Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors
To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGF...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1076524/full |
| _version_ | 1797852459661524992 |
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| author | Yasemin van Heuvel Yasemin van Heuvel Stefanie Schatz Stefanie Schatz Marc Hein Marc Hein Tanya Dogra Daniel Kazenmaier Daniel Kazenmaier Natalie Tschorn Natalie Tschorn Yvonne Genzel Jörn Stitz |
| author_facet | Yasemin van Heuvel Yasemin van Heuvel Stefanie Schatz Stefanie Schatz Marc Hein Marc Hein Tanya Dogra Daniel Kazenmaier Daniel Kazenmaier Natalie Tschorn Natalie Tschorn Yvonne Genzel Jörn Stitz |
| author_sort | Yasemin van Heuvel |
| collection | DOAJ |
| description | To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 106 transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 106 TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 107 TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale. |
| first_indexed | 2024-04-09T19:34:39Z |
| format | Article |
| id | doaj.art-8c77412358714224822d96c752627bca |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-09T19:34:39Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-8c77412358714224822d96c752627bca2023-04-04T16:28:20ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-04-011110.3389/fbioe.2023.10765241076524Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactorsYasemin van Heuvel0Yasemin van Heuvel1Stefanie Schatz2Stefanie Schatz3Marc Hein4Marc Hein5Tanya Dogra6Daniel Kazenmaier7Daniel Kazenmaier8Natalie Tschorn9Natalie Tschorn10Yvonne Genzel11Jörn Stitz12Research Group Medical Biotechnology and Bioengineering, Faculty of Applied Natural Sciences, University of Applied Sciences Cologne, Campus Leverkusen, Cologne, GermanyInstitute of Technical Chemistry, Gottfried Wilhelm Leibniz University Hannover, Hanover, GermanyResearch Group Medical Biotechnology and Bioengineering, Faculty of Applied Natural Sciences, University of Applied Sciences Cologne, Campus Leverkusen, Cologne, GermanyInstitute of Technical Chemistry, Gottfried Wilhelm Leibniz University Hannover, Hanover, GermanyChair of Bioprocess Engineering, Otto-Von-Guericke-University Magdeburg, Magdeburg, GermanyMax Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, GermanyMax Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, GermanyMax Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, GermanyFaculty of Biotechnology, University of Applied Sciences Mannheim, Mannheim, GermanyResearch Group Medical Biotechnology and Bioengineering, Faculty of Applied Natural Sciences, University of Applied Sciences Cologne, Campus Leverkusen, Cologne, GermanyInstitute of Technical Chemistry, Gottfried Wilhelm Leibniz University Hannover, Hanover, GermanyMax Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, GermanyResearch Group Medical Biotechnology and Bioengineering, Faculty of Applied Natural Sciences, University of Applied Sciences Cologne, Campus Leverkusen, Cologne, GermanyTo date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 106 transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 106 TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 107 TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1076524/fullsleeping beauty transposonmRNA transfectionsuspension cellretroviral vectormurine leukemia virus (MLV)stirred-tank bioreactor |
| spellingShingle | Yasemin van Heuvel Yasemin van Heuvel Stefanie Schatz Stefanie Schatz Marc Hein Marc Hein Tanya Dogra Daniel Kazenmaier Daniel Kazenmaier Natalie Tschorn Natalie Tschorn Yvonne Genzel Jörn Stitz Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors Frontiers in Bioengineering and Biotechnology sleeping beauty transposon mRNA transfection suspension cell retroviral vector murine leukemia virus (MLV) stirred-tank bioreactor |
| title | Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors |
| title_full | Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors |
| title_fullStr | Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors |
| title_full_unstemmed | Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors |
| title_short | Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors |
| title_sort | novel suspension retroviral packaging cells generated by transposition using transposase encoding mrna advance vector yields and enable production in bioreactors |
| topic | sleeping beauty transposon mRNA transfection suspension cell retroviral vector murine leukemia virus (MLV) stirred-tank bioreactor |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1076524/full |
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