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HBG1/2 promoter editing in healthy CD34+ cells. We used HDAd5/35++ vectors to express ABE8e either under the control of the phosphoglycerate kinase (PGK) promoter (HDAd-PGK.ABE8e), or the strong EF1 promoter (HDAd-EF1.ABE8e) (Fig.1B). The HDAd vectors also contained a human mgmtP140K expression cassette that allows for enrichment of edited cells after treatment with O6BG/BCNU. To produce HDAdEF1.ABE8e, suppression of ABE8e expression on HDAd producer cells was required. This was achieved by placing the ABE8e gene under the regulation of miR-183-5p, miR-218-5p (32) and mi-vaRNAI (33). The first strategy makes use of two microRNAs (miRNAs), hsa-miR183-5p and hsa-miR218-5p, expressed exclusively by the HDAd producer cells. The second strategy makes use of a miRNA expressed by the helper virus called mivaRNAI. Because these miRNAs are only present during vector production, transgene expression from the HDAd is unimpeded in the transduced target, In addition, we further modified the helper vector by inserting expression cassettes for anti-CRISPR peptides capable of blocking ABE8e activity (Fig.S1) (34). The editing rate of HDAd-ABE8e vectors was tested in human CD34+ cells from two healthy donors at day 3 after transduction (Fig.1C). HDAd-EF1.ABE8e catalyzed on average ~26% of target site - 113 A>G conversions, while HDAd-PGK.ABE8e and HDAd-ABEmax (used in an earlier study (12)), conferred 6.6% and 1.3% editing, respectively. Subsequent studies therefore focused on HDAd-EF1.ABE8e. Due to the wide editing window of ABE8e, in addition to the -113 adenine (shown as A8 at the bottom of Fig.1D), neighboring adenines (A5, A9, A11) can also be converted to guanines. To analyze the effect of editing on reactivation of -globin/HbF expression, HDAd-EF1.ABE8e-transduced CD34+ were subjected to 18 days of erythroid differentiation (ED) with and without O6BG/BCNU in vitro selection (Fig1E). Target site conversion rates were measured at days 7, 13, and 18 of ED (Fig.1F). Starting at about 20% of editing, one time treatment with of O6BG plus 25 or 35M BCNU increased the editing rate to ~60% by day 18 of ED. HDAd-ABE8e HBG1/2 promoter editing, -globin reactivation, and phenotypic improvement in CD34+ cells from -thalassemia and SCD patients. CD34+ cells were from three -thalassemia patients and three SCD patients previously enrolled in mobilization trials. After transduction with HDAd-EF1.ABE8e, cells were subjected to ED with and without in vitro selection (Fig.2A). Rates of -113 A>G conversion reached ~60% by Next Generation Sequencing (NGS) (Fig.2B) or by Sanger sequencing (Fig.S2) in HDAdEF1.ABE8e-transduced cells that were subjected to O6BG/BCNU selection. In ED cultures, the percentages of HbF+ cells in total erythroid cells and enucleated erythroid (NucRed-) cells were significantly higher in the HDAd-EF1.ABE8e-transduced over the untransduced cells (Figs.2C, S3A). Notably, background γ-globin expression in the control vector-transduced samples is triggered by cytokines present in the culture medium (35). In vitro selection at day 3, further increased the percentage