US 12,448,638 B2
Enzyme based system for production of messenger RNA with increased transfection efficiency
Brett Morimoto, Culver City, CA (US); Kayvan Niazi, Culver City, CA (US); Annie Shin, Culver City, CA (US); Lise Geissert, Culver City, CA (US); and Philip T. Liu, Culver City, CA (US)
Assigned to ImmunityBio, Inc., San Diego, CA (US)
Filed by ImmunityBio, Inc., Culver City, CA (US)
Filed on Sep. 12, 2022, as Appl. No. 17/931,352.
Claims priority of provisional application 63/244,990, filed on Sep. 16, 2021.
Prior Publication US 2023/0183770 A1, Jun. 15, 2023
Int. Cl. C12P 19/34 (2006.01); C12N 9/12 (2006.01); C12N 15/70 (2006.01)
CPC C12P 19/34 (2013.01) [C12N 9/1241 (2013.01); C12N 9/1247 (2013.01); C12Y 207/07019 (2013.01)] 7 Claims
 
1. 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.