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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 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.
Author:Yasemin van Heuvel, Stefanie Schatz, Marc Hein, Tanya Dogra, Daniel Kazenmaier, Natalie Tschorn, Yvonne Genzel, Jörn Stitz
Parent Title (English):Frontiers in Bioengineering and Biotechnology
Publisher:Frontiers Media
Document Type:Article
Date of first Publication:2023/06/15
Date of Publication (online):2023/06/15
Tag:Gene Therapy; Murine Leukemia Virus (MLV); Retroviral Vector; Sleeping Beauty Transposon; Stirred-Tank Bioreactor; Suspension Cell; mRNA Transfection
Article Number:1076524
Page Number:10
Institutes:Angewandte Naturwissenschaften (F11)
Dewey Decimal Classification:500 Naturwissenschaften und Mathematik
Open Access:Open Access
OA-Publicationfonds TH Köln:OA-Publicationfonds TH Köln
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International