Alternative nucleic acid molecules containing reduced uracil content and uses thereof

A method of producing a modified mRNA comprising an ORF encoding a polypeptide, the method comprising modifying an mRNA sequence to produce the modified mRNA by replacing at least one codon containing a uracil with a codon that encodes the same amino acid but contains a lower number of uracils,

• wherein at least 95% of uracils in the ORF are 5-methoxyuracils, and
• wherein the uracil content in the ORF is between the theoretical minimum and 150% of the theoretical minimum;
• wherein the level of expression in a mammalian cell of the encoded polypeptide from the mRNA is increased relative to a reference mRNA comprising a reference open reading frame (rORF) encoding the polypeptide,
• wherein at least 95% of uracils in the rORF are 5-methoxyuracils, and wherein the uracil content in the rORF is from about 190% to about 200% of the theoretical minimum.

A composition comprising:

• (a) a lipid nanoparticle comprising a cationic lipid;
• and
• (2) an mRNA encoding a polypeptide, the mRNA comprising:
• (i) a 5′-cap structure;
• (ii) a 5′-UTR;
• (iii) an ORF encoding the polypeptide
• wherein at least 95% uracils in the ORF are 5-methoxyuracils;
• and
• wherein the uracil content in the ORF is between the theoretical minimum and 150% of the theoretical minimum,
• (iv) a 3′-UTR;
• and
• (v) a poly-A region;
• wherein the level of expression in a mammalian cell of the encoded polypeptide from the mRNA is increased relative to a reference mRNA comprising a reference open reading frame (rORF) encoding the polypeptide,
• wherein at least 95% of uracils in the rORF are 5-methoxyuracils, and wherein the uracil content in the rORF is from about 190% to about 200% of the theoretical minimum.