ATS improves circulating fVIII activity levels following BDDpfVIII-transduced HSCT of preimmunized mice. Hemophilia A mice received 6 weekly intravenous injections of recombinant hfVIII (10 U/injection). One week after the last injection, plasma was collected from each mouse (n = 5 per group) and assayed for anti-hfVIII or anti-BDDpfVIII total Ig (ELISA) and inhibitory activity (Bethesda assay). Panel A shows the anti-BDDpfVIII ELISA versus the anti-hfVIII ELISA for each mouse. Panel B shows BDDpfVIII activity following HSCT gene therapy versus pretransplantation anti-hfVIII ELISA titer for each animal. ELISA titer is defined as the reciprocal of the antibody dilution that leads to a signal 3 times background in 15 minutes. Donor cell engraftment (C) and fVIII activity (D) are shown for each animal conditioned with 5.5 Gy TBI + ATS and that received a transplant of 3 × 10⁵ BDDpfVIII-transduced sca-1⁺ cells. Panels E and F show the same analysis for mice conditioned with 35 mg/kg BU + ATS and that received a transplant of 3 × 10⁵ BDDpfVIII-transduced sca-1⁺ cells.