Chapter 4 —— 51 —— cytometry after sub-culturing for 20 days hMSCs initially exposed to the indicated viral vector MOI. (D) AAV donor transductions targeting AAVS1 safe harbor loci in HeLa cells. AAV-HRL.S1GS1 and AdVP.C9KARA were applied at the indicated MOI and, at 3 days posttransduction, EGFP-directed flow cytometry was used to measure the frequency of transgene expressing cells and respective MFI (top and bottom graphs, respectively). Mock-transduced HeLa cells and HeLa cells transduced exclusively with AAV-HRL.S1GS1 provided for controls. Significant differences amongst the marked datasets were assessed with one-way ANOVA followed by Tukey’s test for multiple comparisons; ****P < 0.005 (n = 3 biological replicates). (E) Quantification of HeLa cells stably transduced with AAVHRL.S1GS1. Stable transduction frequencies were measured via EGFP-directed flow cytometry after sub-culturing for 14 days HeLa cells initially exposed to the indicated viral vector MOI. Data are shown as mean ± SD of three biological replicates. (F) AAV donor transductions targeting CCR5 safe harbor loci in hMSCs. The structure of AAV-HRC5GC5 donor is depicted. Horizontal gray bars, DNA homologous to sequences flanking the CCR5 target site of gRNA GC5 (‘homology arms’); gray box, mCherry transgene. AAVHRC5GC5 and AdVP.C9KARA were applied at the specified MOI and, at 3 days posttransduction, mCherry-directed flow cytometry was used to determine the frequency of transgene expressing cells and corresponding MFI (top and bottom graphs, respectively). Mock-transduced hMSCs cells and hMSCs transduced with AAV-HRC5GC5 alone served as controls. Significant differences between the marked datasets were assessed with oneway ANOVA followed by Tukey’s test for multiple comparisons; **P < 0.01 (n = 3 biological replicates). (G) Quantification of HeLa cells stably transduced with AAVHRC5GC5. Stable transduction frequencies were measured via mCherry-directed flow cytometry after sub-culturing for 20 days hMSCs initially treated with indicated viral vector MOI. Numerals above the graph bars correspond to AAV stable transduction frequencies (mean ± SD) normalized to the initial transduction levels. Initial quantification of vector DNA buildup in transduced cells in the presence and absence of AdVPs ruled out an effect of AdVPs per se on AAV vector cell entry (Supplementary Figure S6). Of note, previous experiments have demonstrated that DNA damage response (DDR) proteins (e.g. MRN complex factors MRE11-RAD50-NBS1) associate with incoming AAV vector DNA that presumably hinder transgene expression [47]. Indeed, the dampening of these proteins correlates with increased permissiveness to AAV vector expression in vitro and in vivo [48] and exposing cells to pleiotropic
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