REGULATION OF PROTEIN PRODUCTIVITY BY MICRORNAS IN CHO CELLS

Abstract

MICRORNAS (MIRNAS), A NOVEL CLASS OF SHORT, NON-CODING RNAS WHICH NEGATIVELY REGULATE TARGET GENE EXPRESSION AT POST-TRANSCRIPTIONAL LEVELS, HAVE EMERGED AS PROMISING TARGETS FOR ENGINEERING OF CHINESE HAMSTER OVARY (CHO) CELL FACTORIES TO ENHANCE RECOMBINANT PROTEIN PRODUCTION. TO UNDERSTAND HOW MIRNAS REGULATE PRODUCTIVITY IN CHO CELLS, WE CARRIED OUT NEXT-GENERATION SEQUENCING OF IGG-EXPRESSING CHO SUB-CLONES OF VARYING PRODUCTIVITIES. MIRNAS WHICH WERE FOUND TO BE DIFFERENTIALLY EXPRESSED BETWEEN THE HIGH AND LOW PRODUCERS WERE STABLY TRANSFECTED INTO AN IGG-EXPRESSING SUB-CLONE. STABLY TRANSFECTED POOLS DEMONSTRATED 13-27% INCREASES IN TITER, WHILE SINGLE CELL CLONES EXHIBITED UP TO ~100% INCREASES. CLONES OVER-EXPRESSING MIR-92A SHOWED LOWERED LEVELS OF INSIG1 MRNA, INCREASED INTRACELLULAR CHOLESTEROL CONCENTRATION AND INCREASED RELATIVE GOLGI SURFACE AREA. BY THE USE OF A REPORTER VECTOR ASSAY, INSIG1 WAS VALIDATED AS A TARGET OF MIR-92A. WE POSTULATE THAT MIR-92A ENHANCES PRODU