The impact of the addition of CD4-ADC or CD8-ADC on the efficacy of (CD45.2+CD117)-ADC preconditioning in platelet-specific FVIII gene therapy of HA mice. CD45.2/FVIII^null mice were preconditioned with (CD45.2+CD117)-ADC plus CD8-ADC or CD4-ADC and received 2bF8LV-transduced CD45.1/FVIII^null Sca-1⁺ cells. TBI was used as a control preconditioning regimen in parallel. After ≥3 weeks of BM reconstitution, blood samples were collected from recipients post-HSCT at various time points for chimerism analysis, qPCR analysis, and platelet lysate FVIII activity assay. (A) Schematic diagram of experimental design for 2bF8 gene therapy using (CD45.2+CD117)-ADC plus CD4-ADC or CD8-ADC preconditioning. (B) Leukocyte chimerism analyzed by flow cytometry. (C) Percentage of donor-derived leukocytes at 20 weeks after HSCT. (D) Platelet FVIII expression levels determined by platelet lysate FVIII activity assay. (E) Platelet FVIII expression levels at 21 weeks after HSCT. (F) Average copy number of 2bF8 proviral DNA per cell from peripheral blood leukocytes, as determined by qPCR. Homozygous 2bF8-transgenic mice (LV17/18^Tg+/+) that were generated by our group via 2bF8LV-mediated transgenesis with a known copy number (4 copies per cell) were used as a positive control. (G) Platelet FVIII expression in sequential BMT recipients under ADC-mediated preconditioning. Sixty weeks after HSCT, BM cells were collected from some primary (1°) recipients and transplanted into secondary (2°) recipients under the same conditioning regimen that donors received: (CD45.2+CD117+CD4)-ADC or (CD45.2+CD117+CD8)-ADC. Animals were analyzed monthly. The levels of platelet FVIII expression from the 1° recipients were averaged from the last 3 time points. The 2° recipients’ platelet FVIII levels were averaged from the 3 time points after sequential transplantation. These results demonstrate that the addition of CD4-ADC or CD8-ADC can enhance the efficacy of (CD45.2+CD117)-ADC preconditioning in platelet-specific FVIII gene therapy in HA mice.