a polynucleotide comprising:
 i) a coding sequence for the protein; 
 ii) a primary initiation codon that is upstream of the coding sequence; and 
 iii) one or more secondary initiation codons located within the coding sequence; and 
   b) mutating one or more secondary initiation codons, wherein the mutation results in a decrease in initiation of protein synthesis at the one or more secondary initiation codons resulting in a reduction of ribosomal diversion away from the primary initiation codon,   thereby increasing full-length protein expression efficiency.   
     
     
         2 . The method of  claim 1 , wherein mutating the one or more secondary initiation codons comprises mutating one or more nucleotides such that the amino acid sequence remains unaltered. 
     
     
         3 . The method of  claim 1 , wherein the one or more secondary initiation codons is in the same reading frame as the coding sequence. 
     
     
         4 . The method of  claim 1 , wherein the one or more secondary initiation codons is out-of-frame with the coding sequence. 
     
     
         5 . The method of  claim 1 , wherein the one or more secondary initiation codons is located one or more nucleotides upstream or downstream from a ribosomal recruitment site. 
     
     
         6 . The method of  claim 5 , wherein the ribosomal recruitment site comprises a cap or an IRES. 
     
     
         7 . The method of  claim 1 , wherein the one or more secondary initiation codons is selected from the group consisting of AUG, ACG, GUG, UUG, CUG, AUA, AUC, and AUU. 
     
     
         8 . The method of  claim 1 , wherein more than one secondary initiation codon within the coding sequence is mutated. 
     
     
         9 . The method of  claim 1 , wherein all secondary initiation codons within the coding sequence are mutated. 
     
     
         10 . The method of  claim 1 , wherein mutating the one or more secondary initiation codons comprises mutating a flanking nucleotide to a less favorable nucleotide context. 
     
     
         11 . The method of  claim 1 , wherein mutating the one or more secondary initiation codons does not introduce new initiation codons. 
     
     
         12 . The method of  claim 1 , wherein mutating the one or more secondary initiation codons does not alter usage bias of mutated codons. 
     
     
         13 . The method of  claim 1 , further comprising decreasing the generation of truncated proteins, polypeptide, or peptides other than the full-length encoded protein. 
     
     
         14 . The method of  claim 1 , wherein mutating one or more secondary initiation codons does not introduce miRNA seed sequences, splice donor site, splice acceptor site, or mRNA destabilization elements. 
     
     
         15 . A method of improving full-length protein expression efficiency comprising:
 a) providing a polynucleotide sequence comprising a coding sequence for the protein and one or more miRNA binding sites located within the coding sequence; and   b) mutating the one or more miRNA binding sites, wherein the mutation results in a decrease in miRNA binding at the one or more miRNA binding sites resulting in a reduction of miRNA-mediated down regulation of protein translation, thereby increasing full-length protein expression efficiency.   
     
     
         16 . The method of  claim 15 , wherein mutating the one or more miRNA binding sites comprises mutating one or more nucleotides such that the amino acid sequence remains unaltered. 
     
     
         17 . The method of  claim 15 , wherein mutating the one or more miRNA binding sites comprises mutating one or more nucleotides in a miRNA seed sequence. 
     
     
         18 . The method of  claim 15 , wherein mutating the one or more miRNA binding sites comprises mutating one or more nucleotides such that initiation codons are not introduced into the polynucleotide sequence. 
     
     
         19 . The method of  claim 15 , wherein mutating the one or more miRNA binding sites comprises mutating one or more nucleotides such that rare codons are not introduced into the polynucleotide sequence. 
     
     
         20 . The method of  claim 15 , wherein mutating the one or more miRNA binding sites comprises mutating one or more nucleotides such that additional miRNA seed sequences are not introduced into the polynucleotide sequence. 
     
     
         21 . The method of  claim 15 , wherein the one or more miRNA binding sites is located within the coding sequence. 
     
     
         22 . The method of  claim 15 , wherein the one or more miRNA binding sites is located within the 3′ untranslated region. 
     
     
         23 . The method of  claim 15 , wherein the one or more miRNA binding sites is located within the 5′ leader sequence.