Reduction of CMP-N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy.
Rodent cells, widely used for the industrial production of recombinant human glycoproteins, possess CMP-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac 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 Neu5Gc residues from 4% in the parental cells to less than 1% in the CHO-AsUH2 cell line.
Chenu
Stephane
S
Laboratoire des Sciences du Génie Chimique, CNRS-ENSAIA, 2, av. de la Forêt de Haye, F-54505 Vandoeuvre-lès-Nancy, France.
Grégoire
Anne
A
Malykh
Yanina
Y
Visvikis
Athanase
A
Monaco
Lucia
L
Shaw
Lee
L
Schauer
Roland
R
Marc
Annie
A
Goergen
Jean-Louis
JL
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000201:enzymology / Q000378:metabolism*; D002455:Cell Division; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000096:biosynthesis; D039642:Eukaryotic Initiation Factors; D006001:Glycoconjugates / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009438:Neuraminic Acids / Q000032:analysis / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D013997:Time Factors
D018076:DNA, Complementary; D039642:Eukaryotic Initiation Factors; D006001:Glycoconjugates; D009438:Neuraminic Acids; D016372:RNA, Antisense; D012333:RNA, Messenger; C032592:N-glycolylneuraminic acid; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase