Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks

This study was funded by the Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI12/01097, PI15/02015, PI18/00337 (F.M.) PIE16-00045 (J.A.M.), DTS19/00145 (J.A.M.), and PI18/00330 (K.B.) The CECEyU and CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia provided the following research grants: 2016000073391-TRA, 2016000073332-TRA, PI-57069, and PAIDI-Bio326 (F.M.) and PI0014-2016 (K.B.). K.B. also held a Nicolas Monardes regional Ministry of Health contract (0006/2018). M.T.-M. and N.M.-P. are funded by the Spanish Ministry of Science and Innovation (SMSI) through fellowships FPU16/05467 and FPU17/02268, respectively. M.C.-G is funded by the SMSI through fellowship (PEJ-2018-001760-A). The Ramon y Cajal grant RYC-2015-18382 to P.J.R. founded by the Ministry of Economy and Competitiveness. L.L.-O. is supported by the University of Granada doctoral program (2017). M.C.-G., M.T.-M. and N.M.-P. are University of Granada Biomedicine PhD students.
Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off -target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 30LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.
Spanish ISCIII Health Research Fund
European Commission PI12/01097 PI15/02015 PI18/00337 PIE16-00045 DTS19/00145 PI18/00330
Junta de Andalucia 2016000073391-TRA 2016000073332-TRA PI-57069 PAIDI-Bio326 PI0014-2016
Nicolas Monardes regional Ministry of Health 0006/2018
Spanish Government FPU16/05467
SMSI through fellowship PEJ-2018-001760-A
Spanish Government RYC-2015-18382
Ministry of Economy and Competitiveness
University of Granada doctoral program