Optimization of HLF reporter targeting in CD34⁺ HSPCs. (A) Outline of experimental strategy to optimize reporter integration in CD34⁺ CB cells. (B) Fluorescence-activated cell sorting (FACS) strategy to enrich/deplete HSCs based on CD201 expression from expanded CD34⁺ cells at day 3 of culture with UM171. (C) Selective HLF reporter expression in HSC-containing subfractions of CD34⁺ CB cell cultures. Reporter targeting was carried on day 4 of culture. Total and ZsG⁺ cell counts were determined by FACS on day 3 posttargeting and normalized to 10e4 cells plated in 96-well plates. HR allele frequencies were determined in 1 of 4 replicate wells. One of 5 independent experiments covering 4 biological replicates is shown. Nucleofection program DZ100. (D) Effect of TP53 knockdown on HR and cell yield. CD201⁺ cells were sorted and targeted as in panel C (multiplicity of infection 400 [MOI400]; nucleofection program DZ100) and, as an additional condition, electroporated with RNP and siRNA against TP53. Cell counts were acquired using FACS 3 days posttargeting and normalized to 10e4 plated cells. One of 4 independent experiments covering 4 biological replicates is shown. (E-F) Effect of reporter targeting and TP53 knockdown on cell survival. UM171-expanded CD34⁺ CB cells were sorted based on CD201 expression at day 3 of culture, nucleofected 48 hours postsorting (program CA137), and transduced with rAAV6 (MOI1000) where indicated. (E) Viability was determined at the indicated time points (days 1-3 postnucleofection) using forward scatter (FSC)/side scatter (SSC), annexin V, and 7-AAD, as summarized in the color legend. (F) Live cell counts (normalized to 10e4 plated cells) and ZsG⁺ and HR frequencies are plotted. HR frequencies were determined by ddPCR using remaining cells of pooled replicate wells after FACS analysis. Four technical replicates per time point are plotted. The presented data represent 3 independent experiments across 3 biological replicates.