Recent clinical trial successes of AAV-based gene therapy for hemophilia B utilizing the hyperactive factor (F) IX variant, Padua (Arg338Leu), demonstrate the potential of this strategy to achieve disease ameliorating FIX activity levels at lower vectors doses. However, the underlying molecular mechanisms responsible for the 8-fold increase in specific activity remain unanswered. To address this question, we have undertaken a series of biochemical comparisons between recombinant FIX-Padua and FIX wild-type (WT). We observe FIX-Padua demonstrates increased aPTT-based clotting activity and increased thrombin generation compared to FIX-WT, as both a zymogen and activated (FIXa) enzyme. Furthermore, FIX-Padua and FIX-WT are activated by FXIa at similar rates. Combined, these results suggest that the hyperactivity of FIX-Padua is due to alterations of the activated enzyme and not due to differences in the zymogen or its activation.
We therefore investigated the protease ability of FIXa-Padua and FIXa-WT. In the absence of FVIIIa, FIXa-Padua and FIXa-WT display similar rates of cleavage of peptide substrates. Similarly, FIXa-Padua and FIXa-WT are inactivated by the suicide substrate antithrombin at similar rates. However, when combined with FVIIIa within the intrinsic Xase complex, FIXa-Padua, demonstrates a 3-fold increased catalytic rate (kcat) of FX activation compared to FIXa-WT. Thus, the hyperactivity of FIX-Padua appears to be due to enhanced enzymatic activity with its incorporation into the intrinsic Xase during clotting, and not due to differences in activation or inactivation. The additional fold increase in plasma-based clotting activity may be due to a combination of amplification and feedback reactions occurring in plasma-based assays since the relative difference in clotting activity between FIX-Padua and FIX-WT increases with increasing FX concentration. These biochemical studies support the safety of FIX-Padua as a therapeutic since they suggest that FIX-Padua is regulated similarly to FIX-WT. As such, thrombotic complications would only be anticipated at supratherapeutic FIX activity levels and would be similar for FIX-Padua and FIX-WT at a given activity level. The implication of these results is that the active site of FIXa in the intrinsic Xase is improved by the Padua substitution.
We also observe that most substitutions in a 20 amino acid screen at position 338 result in FIX variants with at least equal activity compared to WT. FIX-Padua is the most active variant, while surprisingly, FIX-WT is one the least functional that does not cause hemophilia. Since Arg-338 is highly conserved within FIX mammalian orthologs, these results suggest that FIX-WT may have evolved to have limited procoagulant activity; indeed, our biochemical studies suggest that the FIX-WT active site is sub-optimal for FX activation. We, therefore, examined if there were additional positions in FIX where amino acid substitutions would lead to variants with increased clotting activity. To direct our search, we focused on positions within the protease domain that were 1) likely structurally important for interactions with FVIIIa and 2) where hemophilia B causing mutations have not been reported. Using this strategy, we identified 3 additional positions where single amino acid substitutions resulted in FIX variants with 2 - 6 fold increased clotting activity compared to FIX-WT. Moreover, the most active novel substitution can be additively combined with the Padua substitution: recombinant FIX of this two amino-acid substituted variant displayed 15-fold increased specific activity compared to FIX-WT and almost 2-fold increased specific activity compared to FIX-Padua. Given the proven benefit of the enhanced activity of FIX-Padua now being employed in clinical trials, novel hyperactive substitutions of FIX could further enhance the safety and efficacy of these therapeutics.
Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; sparK: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Novo Nordisk: Research Funding; Bayer: Consultancy. Arruda:Pfizer: Patents & Royalties, Research Funding.
Asterisk with author names denotes non-ASH members.