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

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 × 107 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 × 107 TU mL−1 in a semi‐perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 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. Graphical and Lay Summary Lentiviral vectors (LVs) are widely used for gene and cell therapies, but process yields are limited by traditional production methods like transient transfection and cytotoxic effects of some expressed vector components. In this work, we established stable suspension producer cell lines for scalable, serum‐free lentivirus production. The obtained producer clones produce LVs pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV‐G) for several weeks in a perfusion stirred‐tank bioreactor process. The purified and concentrated LVs efficiently transduce human hematopoietic stem cells, which are common target cells for ex vivo gene and cell therapies.