Gates Package — page 825

treated and untreated mice in blood cells counts (Fig.S10A), erythroid parameters (Fig.S10B), platelet counts (Fig.S10C), and reticulocyte counts (Fig.S10D) were found. Furthermore, there were no significant differences in the lineage composition of bone marrow cells (Fig.S10E). Moreover, in agreement with the disappearance of vector DNA over time, ABE8e was expressed only transiently, which triggered IgM but not a stable IgG anti-Cas antibody response (Fig.S10F). In summary, in vivo HSC editing with HDAd-EF1.ABE8e was found to be safe and yielded -globin reactivation at levels that are therapeutically relevant. Discussion The approach described here is aimed toward the reactivation of -globin/HbF for the treatment of - thalassemia and SCD. HbF has powerful anti-polymerization properties because its γ-globin subunits form mixed hybrid tetramers of two α-globin chains with one γ-globin and one mutated β-globin chain that are largely excluded from the polymer (40). We used a non-integrating HDAd5/35++ vector that would trigger permanent genetic edits in HSCs through the transient expression of ABE8e and a sgRNA that targets the distal BCL11A binding motif in the HBG1/2 promoters. Studies were performed in ex vivo and in vivo settings in CD46/-YAC transgenic mice. The ex vivo approach yielded > 80% HbF+ RBCs with -globin levels that were 30% of human -globin levels. Notably, this study was done without any in vivo O6BG/BCNU selection. More importantly, we demonstrated that in vivo HSC transduction of CD46/-YAC transgenic mice with a single HDAd-EF1.ABE8e vector and early treatment with O6BG/BCNU resulted in >60% HbF+ RBCs with human -globin levels that were 30% of those of human -globin, in the absence of a competitive background (healthy mice). This outcome is potentially curative in -thalassemia and SCD patients. In individuals with HPFH, HbF makes up approximately 30% of total hemoglobin and is homogeneously distributed in the red-cell population (41, 42). Furthermore, murine ex vivo gene therapy studies with -globin lentivirus vectors demonstrated that long-term amelioration of disease of murine - thalassemia and SCD occurred when approximately 20% gene-modified HSCs repopulated the marrow, HbF levels were ~20% and HbF+ cells constituted two-thirds of the circulating RBCs (43-46). In the absence of -YAC mice with a thalassemia or SCD geno-/phenotype, we tested disease correction using CD34+ cells from -thalassemia and SCD patients. We demonstrated that HDAd-EF1.ABE8e transduction mediated efficient target site editing and reactivation of -globin after erythroid