One-stage Assay Factor VIII Activity Reflects AAV-Derived Factor VIII-Enhanced Thrombin Activation and Predicts Phenotype

Abstract

Hemophilia A (HA) phenotype is predicted by factor VIII (FVIII) activity. Most HA adeno-associated virus (AAV) trials incorporate the B-domain-deleted FVIII-SQ variant and one-stage assay (OSA) FVIII activity exceeds chromogenic substrate assay (CSA) values by 1.5-2-fold. This contrasts with recombinant FVIII-SQ (rFVIII-SQ), suggests altered biochemical properties, and highlights the need to determine which assay reflects hemostatic function. In gene therapy treated mice and SPK-8011 trial participants, AAV-derived FVIII-SQ (AAV-FVIII-SQ) activation and function within the intrinsic tenase enzyme complex was compared to rFVIII-SQ. In both species, AAV-FVIII-SQ demonstrated normal cofactor function and A2-domain stability. In mice, the assay discrepancy persisted without von Willebrand factor (vWF), indicating it is not driven by altered vWF interactions. Both species demonstrated enhanced thrombin-mediated activation of AAV-FVIII-SQ, detectable by OSA but not CSA. Negative binomial regression analysis of 23 SPK-8011 participants, representing 99 cumulative patient-years, trended toward better prediction of annualized bleeding rate with OSA than CSA. In vivo evaluation of AAV-FVIII-SQ in mice demonstrated that OSA activity better correlated with hemostatic function and corresponded to rFVIII-SQ function. These findings support that OSA FVIII activity reflects AAV-FVIII-SQ function within the intrinsic tenase complex, captures enhanced activation not detected by CSA, and best predicts clinical outcome.