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 exp...
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| creator | Chenu, Stephane Grégoire, Anne Malykh, Yanina Visvikis, Athanase Monaco, Lucia Shaw, Lee Schauer, Roland Marc, Annie Goergen, Jean-Louis |
| description | 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. |
| doi_str_mv | 10.1016/S0304-4165(03)00137-5 |
| format | Article |
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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.</description><identifier>ISSN: 0304-4165</identifier><identifier>ISSN: 0006-3002</identifier><identifier>EISSN: 1872-8006</identifier><identifier>DOI: 10.1016/S0304-4165(03)00137-5</identifier><identifier>PMID: 12880951</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Animals ; Antisense RNA ; Base Sequence ; Biochemistry ; Biochemistry, Molecular Biology ; Biotechnology ; Cell Division ; CHO cell ; CHO Cells - enzymology ; CHO Cells - metabolism ; Cloning, Molecular ; CMP-Neu5Ac hydroxylase ; Cricetinae ; DNA, Complementary - biosynthesis ; Eukaryotic Initiation Factors ; Glycoconjugates - chemistry ; Glycoconjugates - metabolism ; Life Sciences ; Mixed Function Oxygenases - biosynthesis ; Mixed Function Oxygenases - genetics ; Mixed Function Oxygenases - metabolism ; Molecular Sequence Data ; N-glycolylneuraminic acid ; Neuraminic Acids - analysis ; Neuraminic Acids - metabolism ; Polymerase Chain Reaction ; Protein Engineering ; RNA, Antisense ; RNA, Messenger - analysis ; Sequence Alignment ; Sialylation ; Time Factors</subject><ispartof>Biochimica et biophysica acta, 2003-07, Vol.1622 (2), p.133-144</ispartof><rights>2003 Elsevier B.V.</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c395t-da99cb96b47ad0abfe1fe56e8d93dca9f28b3d2f18b7b308512aa8a6c91f5d893</citedby><cites>FETCH-LOGICAL-c395t-da99cb96b47ad0abfe1fe56e8d93dca9f28b3d2f18b7b308512aa8a6c91f5d893</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0304-4165(03)00137-5$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,315,781,785,886,3551,27928,27929,45999</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12880951$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.univ-lorraine.fr/hal-02270684$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Chenu, Stephane</creatorcontrib><creatorcontrib>Grégoire, Anne</creatorcontrib><creatorcontrib>Malykh, Yanina</creatorcontrib><creatorcontrib>Visvikis, Athanase</creatorcontrib><creatorcontrib>Monaco, Lucia</creatorcontrib><creatorcontrib>Shaw, Lee</creatorcontrib><creatorcontrib>Schauer, Roland</creatorcontrib><creatorcontrib>Marc, Annie</creatorcontrib><creatorcontrib>Goergen, Jean-Louis</creatorcontrib><title>Reduction of CMP- N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy</title><title>Biochimica et biophysica acta</title><addtitle>Biochim Biophys Acta</addtitle><description>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.</description><subject>Animals</subject><subject>Antisense RNA</subject><subject>Base Sequence</subject><subject>Biochemistry</subject><subject>Biochemistry, Molecular Biology</subject><subject>Biotechnology</subject><subject>Cell Division</subject><subject>CHO cell</subject><subject>CHO Cells - enzymology</subject><subject>CHO Cells - metabolism</subject><subject>Cloning, Molecular</subject><subject>CMP-Neu5Ac hydroxylase</subject><subject>Cricetinae</subject><subject>DNA, Complementary - biosynthesis</subject><subject>Eukaryotic Initiation Factors</subject><subject>Glycoconjugates - chemistry</subject><subject>Glycoconjugates - metabolism</subject><subject>Life Sciences</subject><subject>Mixed Function Oxygenases - biosynthesis</subject><subject>Mixed Function Oxygenases - genetics</subject><subject>Mixed Function Oxygenases - metabolism</subject><subject>Molecular Sequence Data</subject><subject>N-glycolylneuraminic acid</subject><subject>Neuraminic Acids - analysis</subject><subject>Neuraminic Acids - metabolism</subject><subject>Polymerase Chain Reaction</subject><subject>Protein Engineering</subject><subject>RNA, Antisense</subject><subject>RNA, Messenger - analysis</subject><subject>Sequence Alignment</subject><subject>Sialylation</subject><subject>Time