Abstract

Factor VIII (FVIII) gene therapy is a promising approach to potentially permanently and cost-effectively correct the bleeding phenotype of hemophilia A patients and improve patients quality of life. Our group has developed a successful gene therapy approach in which FVIII expression is targeted to platelets. Platelet expressed FVIII protects hemophilic mice from lethal blood loss after vessel injury. Most importantly this therapy does not induce FVIII inhibitory antibodies and is even successful in the treatment of mice with pre-existing high titer inhibitors. Therefore this approach is among the first to hold promise for patients who develop inhibitory antibodies against FVIII that render FVIII replacement therapy ineffective. Levels of platelet expressed FVIII achieved by gene therapy may vary between individuals due to differences in ex vivotransduction and gene expression efficiency. We determined hemostatic efficacy over a wide therapeutic dose range with a novel native whole blood thrombin generation assay.

Tracking the correction of abnormal bleeding phenotypes during the treatment of patients with hemostatic disorders is crucial to evaluate success of therapy. Global coagulation assays in contrast to single clotting factor assays are desirable to better understand the overall hemostatic condition of patients. Here we evaluated thrombin generation using a modified protocol of a recently described whole blood assay. In our native assay we initiated coagulation without the addition of tissue factor. Sole recalcification of whole blood resulted in thrombin generation with high reproducibility. Lag time (LT) determined in blood from C57BL/6 WT mice was 6 ± 0.2 min (Mean ± SEM) , thrombin generation rate was 58 ± 6 nM/min and thrombin peak was 188 ± 7 nM. In contrast, FVIII deficient blood had negligible thrombin generation with 39 ± 7 min LT, 1.4 ± 0.3 nM/min thrombin generation rate and 12 ± 3 nM thrombin peak. Spiking hemophilic blood with increasing concentrations of recombinant FVIII ex vivo resulted in a dose dependent increase in thrombin generation. Reconstitution of hemophilic blood with FVIII to a 1%, 10% and 100% level shortened LT to 19 ± 1, 12 ± 0.3 and 9 ± 0.5 min, respectively. To evaluate efficacy of platelet-derived FVIII we utilized a newly developed transgenic mouse model that expresses high levels of FVIII in platelets. Homozygous mice express platelet FVIII levels corresponding to 20% endogenous FVIII in whole blood. We combined different ratios of FVIII deficient blood with blood from platelet FVIII expressing transgenic mice. At low ratios of transgenic blood, similar to ex vivospiking with recombinant FVIII, thrombin generation parameters were dose-dependent. Remarkably, a corresponding dose of as low as 0.2% platelet-derived FVIII significantly elevated thrombin generation above FVIII deficient blood and had comparable therapeutic efficacy as a 5-fold higher dose of recombinant FVIII (LT, 18 ± 2 vs 19 ± 1). Similarly, efficacy of 1.5% of platelet-derived FVIII compared with the 6.7-fold higher, 10% dose of recombinant FVIII (LT, 13 ± 1 vs 12 ± 0.3). Further increase of thrombin generation was noticed with platelet FVIII expressing transgenic blood ratios corresponding to 2% and 5% FVIII levels (LT, 11 ± 0.3 and 8.7 ± 0.3 min, respectively). Interestingly, our native assay showed that the platelet FVIII expressing transgenic blood ratio corresponding to a FVIII level of only 5% was sufficient to induce maximal thrombin generation, similar to that obtained with undiluted transgenic blood (LT, 8.7 ± 0.6 min). A similar FVIII dose-dependency was identified for additional thrombin generation parameters including endogenous thrombin potential, thrombin peak, peak time and thrombin generation rate.

We conclude that this native whole blood thrombin generation assay could be used to track therapeutic efficacy of hemophilia A treatment. Using this assay, our data indicate that similar to FVIII replacement therapy our previously established platelet targeted FVIII gene therapy approach enhances hemostasis over a wide therapeutic dose level. This is of great importance because levels of platelet expressed FVIII achieved upon gene therapy in mice vary. In agreement with our previous reports our data from native whole blood thrombin generation assay confirm that at lower FVIII dose levels platelet targeted FVIII gene therapy might be more efficient than factor replacement therapy.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.