Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells Tristán Manzano, María Maldonado Pérez, Noelia Justicia Lirio, Pedro Cortijo Gutiérrez, Marina Tristán Ramos, Pablo Blanco Benítez, Carlos Pavlovic, Kristina Aguilar González, Araceli Muñoz Fernández, Pilar Molina Estévez, Francisco Javier Griesche, Valerie Marchal Corrales, Juan Antonio Heras, Sara R. Benabdellah, Karim Martín Molina, Francisco Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 30 long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivatorfree, all-one-one system capable of generating Dox-inducible ATMPs. 2023-09-28T09:58:07Z 2023-09-28T09:58:07Z 2023-06 journal article Tristán-Manzano, M., Maldonado-Pérez, N., Justicia-Lirio, P., Cortijo-Gutierréz, M., Tristán-Ramos, P., Blanco-Benítez, C., ... & Martin, F. (2023). Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells. Molecular Therapy-Nucleic Acids, 32, 322-339.[https://doi.org/10.1016/j.omtn.2023.03.018.] https://hdl.handle.net/10481/84712 10.1016/j.omtn.2023.03.018. eng info:eu-repo/grantAgreement/EC/UE/I18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672 info:eu-repo/grantAgreement/EC/NextGenerationEU/RD21/0017/0004 PI18/00330 PI17/00672 http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional CellPress