Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells

María Tristán-Manzano; Noelia Maldonado-Pérez; Pedro Justicia-Lirio; Marina Cortijo-Gutierréz; Pablo Tristán-Ramos; Carlos Blanco-Benítez; Kristina Pavlovic; Araceli Aguilar-González; Pilar Muñoz; Francisco J Molina-Estevez; Valerie Griesche; Juan Antonio Marchal; Sara R Heras; Karim Benabdellah; Francisco Martin

doi:10.1016/j.omtn.2023.03.018

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells

Mol Ther Nucleic Acids. 2023 Mar 28:32:322-339. doi: 10.1016/j.omtn.2023.03.018. eCollection 2023 Jun 13.

Authors

María Tristán-Manzano¹, Noelia Maldonado-Pérez^{2

3}, Pedro Justicia-Lirio^{1

2}, Marina Cortijo-Gutierréz², Pablo Tristán-Ramos^{2

4}, Carlos Blanco-Benítez^{1

2}, Kristina Pavlovic^{2

5}, Araceli Aguilar-González^{2

6}, Pilar Muñoz^{2

7}, Francisco J Molina-Estevez², Valerie Griesche^{2

8}, Juan Antonio Marchal^{9

10

11

12}, Sara R Heras^{2

4}, Karim Benabdellah², Francisco Martin^{2

3

9

10}

Affiliations

¹ LentiStem Biotech, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), PTS, Avda. de la Ilustración 114, 18016 Granada, Spain.
² Department of Genomic Medicine, Pfizer-University of Granada-Andalusian Regional Government Centre for Genomics and Oncological Research (GENYO), PTS, Av. de la Ilustración 114, 18016 Granada, Spain.
³ Departamento de Bioquimica y Biología Molecular III e Inmunologia, Facultad de Medicina, Universidad de Granada, PTS Granada - Avenida de la Investigación, 11, 18006 Granada, Spain.
⁴ Department de Bioquimica y Biología Molecular II, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain.
⁵ Maimonides Institute of Biomedical Research in Cordoba (IMIBIC), Cellular Therapy Unit, Reina Sofía University Hospital, University of Cordoba, Cordoba, Spain.
⁶ Department of Medicinal & Organic Chemistry and Excellence Research Unit of "Chemistry Applied to Biomedicine and the Environment", Faculty of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain.
⁷ Department de Biología Celular, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva, 18071 Granada, Spain.
⁸ DKFZ. German Cancer Research Center, Heidelberg, Baden-Wurtemberg, Germany.
⁹ Instituto de Investigación Biosanitaria ibs.GRANADA, University Hospitals of Granada - University of Granada, 18016 Granada, Spain.
¹⁰ Excellence Research Unit "Modeling Nature" (MNat), University of Granada, 18016 Granada, Spain.
¹¹ Biopathology and Regenerative Medicine Institute (IBIMER), Center for Biomedicinal Research (CIBM), University of Granada, 18016 Granada, Spain.
¹² Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Biosanitary Research Institute of Granada (ibs.GRANADA), Granada, Spain.

Abstract

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 3' 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 transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

Keywords: MT: Delivery Strategies; advance therapy medicinal products; all-in-one lentiviral vectors; doxycycline; externally controlled; gene therapy; inducible; inducible CAR-T cells; transactivator-free.