Construction of stable packaging cell lines for clinical lentiviral vector production
Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to...
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| Veröffentlicht in: | Scientific reports 2015-03, Vol.5 (1), p.9021-9021, Article 9021 |
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| creator | Sanber, Khaled S. Knight, Sean B. Stephen, Sam L. Bailey, Ranbir Escors, David Minshull, Jeremy Santilli, Giorgia Thrasher, Adrian J. Collins, Mary K. Takeuchi, Yasuhiro |
| description | Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10
6
transducing units/ml can be harvested from the final producer clones, which can be increased to 10
8
TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors. |
| doi_str_mv | 10.1038/srep09021 |
| format | Article |
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6
transducing units/ml can be harvested from the final producer clones, which can be increased to 10
8
TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.</description><identifier>ISSN: 2045-2322</identifier><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/srep09021</identifier><identifier>PMID: 25762005</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>13 ; 13/106 ; 13/31 ; 13/44 ; 38/22 ; 38/77 ; 38/88 ; 38/90 ; 631/61 ; 631/61/201 ; Antibiotics ; Blood cancer ; Cell lines ; Clinical trials ; Cloning ; Cre recombinase ; Cytomegalovirus ; Cytotoxicity ; gag Gene Products, Human Immunodeficiency Virus - genetics ; gag Gene Products, Human Immunodeficiency Virus - metabolism ; Gene Expression ; Gene therapy ; Gene transfer ; Genetic disorders ; Genetic Vectors - genetics ; Genomes ; HEK293 Cells ; HIV-1 - genetics ; HIV-1 - metabolism ; Homologous Recombination ; Humanities and Social Sciences ; Humans ; Lentivirus - genetics ; multidisciplinary ; Packaging ; Plasmids ; pol Gene Products, Human Immunodeficiency Virus - genetics ; pol Gene Products, Human Immunodeficiency Virus - metabolism ; Protein Precursors - genetics ; Protein Precursors - metabolism ; Proteins ; Retroviridae - genetics ; Retroviridae - metabolism ; rev Gene Products, Human Immunodeficiency Virus - genetics ; rev Gene Products, Human Immunodeficiency Virus - metabolism ; Science ; Stem cells ; Transfection ; Vectors (Biology) ; Viral Envelope Proteins - genetics ; Viral Envelope Proteins - metabolism ; Virus Assembly</subject><ispartof>Scientific reports, 2015-03, Vol.5 (1), p.9021-9021, Article 9021</ispartof><rights>The Author(s) 2015</rights><rights>Copyright Nature Publishing Group Mar 2015</rights><rights>Copyright © 2015, Macmillan Publishers Limited. All rights reserved 2015 Macmillan Publishers Limited. All rights reserved</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c504t-2f83f79e8d0fef756122856149d86213a5028366e5ec8622a184b8ba7fd714273</citedby><cites>FETCH-LOGICAL-c504t-2f83f79e8d0fef756122856149d86213a5028366e5ec8622a184b8ba7fd714273</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356972/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356972/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,27903,27904,41099,42168,51554,53769,53771</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25762005$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sanber, Khaled S.</creatorcontrib><creatorcontrib>Knight, Sean B.</creatorcontrib><creatorcontrib>Stephen, Sam L.</creatorcontrib><creatorcontrib>Bailey, Ranbir</creatorcontrib><creatorcontrib>Escors, David</creatorcontrib><creatorcontrib>Minshull, Jeremy</creatorcontrib><creatorcontrib>Santilli, Giorgia</creatorcontrib><creatorcontrib>Thrasher, Adrian J.</creatorcontrib><creatorcontrib>Collins, Mary K.</creatorcontrib><creatorcontrib>Takeuchi, Yasuhiro</creatorcontrib><title>Construction of stable packaging cell lines for clinical lentiviral vector production</title><title>Scientific reports</title><addtitle>Sci Rep</addtitle><addtitle>Sci Rep</addtitle><description>Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10
6
transducing units/ml can be harvested from the final producer clones, which can be increased to 10
8
TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.</description><subject>13</subject><subject>13/106</subject><subject>13/31</subject><subject>13/44</subject><subject>38/22</subject><subject>38/77</subject><subject>38/88</subject><subject>38/90</subject><subject>631/61</subject><subject>631/61/201</subject><subject>Antibiotics</subject><subject>Blood cancer</subject><subject>Cell lines</subject><subject>Clinical trials</subject><subject>Cloning</subject><subject>Cre recombinase</subject><subject>Cytomegalovirus</subject><subject>Cytotoxicity</subject><subject>gag Gene Products, Human Immunodeficiency Virus - genetics</subject><subject>gag Gene Products, Human Immunodeficiency Virus - metabolism</subject><subject>Gene Expression</subject><subject>Gene therapy</subject><subject>Gene transfer</subject><subject>Genetic disorders</subject><subject>Genetic Vectors - genetics</subject><subject>Genomes</subject><subject>HEK293 Cells</subject><subject>HIV-1 - genetics</subject><subject>HIV-1 - metabolism</subject><subject>Homologous Recombination</subject><subject>Humanities and Social