Adeno-associated virus (AAV) vector liver-directed gene therapy (GT) for hemophilia B (HB) is limited by a vector dose–dependent hepatotoxicity. Recently, this obstacle has been partially circumvented by the use of a hyperactive factor IX (FIX) variant, R338L (Padua), which has an 8-fold increased specific activity compared with FIX-WT. FIX-R338L has emerged as the standard for HB GT. However, the underlying mechanism of its hyperactivity is undefined; as such, safety concerns of unregulated coagulation and the potential for thrombotic complications have not been fully addressed. To this end, we evaluated the enzymatic and clotting activity as well as the activation, inactivation, and cofactor dependence of FIX-R338L relative to FIX-WT. We observed that the high specific activity of FIX-R338L requires factor VIIIa (FVIIIa) cofactor. In a potentially novel system using emicizumab, an FVIII-mimicking bispecific antibody, the hyperactivity of both recombinant FIX-R338L and AAV-mediated transgene-expressed FIX-R338L from HB GT subjects was ablated without FVIIIa activity. We conclude that the molecular regulation of activation, inactivation, and cofactor dependence of FIX-R338L is similar to FIX-WT but that the FVIIIa-dependent hyperactivity of FIX-R338L is the result of a faster rate of factor X activation. This mechanism helps mitigate safety concerns of unregulated coagulation and supports the expanded use of FIX-R338L in HB therapy.
Benjamin J. Samelson-Jones, Jonathan D. Finn, Lindsey A. George, Rodney M. Camire, Valder R. Arruda