Chapter 4 —— 57 —— HRLMNAGEX1 led to high and DSB-dependent LMNA protein tagging frequencies in HeLa cell populations, reaching at the uppermost AAV dose 86.5 ± 0.8% and 89.6 ± 2.5% as determined by flow cytometry at 3- and 14days post-transduction, respectively (Figure 4E). The frequencies of LMNA tagging registered in the absence of AdVP.C9KARA were at background levels (Figure 4E). Importantly, the LMNA-tagged cell fractions in populations exposed to the same transduction conditions were similar at early and late timepoints indicating a lack of negative selection against these gene-edited cell populations. Similar results were obtained upon LMNA tagging experiments in hMSCs (Supplementary Figure S12A). These data are, therefore, consistent with the low cytotoxicity profile of the dual viral vector genome-editing system based on viral gene-free AdVP and AAV particles in the herein tested cells as indicated by the stability of LMNA-tagged cell frequencies before and after sub-culturing. Finally, the synthesis and proper location of the fusion mScarlet-I::LMNA protein in cell nuclei was confirmed by direct live-cell fluorescence microscopy (Figure 4F and Supplementary Figure S12B). Taken together, these data indicate that the combined delivery of HDR-based genome editing components by AdVP and AAV particles is a robust and versatile genomic engineering approach that, in principle, can be tailored for generating disease models as well as cellular substrates for drug screens and, eventually, autologous cell therapies.
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