Chapter 4 —— 49 —— respectively). Indeed, in the absence of AdVP.C9KARA, the frequencies of hMSCs and HeLa cells stably transduced with AAV donor DNA were barely above background levels (Figure 2C and E, respectively) as was also grasped via live-cell fluorescence microscopy of hMSC cultures at 19 days posttransduction (Figure 2B, right column). Additional control experiments consisting of exposing cells to AAV-HRL.S1GS1 with and without AdVP.C9KARA confirmed over 90% and background stable transduction frequencies, respectively; whilst those comprising the AAV donor alone or together with a control AdVP.mCherry vector yielded exclusively background stable transduction levels (Supplementary Figure S4). Analogous experiments in hMSCs using AdVP.C9KARA together with AAVHRC5GC5, an AAV co-delivering gRNA GC5 and HR substrates for gene knock-in at the alternative ‘safe harbor’ locus CCR5, also yielded efficient transduction (Figure 2F) and high CRISPR-Cas9-dependent stable transduction levels, i.e. up to 54% of initially transduced cells (Figure 2G). Of note, CRISPR-Cas9-dependent stable transduction levels in primary hMSCs with AdVP.C9KARA and AAV-HRC5GC5 (Figure 2G) compared favorably to those measured in HeLa cells (Supplementary Figure S5, bottom graph). Moreover, albeit by degrees lower than those previously observed using AAV and second-generation AdV vectors (Figure 1A), AAV and viral gene-free AdVP vectors also led to increased AAV transduction levels as measured by reporter-directed flow cytometry at 3 days posttransduction (Figure 2A, D and F, bottom graphs). Based on these data, we next sought to investigate the role of AdVPs per se versus AdVPs combined, or not, with targeted DSB formation on the proficiency of AAV vector transduction as determined by tracing vector DNA amounts and transgene expression levels.
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