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Deciphering key parameters enhancing lentiviral vector producer cells yields
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Resumo(s)
The use of lentiviral vectors (LVs) in gene therapy is expanding, demanding high-quality viral preparations. Producer cell lines for LV production offer robust manufacturing platforms. However, their development is still progressing and more knowledge on the impact of vector components expression levels on vector yields and quality is essential. This work studies the impact of vector cassette expression and stability on vector titer and quality, identifying key parameters in cell line development. Ten heterogeneous LV stable producer clones established through random cassette integration were characterized. The gag-pol and rev cassettes, expressed under the control of constitutive promoters, showed robust expression generating titers of 109 physical particles (P.P.s)/mL. However, Pol and reverse transcriptase expressions were shown to be better indicators of potential functional titers. Envelope and transfer vector expression levels were key to attaining high functional particles yields. The stability analysis of two top clones and their trans-complementation with each genetic cassette further supported this conclusion. The producer LV clones expressed constitutively the 4070A envelope, but the overexpression of the VSV-G envelope increased 30-fold the titer supporting the envelope as key determinant in LV quality. This work further elucidates bottlenecks in LV producer cell line development providing insights for their optimization.
Descrição
Funding Information: M.V.F. acknowledges Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia for PhD fellowship UI/BD/151256/2021 (DOI 10.54499/UI/BD/151256/2021) within the scope of the PhD Program in Bioengineering\u2014 Cell Therapies and Regenerative Medicine. This work was funded by the Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia/Minist\u00E9rio da Ci\u00EAncia, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). A.S.F.-O. acknowledges the Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia for the award of the individual grant SFRH/BD/697002/2013. Publisher Copyright: © 2025 The Author(s)
Palavras-chave
gene therapy lentiviral vectors LV cassettes copy number LV cassettes expression levels LV genetic cassettes LV manufacturing LV stability stable constitutive producer cell line Molecular Medicine Molecular Biology Genetics