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

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 3′LTR 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.

In search of an ideal template for therapeutic genome editing: A review of current developments for structure optimization

“…In contrast to IDLV vectors (Cortijo-Gutiérrez et al, 2021), the possibilities of introducing direct modifications to the AAV donor template for knock-in efficiency stimulation are limited due to the rigid structure parameters of AAV's capsid and genome. Traditionally, it is recommended for homology arms to not exceed 400 bp (Sather et al, 2015).…”

CRISPR Variants for Gene Editing in Plants: Biosafety Risks and Future Directions

“…The detection and evaluation of these off-target effects are crucial for ensuring biosafety. Several genome-wide methods have been developed for this purpose, such as integration-deficient lentiviral vectors (IDLVs) [38]; chromatin immunoprecipitation sequencing (ChIP-seq) [39]; breaks labeling, enrichment on streptavidin and next-generation sequencing (BLESS) [40]; genome-wide unbiased identification of DSBs enabled by sequencing (GUIDE-seq) [41]; differential cellular indexing of transcriptomes via sequencing (DISCOVER-seq) [42]; and genome-wide off-target analysis via two-cell embryo injection (GOTI) [43]. However, each method has limitations; for instance, ID-VLs have a limited off-target detection efficiency of 1%, and BLESS requires a reference genome [44].…”