Chapter 5 —— 103 —— Amendola, 2021). The predictability and safety attributes result from a mitigation of insertional mutagenesis, transgene silencing and/or variegated expression normally associated with genome engineering systems yielding semi-random and random integration profiles, e.g., retroviral vectors and transposons, respectively. Mock-transfected HeLa cells and HeLa cells transfected with a plasmid expressing Cas9 were transduced with AAV-HRS1.A1 and, after sub-culturing in the presence or absence of ouabain, each population was subjected to clonal analysis for the characterization of genome editing events at AAVS1 and ATP1A1. The former characterization involved junction PCR analysis; the latter entailed restriction fragment polymorphism (RFLP) assays (Figure 1C). EGFP-directed flow cytometry at 20 days post-transduction revealed that incubating in ouabain cells initially exposed to Cas9 led to a 2.8-fold increase in the frequency of stably transduced cells, hence, genetically modified cells (Figure 1D). Of notice, amongst the HeLa cell cultures not exposed to Cas9, those untreated with ouabain contained a measurable amount of stably transduced cells (2.3%), whilst those treated with ouabain, as expected, died (Figure 1D). Albeit at low frequencies, there are precedents for programmable nuclease-free gene targeting using AAV HR donors (Spector et al. 2021; Biijani et al. 2022) as well as for a role of inverted repeats, including the AAV ITR, in HR stimulation (Holkers et al. 2012). Yet, the exclusive AAV-HRS1.A1 delivery setup suggests, nonetheless, that the vast majority of stably transduced cells contained random or off-target donor DNA insertions, hence wild-type ATP1A1 alleles, in that they were readily eliminated by ouabain (Figure 1D). Importantly, combining AAV-HRS1.A1 with Cas9 delivery resulted in an ouabain-dependent 2.8-fold increase in the frequency of stably transduced cells, suggesting selection for AAVS1-targeted cells (Figure 1D). Independent experiments involving HeLa cells initially transfected with plasmids expressing Cas9 or an inactive dCas9 protein and, subsequently, equally transduced with AAV-HRS1.A1, yielded similar results, i.e., ouabain-dependent elimination and enrichment of stably transduced cells
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