Effect of insertion of therapeutic transgenes on neighboring genes at AAVS1. (A) Top panel shows a map of 10 neighboring genes on chromosome 19 around AAVS1 site. (B) Bar graph illustrates Q-RT-PCR analysis of 10 neighbor genes after differentiation of corrected α⁰-thal-iPSCs into basophilic erythroblasts (normalized to parental iPSCs). All iPSCs analyzed carried homozygous insertions at AAVS1. mRNA expression levels were calculated using the Delta (δ Ct) method using differentiated uncorrected α⁰-thal-iPSCs as the controls (n = 3). Four genes were activated. The smallest activation was obtained when the α-globin promoter construct was inserted in the same orientation as the PPP1R12C gene. (C) Same as above but data are expressed relative to GAPDH. Expression of the neighboring genes is low compared with GAPDH.