Background We have recently developed a successful gene therapy approach for hemophilia A in which FVIII expression is targeted to platelets by the αIIb promoter. Levels of platelet expressed FVIII (2bF8) achieved by gene therapy may vary between individuals due to differences in ex vivo transduction and gene expression efficiency. Accurate assays to evaluate 2bF8 efficacy are desirable. Objective To compare the hemostatic efficacy of 2bF8 with replacement therapy over a wide therapeutic dose range. Methods Efficacy of 2bF8 was assessed using a new transgenic mouse model expressing high 2bF8 levels (LV18tg). Blood from LV18tg mice or FVIIInull mice infused with recombinant FVIII was mixed with FVIIInull blood at different ratios ex vivo to achieve several concentrations of 2bF8 or plasma FVIII. Samples were evaluated with a novel native whole blood thrombin generation assay (nWB-TGA) that uses recalcified whole blood without the addition of tissue factor to initiate coagulation. Results FVIII dose dependency was observed in all five thrombin generation (TG) parameters. While the total amount of thrombin generated was similar, 2bF8 significantly accelerated TG compared with plasma FVIII. Remarkably, a 10-fold lower dose of 2bF8 than plasma FVIII (0.2% vs 2%) significantly shortened the onset and peak of TG compared with FVIIInull blood. Conclusion Using a new transgenic mouse model we showed that the novel nWB-TGA established here can be used to monitor platelet targeted FVIII gene therapy. The higher therapeutic efficacy of 2bF8 compared with factor replacement therapy seemed to be due to acceleration of TG.
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