The influence of ADC-mediated conditioning on peripheral blood recovery and leukocyte chimerism in HA mice after 2bF8 lentiviral gene delivery to HSCs. CD45.2/FVIII^null mice were preconditioned with CD45.2 and CD117 antibodies conjugated with SAP and transplanted with Sca-1⁺ cells that were isolated from CD45.1/FVIII^null donors and transduced with 2bF8LV. Additional untransduced Sca-1–depleted cells were transplanted into some recipients. TBI (6.6-Gy) preconditioning was used as a control regimen in parallel. After ≥3 weeks of BM reconstitution, blood samples were collected for whole-blood count and flow cytometry analysis of leukocyte chimerism. (A) Schematic diagram of experimental design for 2bF8 gene therapy using (CD45.2+CD117)-ADC preconditioning. Sca-1⁺ cells isolated from CD45.1/FVIII^null donors were transduced with 2bF8LV and transplanted into CD45.2/FVIII^null recipients preconditioned with (CD45.2+CD117)-ADC. Some recipients were cotransplanted with untransduced Sca-1–depleted hematopoietic cells. (B) Leukocyte counts. (C) Platelet counts. (D) Hemoglobin levels. (E) Donor-derived leukocyte chimerism. (F) Percentage of animals with >15% donor-derived leukocytes at 20 weeks after transplantation. (G) Percentage of donor-derived leukocytes at 20 weeks after transplantation. These results show that (CD45.2+CD117)-ADC conditioning does not result in prolonged cytopenias in HA mice and enables engraftment of 2bF8LV-transduced HSCs after platelet-directed gene therapy. *P < .05, **P < .01, ***P < .001, ****P < .0001, ADC vs TBI, 2-way analysis of variance. Bio, biotin; SA, streptavidin.