Platelet FVIII expression in HA mice after 2bF8 lentiviral gene delivery to HSCs under ADC-mediated preconditioning. Blood samples were collected at various time points from 2bF8LV-transduced recipients under (CD45.2+CD117)-ADC–mediated preconditioning or TBI. Leukocytes and platelets were isolated. DNA was purified from leukocytes for PCR and qPCR analysis of proviral DNA. Platelet FVIII expression levels were determined by a chromogenic functional activity assay on platelet lysates. (A) PCR detection of the 2bF8 transgene expression cassette. 2bF8 proviral DNA was detected in leukocytes in all 2bF8LV-transduced recipients. Representative results are shown. FVIII^null and WT mice were used as controls. (B) The average copy number of 2bF8 proviral DNA per cell in leukocytes from 2bF8LV-transduced recipients, as determined by qPCR. Heterozygous 2bF8-transgenic mice (2bF8^Tg+/−) that were generated by our group via embryonic stem cell (ES)–mediated transgenesis with a known copy number (11 copies per cell) were used as a positive control. (C) The platelet FVIII expression levels in platelets from 2bF8LV-transduced recipients. Two of 6 recipients under (CD45.2+CD117)-ADC conditioning that received 2bF8LV-transduced Sca-1 cells failed to achieve platelet FVIII expression. All other recipients had sustained platelet FVIII expression. (D) Percentage of recipients with sustained platelet FVIII expression. (E) Platelet FVIII expression levels at 21 weeks after transplantation. These results demonstrate that sustained platelet FVIII expression can be achieved in HA mice after 2bF8 gene therapy with ADC-mediated preconditioning. *P < .05, ***P < .001, ****P < .0001, ADC vs TBI, 2-way analysis of variance.