Optimization of HDR-dependent GE conditions to functionally correct X-CGD HSPCs and DNA damage response triggered by AAV/DSB. (A) TI CYBB E7-13pA in X-CGD CD34⁺ HSPCs with i53 as indicated. (B) Dot plots of gp91^phox expression in myeloid-differentiated HSPCs gene edited with AAV (E7-13pA and E7-13pA WPRE) or ODN. (C) FACS histograms comparing gp91^phox expression in X-CGD E7 mutation repair by ODN (navy line) aligned with HD (shaded red) and AAV-TI CYBB E7-13pA (orange line), compared with naive control (gray). (D) Representative FACS of gp91^phox expression (top) and DHR assay (bottom) in myeloid-differentiated X-CGD CD34⁺ cells after CYBB E7 ODN mutation repair ±i53 (150 µg/mL). (E) FACS comparison of gp91^phox expression in differentiated X-CGD HSPCs GE with Cas9 RNA or RNP. (F) Percentages of gp91^phox expressing cells with ODN mutation repair of X-CGD HSPCs using Cas9 mRNA ±i53 or RNP +i53 (150 µg/mL). (G) Sequencing shows repaired alleles in HSPCs GE with Cas9 RNA or RNP and ODN ±i53 (150 µg/mL). (H) FACS for phosphorylated H2AX (γH2AX) in HSPCs exposed to AAV alone, or with DSB repaired with AAV or ODN donor at 2 hours. (I) The kinetics of γH2AX expression in HSPCs exposed to AAV with or without DSB and i53 (150 µg/mL). (J) Phosphorylation of H2AX after exposure of HSPCs to AAV (4 different donors) alone or with DSB repair by AAV or ODN. n = 4 experiments. **P < .01, ****P < .0001, paired Student t test. N, naive; SSC, side scatter.