ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Cell and Gene Therapy
Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1648028
Enhancing Lentiviral Production for WAS Gene Therapy: A Comparative Analysis of Stable Producer Cell Lines evaluating Flatware system and Adherent Bioreactors in Perfusion Mode
Provisionally accepted
Parameswari Singh1
Nikki Indresh Lal1Monica Terrao1Sarah Schwingal1Martina Biserni2Florian Aeschimann2Andrea Strauch1
Herbert Dersch1
Angel Jaramillo3
Andreas Gille3
Holger Laux1*- 1CSL Innovation GmbH, Marburg, Germany
- 2CSL Behring, Bern, Switzerland
- 3CSL Behring, Pasadena, United States
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Ex-vivo gene therapies require scalable, high-quality lentivirus (LV) with excellent transduction efficiency. Achieving this involves a synergistic approach combining efficient vector design and LV process optimization. In our study, we evaluated transfection reagents for generating stable producer cell lines from two Tet-off regulated adherent stable LV packaging PCLs, GPRG and GPRTG, to produce lentivirus (LV) to treat Wiskott Aldrich Syndrome (WAS). Stable producer cell lines expressing the WAS transgene or GFP transgene were generated from GPRG and GPRTG PCLs. The GPRTG producer cell line showed 6-fold higher LV titer and resulted in better transduction of CD34+ cells. Further, we optimized the LV production process in continuous perfusion and recirculation mode and compared three technologies: traditional flatware systems, iCELLis™ Nano and scale-X™ Hydro Univercells adherent bioreactors using GPRTG stable producer cell line. Scale-X™ Hydro outperformed iCELLis™ Nano in LV productivity per surface area (TU/cm²). We successfully scaled up LV production from Scale-X™ Hydro (2.4 m²) to Scale-X™ Carbo (10 m²), producing 1.13E+12 TU per 10 m² through 7 harvests using the continuous perfusion process. This process produced LV that efficiently transduced CD34+ cells, achieving a vector copy number (VCN) of upto 4 at a Multiplicity of Infection (MOI) of 10. Our study has successfully established a scalable, cost-effective and robust platform for LV production, demonstrating its potential for clinical applications.
Keywords: ex-vivo gene therapy, Stable packaging cell line (PCL), Lentivirus (LV), stable producer cell line, Adherent bioreactors, Perfusion process, Hematopoietic Stem Cells
Received: 16 Jun 2025; Accepted: 04 Aug 2025.
Copyright: © 2025 Singh, Lal, Terrao, Schwingal, Biserni, Aeschimann, Strauch, Dersch, Jaramillo, Gille and Laux. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Holger Laux, CSL Innovation GmbH, Marburg, Germany
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