Expression of GNE mutant proteins increases CHO intracellular CMP-Neu5Ac levels without impact on bioprocess...

Modulation of various nucleotide sugar levels in cells has been demonstrated as an effective way to alter the composition of N-glycans. Previous studies have demonstrated the ability to impact CMP-Neu5Ac levels by the addition of N-acetylated mannosamine (ManNAc) to culture media. In this study, the relationship between adding varying levels of ManNAc to cell cultures and the impact on both CMP-Neu5Ac levels and cell growth were examined. Increasing the concentration of ManNAc added resulted in higher levels of CMP-Neu5Ac, but negatively impacted cell growth. Through cellular genetic engineering, we sought to devise an alternative method of increasing ManNAc levels without impacting cell growth. The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE) gene is the rate-limiting enzyme in which congenital mutations can cause Sialuria, a rare metabolic disorder characterized by cytoplasmic accumulation and urinary excretion of free sialic acid. A mutant form of the GNE gene, harboring three mutations (D53H, R263I, R266Q), was site-specifically integrated (SSI) into one locus in CHO cells. This mutant protein dramatically increased the intracellular concentrations of CMP-Neu5Ac, reaching the maximal level as with the addition of ManNAc. These data together indicate that the GNE mutants could provide an effective way for substituting the high-cost supplementation of ManNAc without impacting cell growth. The investigation has also demonstrated the feasibility of the dual-landing-pad SSI cell line engineering approach for improving product qualities of biotherapeutics.