135. Optimization of Dual-gRNA Lentiviral Vectors for Targeted Genomic Deletions

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CRISPR-Cas9 technology is a powerful tool for genome editing based therapeutic approaches. Despite its potential, the delivery of Cas9 components into primary cells, such as hematopoietic stem progenitor cells (HSPC), is still a major challenge. In addition, several applications - e.g. the use of Cas9-nickase and the generation of genomic deletions/inversions - require the delivery and expression of two different gRNA. Lentiviral vectors (LV) can efficiently transduce HSPC, but the presence and the orientation of direct repeated elements in the gRNA expression cassettes can potentially trigger recombination events that affect vector stability. To address this issue, we designed different LV encoding for two gRNA pairs, which generate deletions of different size (3 and 13 kb) in the beta-globin gene cluster. gRNA expression was driven by murine and human U6 promoters, having little sequence similarity, to avoid potential recombination events of the LV genome. To further reduce LV rearrangements and optimize expression levels, gRNA cassettes were positioned in different orientations with respect to each other (LV Inward, Tandem and Outward). We compared LV from different vector batches in HCT116 cells and we observed that Tandem configuration resulted in higher viral titers and infectivity, although this difference was not statistically significant. Analysis of proviral integrity on genomic DNA of transduced cells showed the intact dual-gRNA cassette and no sign of recombination for all the LV configurations. To understand if different orientations could affect gRNA expression (measured by RT-qPCR) and consequently deletion/inversion efficiency, we co-transduced erythroleukemic K562 cells with LV-Cas9-Blast and the three different LV. Surprisingly, LV Inward showed poor gRNA expression, negligible deletion and inversion frequency and low efficiency of InDel formation at each gRNA target site (2.4 % and 6.8%). Conversely, LV Tandem and Outward configurations allowed efficient cell transduction and significant and comparable levels of gRNA expression. As a results, we observed a good deletion frequency (11.0% ±1.8 of total alleles for LV Tandem and 12.3 % ± 2.5 for LV Outward), a lower proportion of inversions events (5.4% ±1.3 for LV Tandem and 3.8 % ± 0.4 for LV Outward) and up to 58% of InDels events at each gRNA target sites. Comparable results were also obtained in adult HSPC-derived erythroid cell line (HUDEP-2). Overall, our study indicates LV Tandem and LV Outward as promising tools for genome editing of primary cells; both vectors are now being evaluated in primary HSPC.