Chenu Stephane, Grégoire Anne, Malykh Yanina, Visvikis Athanase, Monaco Lucia, Shaw Lee, Schauer Roland, Marc Annie, Goergen Jean-Louis
Laboratoire des Sciences du Génie Chimique, CNRS-ENSAIA, 2, av. de la Forêt de Haye, F-54505 Vandoeuvre-lès-Nancy, France.
Biochim Biophys Acta. 2003 Jul 23;1622(2):133-44. doi: 10.1016/s0304-4165(03)00137-5.
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.
啮齿动物细胞被广泛用于重组人糖蛋白的工业化生产，其具有CMP-N-乙酰神经氨酸羟化酶（CMP-Neu5Ac羟化酶；EC 1.14.13.45），该酶是唾液酸N-羟乙酰神经氨酸（Neu5Gc）形成过程中的关键酶。这种酶在人类中不以活性形式表达，有证据表明重组治疗性糖蛋白中Neu5Gc的存在可能引发免疫反应。因此，这项工作的目的是使用合理的反义RNA方法降低中国仓鼠卵巢（CHO）细胞系中CMP-Neu5Ac羟化酶的活性，从而降低所得糖缀合物中Neu5Gc的含量。为此，仓鼠羟化酶的cDNA被部分克隆并测序。基于小鼠和仓鼠cDNA的序列，从初步的体外翻译试验中选择了最佳的反义RNA片段。与亲代细胞系相比，用源自小鼠CMP-Neu5Ac羟化酶cDNA的199 bp反义片段转染的新菌株（CHO-AsUH2）的羟化酶活性降低了80%。对细胞自身糖缀合物中存在的唾液酸进行分析发现，Neu5Gc残基的百分比从亲代细胞中的4%降至CHO-AsUH2细胞系中的不到1%。
