Lent-On-Plus: An all-in-one transactivator-free doxycycline inducible LVs ready for clinical applications

C Blanco-Benítez(1,2) M Tristán-Manzano(1) P Justicia-Lirio(1,2) M E Calleja-Cervantes(3,4) J R Rodriguez-Madoz(3,6,7) F Prosper(3,5,6,7) H Boulaiz(8) F Martin(2)

1:LentiStem Biotech. GENYO. Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional Government.PTS Granada - Avenida de la Ilustración, 114 - 18016 GRANADA, Spain.; 2:Departamento de bioquímica y biología molecular iii e inmunología. Facultad de medicina. Universidad de Granada. Av. de la Investigación, 11, 18016 Granada.; 3:Hemato-Oncology Program. Cima Universidad de Navarra. Pamplona, Spain.; 4:Computational BiologyProgram. Cima Universidad de Navarra. Pamplona, Spain; 5:Hematology and Cell Therapy Department. ClinicaUniversidad de Navarra. Pamplona, Spain.; 6:Instituto de Investigación Sanitaria de Navarra (IdiSNA). Pamplona, Spain.; 7:Centro de Investigación Biomédica en Red de Cancer (CIBERONC). Madrid, Spain; 8:Departamento de Anatomía y Embriología Humana. Facultad de medicina. Universidad de Granada. Av. de la Investigación, 11, 18016 Granada.

Tet-On systems induced by tetracycline and its derivatives are used for a variety of applications, including gene therapy. However, most of these Tet-On systems use rtTAtransactivator proteins (a TetR-VP16 chimera), which constitute a safety issue, since VP16 has been shown to cause different types of cellular toxicities by sequestering transcription factors and by activating cellular genes due to the binding of the TetR-VP16 protein to pseudo-TetO sites. In the present study we analysed the suitability of the Lent-On-Plus system (without transactivators) for clinical applications by studying doxycycline responsiveness, stability of the transgene over time and potential alterations of the cells RNA content on induced cells.For comparison, the same analysis was performed using the state of the art Tet-On 3G LVs from Takara bio. Our results show that the minimum dose necessary to achieve maximum GFP expression with the Lent-On-Plus system is 0.1ng/ml, while the Tet-On 3G system requires up to 3µg/ml, although the systems start to respond at 0.01ng/ml and 10ng/ml respectively. Additionally, transgene expression was stable for over 60 days in culture in cells transduced with the Lent-On-Plus system while around half of those generated with the Tet-On 3G LVs were silenced. More importantly, transcriptomic RNA-Seq analysis showed a large number of altered genes in cells generated with Tet-On 3G LVs, while these changes were minimal on cells generated with the Lent-On-Plus LVs.All this data together points to the Lent-On-Plus LVs as a safer alternative for gene therapy applications.