Generation of stable suspension producer cell lines for serum-free lentivirus production.

Publisher: Wiley-VCH Verlag Country of Publication: Germany NLM ID: 101265833 Publication Model: Print Cited Medium: Internet ISSN: 1860-7314 (Electronic) Linking ISSN: 18606768 NLM ISO Abbreviation: Biotechnol J Subsets: MEDLINE

Original Publication: Weinheim : Wiley-VCH Verlag, c2006-

Lentivirus*/genetics ; Bioreactors* ; Genetic Vectors*/genetics; Humans ; Culture Media, Serum-Free ; Cell Line ; Cell Culture Techniques/methods ; Virus Cultivation/methods ; HEK293 Cells ; Transfection/methods

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 10 ⁷  TU mL ^-1  in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 10 ⁷  TU mL ^-1  in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 10 ⁷  TU mL ^-1  day ^-1  for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34 ⁺ cells, reducing the LV quantities required for gene and cell therapy applications.
(© 2024 CSL Innovation GmbH and The Authors. Biotechnology Journal published by Wiley‐VCH GmbH.)

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CSL Limited

Keywords: cell line development; gene and cell therapy; hematopoietic stem cells; lentiviral vector; lentiviral vector production; stable suspension producer cell line

0 (Culture Media, Serum-Free)

Date Created: 20240508 Date Completed: 20240508 Latest Revision: 20240508

20240509

10.1002/biot.202400090

38719592