Reduction of CMP-N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy

被引:31
作者
Chenu, S
Grégoire, A
Malykh, Y
Visvikis, A
Monaco, L
Shaw, L
Schauer, R
Marc, A
Goergen, JL
机构
[1] ENSAIA, CNRS, Lab Sci Genie Chim, F-54505 Vandoeuvre Les Nancy, France
[2] Univ Kiel, Inst Biochem, D-24098 Kiel, Germany
[3] Univ H Poincare, Fac Pharm, F-54000 Nancy, France
[4] Ist Sci San Raffaele, DIBIT, I-20132 Milan, Italy
来源
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS | 2003年 / 1622卷 / 02期
关键词
CMP-Neu5Ac hydroxylase; sialylation; N-glycolylneuraminic acid; CHO cell; antisense RNA;
D O I
10.1016/S0304-4165(03)00137-5
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Rodent cells, widely used for the industrial production of recombinant human glycoproteins, possess CMP-N-acetylneuraminic acid hydroxylase (CMP-Ncu5Ac hydroxylase; EC 1.14.13.45) which is the key enzyme in the formation of the sialic acid, N-glycolylneuraminic acid (Neu5Gc). This enzyme is not expressed in an active form in man and evidence suggests that the presence of Neu5Gc in recombinant therapeutic glycoproteins may elicit an immune response. The aim of this work was, therefore, to reduce CMP-Neu5Ac hydroxylase activity in a Chinese Hamster Ovary (CHO) cell line, and thus the Neu5Gc content of the resulting glycoconjugates, using a rational antisense RNA approach. For this purpose, the cDNA of the hamster hydroxylase was partially cloned and sequenced. Based on the sequence of the mouse and hamster cDNAs, optimal antisense RNA fragments were selected from preliminary in vitro translation tests. Compared to the parental cell line, the new strain (CHO-AsUH2), which was transfected with a 199-bp antisense fragment derived from the mouse CMP-Neu5Ac hydroxylase cDNA, showed an 80% reduction in hydroxylase activity. An analysis of the sialic acids present in the cells' own glycoconjugates revealed a decrease in the percentage of Neu5Ge residues from 4% in the parental cells to less than 1% in the CHO-AsUH2 cell line. (C) 2003 Elsevier B.V. All rights reserved.
引用
收藏
页码:133 / 144
页数:12
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