Factor IX Padua: From Biochemistry to Gene Therapy

Novel approaches to enhance the biologic activity of therapeutic proteins have the potential to improve protein- and gene-based therapy for hemophilia. We have identified the variant Factor IX Padua (FIX) (R338L) with 8-fold increase in specific activity compared to wild-type FIX as well as additional strategies to identify other modifications with a positive effect on the biological activity of FIX. FIX-Padua is already in early phase gene therapy clinical trials for hemophilia B. However, it is notable that the field is moving forward even though the molecular basis for its enhanced function has remained elusive. The increased specific activity of FIX Padua compared to FIX wild-type resides in the activated protease as purified recombinant FIX Padua displays enhanced clotting activity as both a zymogen and activated protease. This augmentation of FIX Padua zymogen and protease is observed in both clotting and thrombin generation assays. However, preliminary biochemical characterization suggests that that the increased activity is most pronounced in plasma-based assays, while differences in enzyme kinetic parameters measured in reconstituted systems are more modest. Intriguingly, we have found that most amino acid substitutions at position 338, result in a FIX variant with comparable or enhanced clotting activity with the Padua substitution resulting in the most active variant, suggesting that R338 in FIX wild-type forms an unfavorable interaction that can be relieved by most amino acid substitutions. The wild-type variant is actually the least active variant at this position that is not known to cause hemophilia B. Since, R338 is strictly conserved among mammalian FIX orthologues, there may be an evolutionary pressure to maintain the unfavorable interactions of R338 and limit FIX activity. The corollary to this speculation is that other FIX mutations that relieve deleterious interactions will also increase clotting activity. The characterization of FIX Padua suggests small biochemical improvements may result in substantial increases in plasma based clotting activity. Promising preclinical studies on efficacy and safety, including thrombogenicity and immunogenicity, in small and large animal models provide the basis for translational studies using these proteins. These studies support the concept that the thrombotic risk of FIX Padua activity is similar to FIX wild-type activity. The immunogenicity of FIX Padua is comparable to FIX wild-type in either an adeno-associated virus-based muscle- or liver-directed gene therapy in canine models of hemophilia B. In the last 18 months, results from first 10 men with severe hemophilia B enrolled in two ongoing AAV liver-directed gene therapy clinical trials using a FIX Padua as a transgene were reported. No subject in either study developed inhibitors to FIX Padua or thrombotic complications. In subjects with sustained FIX Padua expression, FIX activity was greater than 10%. These promising early phase results demonstrate the potential of utilizing variants with increased specific activity in gene therapy allowing for lower therapeutic vector doses. It remains to be seen if curative factor levels can be safely achieved with further vector refinements including improved FIX variants. Disclosures Arruda: Pfizer: Research Funding.