Enzyme based system for production of messenger RNA with increased transfection efficiency

A method for in vitro synthesis of mRNA, the method comprising:

• transfecting a first plurality of competent bacterial cells with a first plasmid vector comprising an arabinose promoter linked to a nucleic acid sequence encoding T7 RNA polymerase, wherein the first plasmid vector comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:
• 19;
• transfecting a second plurality of competent bacterial cells with a second plasmid vector comprising at least one arabinose promoter linked to a nucleic acid sequence encoding vaccinia virus capping enzyme (VVCE), wherein the second plasmid vector comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:
• 20;
• transfecting a third plurality of competent bacterial cells with a third plasmid vector comprising a UUG start codon, an arabinose promoter linked to a nucleic acid sequence encoding polyadenosine (poly (A)) polymerase, wherein the third plasmid vector comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:
• 21;
• expressing and purifying T7 RNA polymerase from the first plurality of competent bacterial cells;
• expressing and purifying VVCE from the second plurality of competent bacterial cells;
• expressing and purifying poly (A) polymerase from the third plurality of competent bacterial cells;
• adding linearized plasmid DNA to a first composition comprising the purified T7 RNA polymerase to produce RNA transcripts;
• adding the RNA transcripts to a second composition comprising the purified VVCE to produce capped RNA transcripts;
• adding the capped RNA transcripts to a third composition comprising the purified poly (A) polymerase to produce polyadenylated capped mRNA transcripts;
• and
• purifying mRNA from the third composition.