Evaluating clinically translatable conditioning for platelet gene therapy in murine hemophilia A with inhibitors

Platelet gene therapy is effective in hemophilia A (HA) mice even with inhibitors. Fludarabine (Flu), along with busulfan (Bu) or melphalan (Mel), preconditioning has been shown to be highly effective for hematopoietic stem cell transplantation in the clinic. To evaluate the efficacy of Bu–Flu and Mel–Flu preconditioning in platelet gene therapy of HA with inhibitors. Bu–Flu and Mel–Flu were used to condition HA mice preimmunized with recombinant human factor (F)VIII. An optimal 660 centigray total body irradiation was used as a control regimen in parallel. Platelet–FVIII expression was introduced by transplantation of 2bF8 lentivirus (LV)-transduced hematopoietic stem cells. Animals were analyzed by fluorescence-activated cell sorting, quantitative polymerase chain reaction, FVIII assays, and tail bleeding tests. Bu–Flu, but not Mel–Flu, enabled successful 2bF8 gene therapy. All recipients achieved >55% chimerism post hematopoietic stem cell transplantation in both Bu–Flu and 660 centigray groups, with comparable copy numbers of 2bF8 cassette and the platelet–FVIII levels. The bleeding phenotype was rescued in 2bF8LV-transduced recipients. FVIII inhibitor titers declined with time, with comparable disappearance time of inhibitors between the 2 groups. When animals were rechallenged with recombinant human FVIII after the titers dropped to undetectable levels, no inhibitors were detected in 2bF8LV-transduced recipients. In contrast, all untransduced transplanted control mice produced inhibitors. These data demonstrate that immune tolerance was established in 2bF8LV-transduced primed HA mice under Bu–Flu conditioning. Bu–Flu preconditioning allows for successfully introducing platelet–FVIII expression to restore hemostasis and induce immune tolerance in primed HA mice, suggesting that this approach is a promising clinically translatable strategy for gene therapy of HA with inhibitors.