Gates Package — page 839

Fig.2. In vitro editing studies with CD34+ cells from -thalassemia and SCD patients. A) Schematic of the experiment. G-CSF/Plerixafor mobilized CD34+ cells from three - thalassemia (Thal) patients were used. SCD CD34+ cells from fresh peripheral blood were isolated post blood transfusion of three patients (one 0/S, two S/S patients). CD34+ cells were transduced with HDAd-EF1a.ABE8e or left untransduced (UNTD). Cells were then subjected to erythroid differentiation for 18 days. Aliquots were collected at the indicated time points. On Day 3, one set of ED cells was treated once with O6BG/BCNU for in vitro selection. (Only from second S/S patient, we obtained sufficient numbers of CD34+ cell to perform in vitro selection). B) Editing rate of the -113 A target site and percentage of reads with indels analyzed by NGS for the thalassemia samples (left panel) and SCD samples (right panel). Note the different scale on the y-axes for on-target editing and indels. C) Flow cytometry analysis for -globin/HbF at the end of ED in total cells and in differentiated enucleated erythroid (NucRed-) cells in Thal and SCD samples. D) Measurement of globin protein chains by HPLC. Shown is the percentage of human -globin relative to human - and a-globin chains in Thal samples (N=3), two SCD (S/S) samples without selection, and one SCD (S/S) sample with selection. Statistical analyses of the data from the Thal samples were performed using two-way ANOVA.