TY - JOUR U1 - Wissenschaftlicher Artikel A1 - van Heuvel, Yasemin A1 - Schatz, Stefanie A1 - Hein, Marc A1 - Dogra, Tanya A1 - Kazenmaier, Daniel A1 - Tschorn, Natalie A1 - Genzel, Yvonne A1 - Stitz, Jörn T1 - Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors JF - Frontiers in Bioengineering and Biotechnology N2 - 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. KW - Sleeping Beauty Transposon KW - mRNA Transfection KW - Suspension Cell KW - Retroviral Vector KW - Murine Leukemia Virus (MLV) KW - Stirred-Tank Bioreactor KW - Gene Therapy Y1 - 2023 UN - https://nbn-resolving.org/urn:nbn:de:hbz:832-epub4-21473 SN - 2296-4185 SS - 2296-4185 U6 - https://doi.org/10.3389/fbioe.2023.1076524 DO - https://doi.org/10.3389/fbioe.2023.1076524 VL - 11 SP - 10 S1 - 10 PB - Frontiers Media S.A. ER -