CHINESE-HAMSTER OVARY CELLS WITH CONSTITUTIVELY EXPRESSED SIALIDASE ANTISENSE RNA PRODUCE RECOMBINANT DNASE IN BATCH CULTURE WITH INCREASEDSIALIC-ACID

Under some cell culture conditions, recombinant glycoprotein therapeut ics expressed in Chinese hamster ovary (CHO) cells lose sialic acid du ring the course of the culture (Sliwkowski et al., 1992; Munzert et at ., 1996). A soluble sialidase of CHO cell origin degrades the expresse d recombinant protein and has been shown to be released into the cultu re fluid as the viability of the cells decreases. To reduce the levels of the sialidase and to prevent desialylation of recombinant protein, a CHO cell line has been developed that constitutively expresses sial idase antisense RNA. Several antisense expression vectors were prepare d using different regions of the sialidase gene. Co-transfection of th e antisense constructs with a vector conferring puromycin resistance g ave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5' 474 bp coding seg ment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type va lues. To test if this level of sialidase would lead to increased siali c acid content of an expressed recombinant protein, the 474 antisense clone was employed as a host for expression of human DNase as a model glycoprotein. The sialic acid content of the DNase produced in the ant isense cultures was compared with material made in the wild-type paren tal cell line. About 20-37% increase in sialic acid content, or 0.6-1. 1 mole of additional sialic acid out of a total of 3.0 mole on the pro duct, was found on the DNase made in the antisense cell lines. (C) 199 8 John Wiley & Sons, Inc.