Sciences</subject><subject>Humans</subject><subject>Lentivirus - genetics</subject><subject>multidisciplinary</subject><subject>Packaging</subject><subject>Plasmids</subject><subject>pol Gene Products, Human Immunodeficiency Virus - genetics</subject><subject>pol Gene Products, Human Immunodeficiency Virus - metabolism</subject><subject>Protein Precursors - genetics</subject><subject>Protein Precursors - metabolism</subject><subject>Proteins</subject><subject>Retroviridae - genetics</subject><subject>Retroviridae - metabolism</subject><subject>rev Gene Products, Human Immunodeficiency Virus - genetics</subject><subject>rev Gene Products, Human Immunodeficiency Virus - metabolism</subject><subject>Science</subject><subject>Stem cells</subject><subject>Transfection</subject><subject>Vectors (Biology)</subject><subject>Viral Envelope Proteins - genetics</subject><subject>Viral Envelope Proteins - 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genetics</topic><topic>gag Gene Products, Human Immunodeficiency Virus - metabolism</topic><topic>Gene Expression</topic><topic>Gene therapy</topic><topic>Gene transfer</topic><topic>Genetic disorders</topic><topic>Genetic Vectors - genetics</topic><topic>Genomes</topic><topic>HEK293 Cells</topic><topic>HIV-1 - genetics</topic><topic>HIV-1 - metabolism</topic><topic>Homologous Recombination</topic><topic>Humanities and Social Sciences</topic><topic>Humans</topic><topic>Lentivirus - genetics</topic><topic>multidisciplinary</topic><topic>Packaging</topic><topic>Plasmids</topic><topic>pol Gene Products, Human Immunodeficiency Virus - genetics</topic><topic>pol Gene Products, Human Immunodeficiency Virus - metabolism</topic><topic>Protein Precursors - genetics</topic><topic>Protein Precursors - metabolism</topic><topic>Proteins</topic><topic>Retroviridae - genetics</topic><topic>Retroviridae - metabolism</topic><topic>rev Gene Products, Human Immunodeficiency Virus - genetics</topic><topic>rev Gene Products, Human Immunodeficiency Virus - metabolism</topic><topic>Science</topic><topic>Stem cells</topic><topic>Transfection</topic><topic>Vectors (Biology)</topic><topic>Viral Envelope Proteins - genetics</topic><topic>Viral Envelope Proteins - metabolism</topic><topic>Virus Assembly</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sanber, Khaled S.</creatorcontrib><creatorcontrib>Knight, Sean B.</creatorcontrib><creatorcontrib>Stephen, Sam L.</creatorcontrib><creatorcontrib>Bailey, Ranbir</creatorcontrib><creatorcontrib>Escors, David</creatorcontrib><creatorcontrib>Minshull, Jeremy</creatorcontrib><creatorcontrib>Santilli, Giorgia</creatorcontrib><creatorcontrib>Thrasher, Adrian J.</creatorcontrib><creatorcontrib>Collins, Mary K.</creatorcontrib><creatorcontrib>Takeuchi, Yasuhiro</creatorcontrib><collection>Springer Nature Open Access Journals</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>PML(ProQuest Medical Library)</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Scientific reports</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sanber, Khaled S.</au><au>Knight, Sean B.</au><au>Stephen, Sam L.</au><au>Bailey, Ranbir</au><au>Escors, David</au><au>Minshull, Jeremy</au><au>Santilli, Giorgia</au><au>Thrasher, Adrian J.</au><au>Collins, Mary K.</au><au>Takeuchi, Yasuhiro</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Construction of stable packaging cell lines for clinical lentiviral vector production</atitle><jtitle>Scientific reports</jtitle><stitle>Sci Rep</stitle><addtitle>Sci Rep</addtitle><date>2015-03-12</date><risdate>2015</risdate><volume>5</volume><issue>1</issue><spage>9021</spage><epage>9021</epage><pages>9021-9021</pages><artnum>9021</artnum><issn>2045-2322</issn><eissn>2045-2322</eissn><abstract>Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10
6
transducing units/ml can be harvested from the final producer clones, which can be increased to 10
8
TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>25762005</pmid><doi>10.1038/srep09021</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 13 13/106 13/31 13/44 38/22 38/77 38/88 38/90 631/61 631/61/201 Antibiotics Blood cancer Cell lines Clinical trials Cloning Cre recombinase Cytomegalovirus Cytotoxicity gag Gene Products, Human Immunodeficiency Virus - genetics gag Gene Products, Human Immunodeficiency Virus - metabolism Gene Expression Gene therapy Gene transfer Genetic disorders Genetic Vectors - genetics Genomes HEK293 Cells HIV-1 - genetics HIV-1 - metabolism Homologous Recombination Humanities and Social Sciences Humans Lentivirus - genetics multidisciplinary Packaging Plasmids pol Gene Products, Human Immunodeficiency Virus - genetics pol Gene Products, Human Immunodeficiency Virus - metabolism Protein Precursors - genetics Protein Precursors - metabolism Proteins Retroviridae - genetics Retroviridae - metabolism rev Gene Products, Human Immunodeficiency Virus - genetics rev Gene Products, Human Immunodeficiency Virus - metabolism Science Stem cells Transfection Vectors (Biology) Viral Envelope Proteins - genetics Viral Envelope Proteins - metabolism Virus Assembly |
| title | Construction of stable packaging cell lines for clinical lentiviral vector production |
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