Abstract Number: OC 75.3
Meeting: ISTH 2021 Congress
Background: Current hemophilia A (HA) gene therapy trials target hepatocyte expression of human B-domain deleted FVIII (BDD-hFVIII). Multiple trials have demonstrated OS-determined FVIII:C is approximately 1.6-fold higher than CS-determined FVIII:C for transgene-derived BDD-hFVIII, wherein 1 trial reported plasma BDD-hFVIII antigen concentrations (FVIII:Ag) correlate with CS-determined FVIII:C. It remains unclear: 1) which assay best predicts in vivo hemostatic function of hepatocyte-derived BDD-hFVIII and 2) what the molecular etiology of this discrepancy is.
Aims: We investigated a possible biological basis for the observed discrepancy in OS versus CS assay-determined FVIII:C for hepatocyte-derived BDD-hFVIII.
Methods: Male HA/CD4KO mice were infused with AAV8 vectors for hepatocyte expression of BDD-hFVIII. Human hepatocyte-like and human endothelial-like cell lines were transfected with BDD-hFVIII. FVIII:C was measured by OS clotting assay (TriniCLOT Automated aPTT reagent/STart analyzer) and by Chromogenix COAMATIC Factor VIII kit. FVIII:Ag was determined by ELISA (Factor VIII Paired Antibody Set). BDD-hFVIII FVIII:C and FVIII:Ag of AAV-treated mouse plasma and of transfected cell media were calibrated against a standard curve of recombinant BDD-hFVIII purified from BHK cells spiked into HA mouse plasma and conditioned media, respectively.
Results: Preliminary data from in vitro transfection of hepatocyte-like cells demonstrated higher OS- than CS-determined FVIII:C, while OS- and CS-determined FVIII:C of endothelial-like cells did not differ. Consistent with observations in human HA gene therapy trials, OS-determined FVIII:C of hepatocyte-derived BDD-hFVIII from AAV-treated mice was 1.6-fold higher than CS-determined FVIII:C (Figure 1), and plasma FVIII:Ag better correlated with CS-determined FVIII:C (Figure 2).
Conclusions: Recapitulation of the hepatocyte-derived BDD-hFVIII OS/CS assay discrepancy and correlation of FVIII:Ag with CS-determined FVIII:C in mice demonstrates this observation is not species-specific and suggests a biological basis for the differences in measured FVIII:C by different assays. These in vitro and in vivo assays that recapitulate the OS/CS discrepancy provide an experimental system to understand the biological basis of these differences.
To cite this abstract in AMA style:Sternberg AR, J Davidson R, J Samelson-Jones B, A George L. In vitro and In vivo Models to Understand One-stage and Chromogenic Factor VIII Activity Assay Discrepancy of Hepatocyte-derived Factor VIII [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/in-vitro-and-in-vivo-models-to-understand-one-stage-and-chromogenic-factor-viii-activity-assay-discrepancy-of-hepatocyte-derived-factor-viii/. Accessed January 24, 2022.
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