Abstract Number: OC 01.3
Meeting: ISTH 2022 Congress
Background: Hemophilia A (HA) AAV-mediated gene transfer clinical trials demonstrated transgene-derived FVIII:C is ~2-fold higher by one-stage clotting assay (OSA) than chromogenic assay (CSA). Plasma FVIII antigen (FVIII:Ag) concentrations correlate with CSA-determined FVIII:C. The underlying mechanism is unclear and curious, particularly because this is not observed with recombinant proteins of the same amino acid sequence. Possible hypotheses that may explain the OSA vs CSA-determined FVIII:C differences include enhanced activation (e.g. altered vWF affinity or enhanced thrombin or FXa cleavage) or, alternatively, differences in the function of the transgene-derived FVIIIa species. Understanding the mechanism(s) of the OSA/CSA discrepancy is necessary to determine which assay best represents in vivo AAV-derived FVIII hemostatic efficacy and thereby patient management.
Aims: We sought to interrogate the mechanism of the OSA/CSA discrepancy of transgene-derived FVIII.
Methods: Male HA/CD4KO or HA/vWF-/- mice were infused with AAV8-BDD-hFVIII (1E11-5E11 vg/mouse). FVIII:C was measured by OSA and CSA against a standard curve of recombinant BDD-hFVIII (rFVIII) purified from BHK cells reconstituted in mouse plasma. Thrombin generation assays (TGA) and 2-stage clotting assays (2SA) were also performed.
Results: Like humans, transgene-derived FVIII:C in mice determined by OSA was ~2-fold CSA-determined FVIII:C, and FVIII:Ag closely correlated with CSA FVIII:C. Consistent with prior human studies, shortened lag time in a TGA was recapitulated in plasma from AAV-treated HA mice. The OSA/CSA discrepancy was maintained in a vWF-free system. FVIII:C determined by 2SA was higher for transgene-derived FVIII compared to rFVIII.
Conclusion(s): These data demonstrate the OSA/CSA discrepancy is observed in humans and mice, and therefore not species specific. The discrepency was maintained in the absence of vWF, supporting that it is not related to interactions with vWF. Interestingly, FVIII:C by 2SA is higher for transgene-derived versus recombinant FVIII, suggesting gene therapy derived FVIIIa has enhanced function relative to rFVIII.
To cite this abstract in AMA style:Sternberg A, Samelson-Jones B, George L. Elucidating the Mechanism Behind the AAV-Derived Factor VIII Assay Discrepancy [abstract]. https://abstracts.isth.org/abstract/elucidating-the-mechanism-behind-the-aav-derived-factor-viii-assay-discrepancy/. Accessed November 29, 2023.
« Back to ISTH 2022 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/elucidating-the-mechanism-behind-the-aav-derived-factor-viii-assay-discrepancy/