Chapter 4 —— 48 —— These cumulative datasets are consistent with the fact that although E1- and E2A-deleted AdVs encode less viral regulatory functions than their firstgeneration, E1-deleted, counterparts, ‘leaky’ viral gene expression is nonetheless detected at high vector doses [11,43]. Importantly, transduction of hMSCs with viral gene-free AdVPs did not impair their differentiation capacity into osteoblasts as assessed by the detection of calcium deposits upon incubation in osteogenic differentiation medium and alizarin red S staining (Supplementary Figure S3). Gene targeting experiments using AdVP.C9KARA and AAV-HRL.S1GS1, an AAV co-delivering gRNA GS1 and matching AAVS1-tailored HR templates, led to robust transduction of hMSCs (Figure 2A and B, left column) as well as HeLa cells (Figure 2D). Crucially, this co-transduction scheme yielded CRISPR-Cas9-dependent stable transduction frequencies of up to ∼80% and 90% at end-point hMSC and HeLa cell cultures (Figure 2C and E, homologous to sequences flanking the AAVS1 target site of gRNA GS1 (‘homology arms’); gray box, EGFP transgene; orange boxes, GS1 target sequence. hMSCs were exposed to AAV-HRS1 or to AAV-HMEJS1 together with AdVs encoding AAVS1-targeting CRISPRCas9 complexes (Cas9:GS1) at the indicated multiplicity-of-infection (MOI). TU and GC, transducing units and genome copies, respectively. AAV donor transduction was determined by quantifying EGFP-positive cells and corresponding mean fluorescence intensity (MFI) values by flow cytometry at 3 days post-transduction (top and bottom graphs, respectively). Mock-transduced hMSCs and hMSCs exposed exclusively to AAV donors served as controls. (B) AAV donor expression imaging. Representative direct fluorescence microscopy images of hMSCs transduced with AAV-HRS1 or AAV-HMEJS1 at a single MOI and different amounts of AdVs encoding Cas9:GS1 complexes or with AAV donors alone. Images were acquired at 2 days post-transduction (C) AAV stable transduction levels. Stable transduction frequencies were assessed by EGFP-directed flow cytometry after sub-culturing for 20 days hMSCs initially exposed to the indicated viral vector MOI. Data are presented as mean ± standard deviation (SD) of three biological replicates. Significant differences between the defined datasets were determined with oneway analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons; ***P < 0.001; ****P < 0.0001; P > 0.05 was considered non-significant (ns).
RkJQdWJsaXNoZXIy MTk4NDMw