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.

Keywords: IDLV; gene delivery; gene editing; gene expression; off-targets.

Figure 1

The IS2 element enhances expression levels inside the LTR, while the SAR element is more effective outside the LTR. (A) Schematic diagram of different constructs used in this study. (B) Representative plots of 293T cells transduced with SE, SE-IS2 (in-LTR), SE-IS2, SE-SAR, and SE-HS4_650 with an MOI = 0.4 and analysed 72 h after transduction, showing the percentage of eGFP+ cells and MFI. (C) Graphs showing the percentage of eGFP+ cells (left) and the eGFP expression levels (MFI, right) of 293T cells transduced with the different IDLVs relative to the values for SEs. Relative viral episomes in transduced cells are shown for each IDLV at the bottom of the graph on the left. (D) Graphs showing the estimated efficiency of the different constructs measured as the percentage of eGFP+ cells (left) or MFI (right) relative to the amount of viral episomes (as detailed in Materials and Methods). All graphs represent mean of at least three separate experiments, and error bars indicate standard error of the mean (SEM); *** = p < 0.001; ** = p < 0.05; and two-tailed unpaired Student’s t-test.

Figure 2

Effect of engineered genetic elements and position on IDLV-driven expression in hiPSCs. (A) Representative bright-field and EGFP fluorescence images of hiPSCs used in this study. (B) Representative plots of hiPSCs transduced with SE, SE-IS2 (in-LTR), SE-IS2, SE-SAR, and SE-HS4_650 with an MOI = 3 and analysed 72 h after transduction. (C) Graphs showing the percentage of eGFP+ cells (left) and the relative eGFP expression levels (MFI, right) of hiPSCs cells transduced with the different IDLVs relative to the values for SE. The relative viral episomes/transduced cell is shown for each IDLV below the graph on the left. (D) The graphs show the estimated efficiency of the different constructs relative to the amount of viral episomes (as detailed in Materials and Methods). All graphs represent mean of at least three separate experiments; error bars indicate standard error of the mean (SEM); *** = p < 0.001; ** = p < 0.05; and two-tailed unpaired Student’s t-test.

Figure 3

Presence of IS2 enhances the capture of IDLVs in DSBs. (A) Schematic diagram of the two constructs used in this study. (B) Workflow followed and experimental schema for IDLV gene trapping, 293T cell transduction with IDLV expressing GFP, followed by treatment with etoposide (8 µM). (C) Graph showing intracellular IDLV yield after transduction with the same multiplicity of infection (MOI = 4). (D) Representative experiment showing the increase in the percentage of eGFP expression, reflecting episomal relaxation due to etopside treatment, and the increased levels of eGFP labelling 36 d after transduction, which were further enhanced upon transduction with SE-IS2 (in-LTR). (E) IDLV capture by DSBs relative to the initial amount of viral genome episomes in the cells. The graphs represent mean of at least three separate experiments, while error bars indicate standard error of the mean (SEM); * = p < 0.01; two-tailed unpaired Student’s t-test.

Figure 4

Presence of IS2 increases the sensitivity of IDLVs to DSB capture induced by sequence specific programmable nucleases. (A) (top) Workflow followed and experimental scheme for IDLV gene trapping; Jurkat cells were transduced with IDLV-expressing eGFP (MOI = 4), followed by electroporation with ribonucleoproteins (RNPs; gRNA + CAS9 proteins); (bottom left) CRISPR/Cas9-targeting TRAC locus showing the gRNA sequences used in this study; (bottom right) Sanger sequencing of bulk population showing the genome editing efficiency of the targeted locus. (B) Representative plots of Jurkat cells transduced with SE and SE-IS2 (LTR), nucleofected or not with RNPs targeting the T-cell receptor (TCR) locus 5 and 42 days after nuncleofection. (C) Relative vector DNA genomes in IDLV-transduced cells 72 h after transduction. (D) The graph shows the estimated capture efficiency of SE-IS2 (in-LTR) IDLVs relative to SE IDLVs either in the TCR locus (in target CD3⁺) or outside the TCR locus (off-target and random integration CD3⁻). (E) Capture efficiency after normalization to the intracellular IDLVs. All graphs represent mean of at least three separate experiments; error bars indicate standard error of the mean (SEM); * = p < 0.01, ** = p < 0.05; and two-tailed unpaired Student’s t-test.