Self-inactivating helper virus for the production of high-capacity adenoviral vectors
Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (Ψ) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and...
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| Veröffentlicht in: | Gene therapy 2011-11, Vol.18 (11), p.1025-1033 |
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| creator | Gonzalez-Aparicio, M Mauleon, I Alzuguren, P Bunuales, M Gonzalez-Aseguinolaza, G San Martín, C Prieto, J Hernandez-Alcoceba, R |
| description | Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (Ψ) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which Ψ is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers >10
10
infectious units and |
| doi_str_mv | 10.1038/gt.2011.58 |
| format | Article |
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10
infectious units and <0.1% HV contamination. The residual HVs lacked Ψ and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of HC-Ads.</description><identifier>ISSN: 0969-7128</identifier><identifier>EISSN: 1476-5462</identifier><identifier>DOI: 10.1038/gt.2011.58</identifier><identifier>PMID: 21525953</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/326/596/1278 ; 631/326/596/2561 ; 631/45/607/1170 ; Adenoviridae - genetics ; Adenovirus ; Adenoviruses ; Adherent cells ; Analysis ; Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy ; Applied cell therapy and gene therapy ; Biological and medical sciences ; Biomedical and Life Sciences ; Biomedicine ; Biotechnology ; Cell Biology ; Contamination ; Doxycycline - pharmacology ; enabling-technologies ; Encapsidation ; Expression vectors ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Gene Therapy ; Genetic Therapy - methods ; Genetic Vectors - chemical synthesis ; Genomes ; Health aspects ; Health. Pharmaceutical industry ; Helper Viruses - genetics ; Human Genetics ; Industrial applications and implications. Economical aspects ; Integrases - administration & dosage ; Medical sciences ; Nanotechnology ; Nuclear transport ; Packaging ; Physiological aspects ; Recombinase ; Tamoxifen - analogs & derivatives ; Tamoxifen - pharmacology ; Transfusions. Complications. Transfusion reactions. Cell and gene therapy ; Vector space ; Viral proteins ; Viruses</subject><ispartof>Gene therapy, 2011-11, Vol.18 (11), p.1025-1033</ispartof><rights>Macmillan Publishers Limited 2011</rights><rights>2015 INIST-CNRS</rights><rights>COPYRIGHT 2011 Nature Publishing Group</rights><rights>Macmillan Publishers Limited 2011.</rights><rights>Copyright Nature Publishing Group Nov 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c630t-55ed055f0da4c590afccdf33be7d07a90c68b35a88327900d832ad05507f05fd3</citedby><cites>FETCH-LOGICAL-c630t-55ed055f0da4c590afccdf33be7d07a90c68b35a88327900d832ad05507f05fd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/gt.2011.58$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/gt.2011.58$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27923,27924,41487,42556,51318</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=24729194$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21525953$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gonzalez-Aparicio, M</creatorcontrib><creatorcontrib>Mauleon, I</creatorcontrib><creatorcontrib>Alzuguren, P</creatorcontrib><creatorcontrib>Bunuales, M</creatorcontrib><creatorcontrib>Gonzalez-Aseguinolaza, G</creatorcontrib><creatorcontrib>San Martín, C</creatorcontrib><creatorcontrib>Prieto, J</creatorcontrib><creatorcontrib>Hernandez-Alcoceba, R</creatorcontrib><title>Self-inactivating helper virus for the production of high-capacity adenoviral vectors</title><title>Gene therapy</title><addtitle>Gene Ther</addtitle><addtitle>Gene Ther</addtitle><description>Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (Ψ) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which Ψ is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers >10
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infectious units and <0.1% HV contamination. The residual HVs lacked Ψ and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of HC-Ads.</description><subject>631/326/596/1278</subject><subject>631/326/596/2561</subject><subject>631/45/607/1170</subject><subject>Adenoviridae - genetics</subject><subject>Adenovirus</subject><subject>Adenoviruses</subject><subject>Adherent cells</subject><subject>Analysis</subject><subject>Anesthesia. Intensive care medicine. Transfusions. 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Economical aspects</topic><topic>Integrases - administration & dosage</topic><topic>Medical sciences</topic><topic>Nanotechnology</topic><topic>Nuclear transport</topic><topic>Packaging</topic><topic>Physiological aspects</topic><topic>Recombinase</topic><topic>Tamoxifen - analogs & derivatives</topic><topic>Tamoxifen - pharmacology</topic><topic>Transfusions. Complications. Transfusion reactions. 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To avoid HV encapsidation, its packaging signal (Ψ) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which Ψ is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers >10
10
infectious units and <0.1% HV contamination. The residual HVs lacked Ψ and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of HC-Ads.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>21525953</pmid><doi>10.1038/gt.2011.58</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Gene therapy, 2011-11, Vol.18 (11), p.1025-1033 |
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| subjects | 631/326/596/1278 631/326/596/2561 631/45/607/1170 Adenoviridae - genetics Adenovirus Adenoviruses Adherent cells Analysis Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy Applied cell therapy and gene therapy Biological and medical sciences Biomedical and Life Sciences Biomedicine Biotechnology Cell Biology Contamination Doxycycline - pharmacology enabling-technologies Encapsidation Expression vectors Fundamental and applied biological sciences. Psychology Gene Expression Gene Therapy Genetic Therapy - methods Genetic Vectors - chemical synthesis Genomes Health aspects Health. Pharmaceutical industry Helper Viruses - genetics Human Genetics Industrial applications and implications. Economical aspects Integrases - administration & dosage Medical sciences Nanotechnology Nuclear transport Packaging Physiological aspects Recombinase Tamoxifen - analogs & derivatives Tamoxifen - pharmacology Transfusions. Complications. Transfusion reactions. Cell and gene therapy Vector space Viral proteins Viruses |
| title | Self-inactivating helper virus for the production of high-capacity adenoviral vectors |
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