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.Spanish ISCIII Health Research FundEuropean Union (EU) PI18/00337
PI21/00298
RD21/0017/0004
PI18/00330
PI17/00672Red TerAvJunta de Andalucia FEDER/European Cohesion Fund (FSE) for AndalusiaSpanish Government PI18/00337
PI21/00298European Union-NextGenerationEU - Maria Zambrano Senior Program RD21/0017/0004
PI18/00330
PI17/00672Ministry of Health 2016000073332-TRA
PI-57069
CARTPI-0001-201
PE-CART-0031-2020
PI-0014-2016
PECART-0027-2020
ProyExcel_00875
PEER-0286-2019European Cooperation in Science and Technology (COST) 00123009/SNEO-20191072MINECO - European Regional Development Fund PLEC2021-008094Spanish Government 0006/2018FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades CA21113Spanish Government SAF2015-71589-PMCI RYC-2016-21395German Research Foundation (DFG) PY20_00619 y A-CTS-28_UGR20Biomedicine Program of the University of Granada (Spain) FPU16/05467
FPU17/02268
FPU17/04327
DIN2018-010180
DIN2020-011550
PEJ-2018-001760-
