@misc{10481/70733,
year = {2021},
month = {8},
url = {http://hdl.handle.net/10481/70733},
abstract = {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.},
organization = {Spanish ISCIII Health Research Fund},
organization = {European Commission	PI12/01097	
PI15/02015	
PI18/00337	
PIE16-00045	
DTS19/00145	
PI18/00330},
organization = {Junta de Andalucia	2016000073391-TRA	
2016000073332-TRA	
PI-57069	
PAIDI-Bio326	
PI0014-2016},
organization = {Nicolas Monardes regional Ministry of Health	0006/2018},
organization = {Spanish Government	FPU16/05467},
organization = {SMSI through fellowship	PEJ-2018-001760-A},
organization = {Spanish Government	RYC-2015-18382},
organization = {Ministry of Economy and Competitiveness},
organization = {University of Granada doctoral program},
publisher = {MDPI},
keywords = {IDLV},
keywords = {Gene delivery},
keywords = {Gene expression},
keywords = {Gene editing},
keywords = {Off-targets},
title = {Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks},
doi = {10.3390/pharmaceutics13081217},
author = {Cortijo Gutiérrez, Marina and Sánchez Hernández, Sabina and Tristán Manzano, María and Maldonado Pérez, Noelia and López Onieva, Lourdes and Real Luna, Pedro José and Marchal Corrales, Juan Antonio and Martín Molina, Francisco and Benabdellah, Karim},
}