Antisense strategies for glycosylation engineering of Chinese hamster ovary (CHO) cells

Abstract

Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities.

Inhibition of sodium butyrate‐induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase‐3

“…Antisense strategy can be performed as transcription of antisense full sequence or partial fragment of target gene, or as introduction of an exogenous antisense oligonucleotide. Despite the time-consuming, laborious process in specific gene knockout, the transcription of antisense sequence is preferred to the introduction of an exogenous antisense oligonucleotide because of its efficiency and low cost (Prati et al, 1998). Furthermore, although depending on both the sense and antisense sequences, the expression of antisense RNA of caspase seems to be able to achieve complete inhibition of apoptotic cell death at a low level of expression of antisense RNA, removing the negative effect on growth rate and productivity originated from the synthesis of large quantities of survival protein (Singh et al, 1997).…”

Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation

“…The glycosylation patterns of proteins produced by CHO cells are very similar to those of many human plasma glycoproteins and as a result CHO cells are one of the most frequently used cell lines for recombinant protein production (Jenkins et al, 1996). Approaches have also been made to direct glycosylation patterns of CHO cells either by transfecting cells to produce specific glycosyltransferases that are not usually expressed (Lee et al, 1989;Minch et al, 1995;Prati et al, 1998;Sburlati et al, 1998) or by supplementing the cell culture medium with precursors such as N-acetylmannosamine (Gu and Wang, 1998).…”

Engineering of coordinated up‐ and down‐regulation of two glycosyltransferases of the O‐glycosylation pathway in Chinese hamster ovary (CHO) cells

“…Therefore, glycosylation engineering of animal cells and, in particular, of Chinese hamster ovary (CHO) cells, the preferred applied cell line for production of therapeutic glycoproteins, provides an enabling technology for generating potentially useful new glycoprotein products. The glycosylation machinery of CHO cells has been extensively characterized and can be engineered to modify glycosylation profiles by transfection with genes encoding glycosyltransferases and glycosidases or by inhibiting particular oligosaccharide biosynthesis enzymes by antisense technology Ferrari et al, 1998, Monteith andLevin, 1999;Prati et al, 1998: Roush, 1997.…”