Chapter 4 —— 45 —— to facilitate delivery into a range of human cell types broader than that engaged by their prototypic AAV type-2 capsid counterparts [42]. In this regard, AAV transduction experiments in hMSCs revealed efficient transfer of donor constructs by these pseudotyped AAV particles, i.e. ∼80% EGFPpositive hMSCs (Figure 1A). Interestingly, co-transduction of hMSCs with AAV and AdV vectors delivering AAVS1-targeting donor templates and CRISPR-Cas9 complexes, respectively, led to a clear AdV-dependent increase in AAV transduction as assessed through flow cytometry-based quantification of reporter-positive cells (i.e. 90–100%) and expression levels per cell (Figure 1A, top and bottom graphs, respectively) as well as via direct visualization of reporter-positive hMSCs using fluorescence microscopy (Figure 1B). Potential cause for this AdV-dependent AAV transduction enhancement is the expression of AAV helper functions from AdV genomes. Related to this aspect, wild-type AAVs (Dependoparvovirus genus) rely on unrelated viruses (e.g. adenoviruses) as helpers for the completion of their lytic infection cycle [12]. A well-established AAV helper function encoded in E1- and E2A-deleted AdV genomes is that of the E4ORF6 protein whose ‘leaky’ expression is known to buildup in an AdV dose-dependent manner [43]. The E4ORF6 helper function [44] has been implicated in converting incoming single-stranded AAV genomes into transcriptionally active doublestranded DNA [45,46]. Hence, it is likely that the AdV-dependent AAV transduction enhancing effect is contributed by the introduction of AAV helper functions into target cells. Regardless, upon episomal AAV DNA elimination through hMSC sub-culturing, CRISPR-Cas9-dependent stable transduction levels were variable and low (Figure 1C). In fact, these levels were not significantly higher than those detected in cultures initially exposed exclusively to AAV-HRS1 or AAV-HMEJS1, respectively (Figure 1C). Based on the previous data, we next sought to improve the dual viral vector genome-editing system by assembling and testing high-capacity AdVPs encoding the high-specificity Cas9 nuclease SpCas9KARA [35] alone (i.e. AdVP.C9KARA) or the optimized high-specificity eCas94NLS nuclease [30]
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