Factor VIII activity after AAV gene therapy. (A) AAV5-BDD-FVIII (valoctocogene roxaparvovec) is infused intravenously into a patient with severe hemophilia A. The vector-gene targets the hepatocyte and is expressed and secreted into the circulation. (B) Transgene-produced FVIII measured by the OS clot assay, indicated by the dark line, is 1.6-fold higher than FVIII activity measured by CS assay, indicated by the light line. (C) Kinetic bias leads to higher FVIII activity by OS than CS activity by gene-produced FVIII compared with native (wild-type) FVIII, as the former, but not the latter, accelerates early factor Xa formation, leading to earlier visible clot formation in the OS assay, which is read within the first 1 to 2 minutes of the coagulation reaction, whereas gene-produced FVIII and native FVIII show similar clot formation in the CS assay, which measures FVIII activity by color change after a 5-minute incubation, and is unaffected by the early reaction resulting in lower FVIII activity than by the OS assay. For panel C, see Figure 6 in the article by Rosen et al that begins on page 2524.