September 24, 2020

Collaboration with BioMarin – Study on FVIII gene therapy assay discrepancy now published in Blood!

We have been working with BioMarin in order to identify a cause of dicrepancy between one stage assays and chromogenic assays when analysing transgene-produced FVIII-SQ (AAV5-FVIII-SQ). The results from this exciting study are now published in Blood.

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Activity of Transgene-Produced B-Domain Deleted Factor VIII in Human Plasma Following AAV5 Gene Therapy

Steffen Rosen, Stefan Tiefenbacher, Mary Robinson, Mei Huang, Jaydeep Srimani, Donnie Mackenzie, Terri Christianson, John Pasi, Savita Rangarajan, Emily Symington, Adam Giermasz, Glenn F Pierce, Benjamin Kim, Stephen J Zoog, Christian Vettermann

Article available online and in print

Key Points

  • Higher OS than CS activity for AAV5-FVIII-SQ is likely caused by accelerated early FXa formation, resulting in a kinetic bias between assays
  • Specific activity (IU/mg) remains comparable between transgene-produced and recombinant FVIII-SQ in the CS assay, but not in the OS assay.
Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically utilize a B-domain deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) than chromogenic-substrate (CS) assays, while recombinant FVIII-SQ products have lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (IU/mg) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assays kits and clinical laboratories, suggesting intrinsic molecular features as a potential root cause. Further experiments in two participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from non-hemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate endpoint to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on (NCT02576795 and NCT03370913).