Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors

• Published in: Frontiers in bioengineering and biotechnology Vol. 11; p. 1076524
• Main Authors: van Heuvel, Yasemin, Schatz, Stefanie, Hein, Marc, Dogra, Tanya, Kazenmaier, Daniel, Tschorn, Natalie, Genzel, Yvonne, Stitz, Jörn
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 04.04.2023
• Subjects: Bioengineering and Biotechnology; mRNA transfection; murine leukemia virus (MLV); retroviral vector; sleeping beauty transposon; stirred-tank bioreactor; suspension cell; murine leukemia virus (MLV); stirred-tank bioreactor; retroviral vector; sleeping beauty transposon; mRNA transfection; gene therapy; suspension cell
• ISSN: 2296-4185; 2296-4185
• DOI: 10.3389/fbioe.2023.1076524

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 × 10 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 × 10  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 × 10  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.