Fig. 4: Multiplex CD33/HBG ABE8e editing of human CD34⁺ cells and GO selection for multiplex edits in vitro.

a Schematic of HBG −175 ABE8e target site. Adenines in the editing window are marked in bold red and PAM in light red. b HBG ABE8e editing efficiency measured in human mPB CD34⁺ HSPCs after treatment by mock electroporation (unedited, UE) or by HBG ABE8e mRNA. Results are from one representative donor. c HBG editing efficiency in human mPB CD34⁺ HSPCs treated by mock electroporation (UE), by HBG ABE8e mRNA (single) or by CD33/HBG ABE8e mRNA (multiplex). Results are from 4 different donors, 4 independent experiments. d CD33 editing efficiency in human mPB CD34+ HSPCs treated by mock EP (UE) or by CD33/HBG ABE8e mRNA (multiplex). Results are from 5 different donors, 5 independent experiments. e Colony-forming potential of mPB CD34⁺ cells treated by multiplex ABE8e editing. Results are from 1 representative donor, 3 technical replicates. M=macrophages, G=granulocytes, GM=granulocyte/macrophage, GEMM=granulocyte/erythrocyte/macrophage/monocyte BFU-E=erythroid. f Frequency of colony-forming cells displaying unedited (UE), edits at each gene target, or edits at both gene targets in the same colony. Results are from 2 different human donors, 2 independent experiments. g Frequency of colony-forming cells with unedited (UE), monoallelic (mono) or biallelic (bi) CD33 edits, and accompanying edits (0 to 4) at the HBG target site in two different donors. Number of colonies analyzed is shown on top. N/A=Not Applicable. h Unedited (UE) or multiplex edited ML1 cells treated for 6 consecutive days with 10 pg/mL Gemtuzumab Ozogamicin (GO) starting at 5 days post-electroporation. Viable count was determined by trypan blue staining. Results are from 1 representative experiment. i, Editing efficiency measured at the CD33 (left) or HBG (right) gene targets in multiplex edited ML1 cells from h treated or not with GO at 8 days post GO removal (14 days post-electroporation). All error bars in this figure show ±SEM. Source data are provided as a Source Data file.