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

authored by

Yasemin van Heuvel, Stefanie Schatz, Marc Hein, Tanya Dogra, Daniel Kazenmaier, Natalie Tschorn, Yvonne Genzel, Jörn Stitz

Abstract

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.

Organisation(s)

Institute of Technical Chemistry

External Organisation(s)

TH Köln - University of Applied Sciences
Otto-von-Guericke University Magdeburg
Max Planck Institute for Dynamics of Complex Technical Systems
Mannheim University of Applied Sciences

Type

Article

Journal

Frontiers in Bioengineering and Biotechnology

Volume

11

ISSN

2296-4185

Publication date

04.04.2023

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Biotechnology, Bioengineering, Histology, Biomedical Engineering

Electronic version(s)

https://doi.org/10.3389/fbioe.2023.1076524 (Access: Open)