Factors</subject><issn>0304-4165</issn><issn>0006-3002</issn><issn>1872-8006</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1u1DAQxy1ERbeFRwD5hOghxY7jxD6h1Qoo0ragAmfLsSe7RonT2s6KvEEfu97uqhyxRvJo_JsPzx-ht5RcUkLrjz8JI1VR0Zp_IOyCEMqagr9ACyqashCE1C_R4hk5RWcx_iH5cMlfoVNaCkEkpwv0cAt2MsmNHo8dXl3_KPBNoQ2kufcwBT047wzWxlm8nW0Y_869jpADye1cmrHzGPzGeYAAFq-22cvPWz3EBAGPOx1mbKDvI56i8xusfbbkIvgIxe3NEscUdILN_BqddLqP8OZ4n6PfXz7_Wl0V6-9fv62W68IwyVNhtZSmlXVbNdoS3XZAO-A1CCuZNVp2pWiZLTsq2qZlRHBaai10bSTtuBWSnaOLQ92t7tVdcEOeUI3aqavlWu1jpCwbUotqRzP7_sDehfF-gpjU4OL-N9rDOEXVsEryvOMM8gNowhhjgO65MiVqL5d6kkvttVCEqSe5FM95744NpnYA-y_rqE8GPh0AyCvZOQgqGgfegHUBTFJ2dP9p8QiotabL</recordid><startdate>20030723</startdate><enddate>20030723</enddate><creator>Chenu, Stephane</creator><creator>Grégoire, Anne</creator><creator>Malykh, Yanina</creator><creator>Visvikis, Athanase</creator><creator>Monaco, Lucia</creator><creator>Shaw, Lee</creator><creator>Schauer, Roland</creator><creator>Marc, Annie</creator><creator>Goergen, Jean-Louis</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>1XC</scope></search><sort><creationdate>20030723</creationdate><title>Reduction of CMP- N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy</title><author>Chenu, Stephane ; Grégoire, Anne ; Malykh, Yanina ; Visvikis, Athanase ; Monaco, Lucia ; Shaw, Lee ; Schauer, Roland ; Marc, Annie ; Goergen, Jean-Louis</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c395t-da99cb96b47ad0abfe1fe56e8d93dca9f28b3d2f18b7b308512aa8a6c91f5d893</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Animals</topic><topic>Antisense RNA</topic><topic>Base Sequence</topic><topic>Biochemistry</topic><topic>Biochemistry, Molecular Biology</topic><topic>Biotechnology</topic><topic>Cell Division</topic><topic>CHO cell</topic><topic>CHO Cells - enzymology</topic><topic>CHO Cells - metabolism</topic><topic>Cloning, Molecular</topic><topic>CMP-Neu5Ac hydroxylase</topic><topic>Cricetinae</topic><topic>DNA, Complementary - biosynthesis</topic><topic>Eukaryotic Initiation Factors</topic><topic>Glycoconjugates - chemistry</topic><topic>Glycoconjugates - metabolism</topic><topic>Life Sciences</topic><topic>Mixed Function Oxygenases - biosynthesis</topic><topic>Mixed Function Oxygenases - genetics</topic><topic>Mixed Function Oxygenases - metabolism</topic><topic>Molecular Sequence Data</topic><topic>N-glycolylneuraminic acid</topic><topic>Neuraminic Acids - analysis</topic><topic>Neuraminic Acids - metabolism</topic><topic>Polymerase Chain Reaction</topic><topic>Protein Engineering</topic><topic>RNA, Antisense</topic><topic>RNA, Messenger - analysis</topic><topic>Sequence Alignment</topic><topic>Sialylation</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chenu, Stephane</creatorcontrib><creatorcontrib>Grégoire, Anne</creatorcontrib><creatorcontrib>Malykh, Yanina</creatorcontrib><creatorcontrib>Visvikis, Athanase</creatorcontrib><creatorcontrib>Monaco, Lucia</creatorcontrib><creatorcontrib>Shaw, Lee</creatorcontrib><creatorcontrib>Schauer, Roland</creatorcontrib><creatorcontrib>Marc, Annie</creatorcontrib><creatorcontrib>Goergen, Jean-Louis</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Hyper Article en Ligne (HAL)</collection><jtitle>Biochimica et biophysica acta</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chenu, Stephane</au><au>Grégoire, Anne</au><au>Malykh, Yanina</au><au>Visvikis, Athanase</au><au>Monaco, Lucia</au><au>Shaw, Lee</au><au>Schauer, Roland</au><au>Marc, Annie</au><au>Goergen, Jean-Louis</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Reduction of CMP- N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy</atitle><jtitle>Biochimica et biophysica acta</jtitle><addtitle>Biochim Biophys Acta</addtitle><date>2003-07-23</date><risdate>2003</risdate><volume>1622</volume><issue>2</issue><spage>133</spage><epage>144</epage><pages>133-144</pages><issn>0304-4165</issn><issn>0006-3002</issn><eissn>1872-8006</eissn><abstract>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.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>12880951</pmid><doi>10.1016/S0304-4165(03)00137-5</doi><tpages>12</tpages></addata></record> |
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| language | eng |
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| subjects | Animals Antisense RNA Base Sequence Biochemistry Biochemistry, Molecular Biology Biotechnology Cell Division CHO cell CHO Cells - enzymology CHO Cells - metabolism Cloning, Molecular CMP-Neu5Ac hydroxylase Cricetinae DNA, Complementary - biosynthesis Eukaryotic Initiation Factors Glycoconjugates - chemistry Glycoconjugates - metabolism Life Sciences Mixed Function Oxygenases - biosynthesis Mixed Function Oxygenases - genetics Mixed Function Oxygenases - metabolism Molecular Sequence Data N-glycolylneuraminic acid Neuraminic Acids - analysis Neuraminic Acids - metabolism Polymerase Chain Reaction Protein Engineering RNA, Antisense RNA, Messenger - analysis Sequence Alignment Sialylation Time Factors |
| title | Reduction of CMP- N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy |
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