Targeting codon-optimized FIX-Padua expression to highly primed FIX^nullmice (the inhibitor model). Both donor and recipient FIX^null mice were immunized with rhFIX at 200 U/kg in the presence of IFA twice with a 3-week interval to induce the development of anti-FIX antibodies. Two weeks after the second immunization, anti-FIX inhibitor titers were determined by Bethesda assay, and anti-FIX total IgG titers were determined by ELISA. Sca-1⁺ cells isolated from FIX-primed donors were transduced with 2bCoF9R338L LV and transplanted into FIX-primed recipients under either a lethal 11-Gy or a sublethal 6.6-Gy TBI. Four weeks after HSCT, blood samples were collected from various time points for assays. (A) Diagram of experimental design. FIX^null mice with a B6-129S mixed background were used. (B) Average copy number of 2bCoF9R338L proviral DNA per cell in transduced FIX-primed recipients during the study period. The copy number was determined by qRT-PCR. (C) Average copy number of 2bCoF9R338L proviral DNA per cell in each rhFIX-primed recipient. Individual mice were analyzed more than once during the study, and the average copy number was calculated. (D) Platelet-FIX:Ag expression levels in 2bCoF9R338L-transduced FIX-primed recipients during the study period. Platelets were isolated and lysed in 0.5% CHAPS. Platelet-FIX:Ag levels in platelet lysates were determined by ELISA. (E) Platelet-FIX activity (platelet-FIX:C) levels in 2bCoF9R338L-transduced FIX-primed recipients during the study period. Platelet-FIX:C levels in platelet lysates were quantified by a chromogenic assay. (F) Average platelet-FIX expression in 2bCoF9R338L-transduced FIX-primed recipients. Individual mice were analyzed at various time points, and the average FIX expression level was calculated. (G) The ratio of platelet-FIX:C to platelet-FIX:Ag during the study period. (H) Average platelet-FIX:C expression levels in sequential transplantation recipients. Four primary (1°) recipients from the 6.6-Gy group that received 2bCoF9R338L-transduced Sca-1⁺ cells were euthanized at 63 weeks after HSCT, and BM mononuclear cells isolated from the 1° recipients were transplanted into the 2° recipients that were also primed with rhFIX plus IFA and preconditioned with 6.6 Gy TBI plus bortezomib 1 mg/kg by IV administration. Platelet-FIX:C levels in recipients were monitored. Individual mice were analyzed more than once, and the average FIX expression level was calculated. (I) Sustained FIX expression in the 2° recipients during the study period. Data are presented as mean ± SD. ***P < .001.