In vitro and in vivo expansion of CD33/HBG promoter-edited HSPCs with Mylotarg

We developed an <i>in vivo</i> HSC gene therapy approach that consists of HSC mobilization and intravenous injection of HSC-tropic HDAd vectors. To achieve therapeutically relevant numbers of corrected cells, we incorporated <i>in vivo</i> expansion of transduced cells. We used an HDAd vector for a multiplex adenine base editing approach to (1) remove the region within CD33 that is recognized by gemtuzumab ozogamicin (GO) (Mylotarg), and (2) create therapeutic edits within the HBG1/2 promoters to reactivate γ-globin/HbF. <i>In vitro</i> studies with HDAd-transduced human CD34⁺ cells showed editing of both targeted sites and a 2- to 3-fold GO-mediated expansion of edited erythroid/myeloid progenitors. After erythroid <i>in vitro</i> differentiation, up to 40% of erythrocytes were HbF positive. For <i>in vivo</i> studies, mice were transplanted with human CD34⁺ cells. After engraftment, HSCs were mobilized with G-CSF/AMD3100 followed by an intravenous HDAd injection and GO-mediated <i>in vivo</i> selection. Two months later, editing in human cells within the bone marrow was significantly higher in GO-treated mice. The percentage of HbF⁺ human erythroid cells was 2.5-fold greater compared with untreated mice. These data indicate that <i>in vivo</i> GO selection can increase edited erythroid cells.