Gates Package — page 837

Fig.1. In vitro studies with HDAd-ABE8e vectors in CD34+ cells from healthy donors. A) Reactivation of -globin by HBG1/2 base editor. In adult erythroid cells, expression of -globin is inhibited by a number of repressor proteins including BCL11A that binds within the promoter of the two copies of the HBG gene (HBG1/Gand HBG2/A). This directs the action of the strong -globin locus control region (LCR) toward expression of -globin. LCR activity can be switched back to the -globin by targeting the BCL11A binding motif using an adenine base editor. Expected outcomes of editing the -118 to -113 TGACCA BCL11A binding motif by ABE8e are shown on the bottom. In addition to the target -113 A>G conversion, bystander editing of neighboring adenines. The -116 A>G conversion would further destroy the BCL11A binding motif (underlined blue). In the absence of the -110 A>G bystander conversion, a GATA motif would be created (underlined red). B) The vectors contain an HBG1/2 sgRNA and the ABE8e gene linked to miRNA regulatory elements. The ABE8e gene is either under the control of the PGK promoter (HDAd-PGK.ABE8e) or the EF1a promoter (HDAd-EF1a.ABE8e). The vectors also contain a PGK-mgmtP140K cassette for O6BG/BCNU selection. The IR and frt sequences are remnants from previous vectors that were integrated by SB100x transposase. They are irrelevant for this study that does not employ the SB100x integrating system. C) Editing rate in CD34+ cells from three healthy, G-CSF-mobilized donors. Cells were transduced at an MOI of 2000 vp/cell and three days later, genomic DNA was analyzed by Sanger sequencing for the -113A>G conversion. ABEmax is an early adenine base editor version. D) Sequences of the top 12 edited alleles in CD34+ cells from donor #1 after transduction with HDAd-EF1a.ABE8e. The target site -113 A (A8 in the sequence shown in the lower panel) is marked by a green box. Editable window in the spacer by ABE8e is highlighted in orange. E, F) In vitro transduction studies with donor #1 CD34+ cells that were subjected to erythroid differentiation (ED) and one cycle of O6BG/BCNU selection. E) Schematic of the experiment. F) Percentage of -113A>G conversion at different time points without or with O6BG + 15, 25, 35mM BCNU added at day 3. The standard deviation was less than 10% for all time points. n=3 technical replicates.