FIX-R338L (FIX Padua) as a Successful Alternative for the Treatment of Canine Severe Hemophilia B. (2009)

Abstract Abstract 694 Hemophilia B (HB) is a severe bleeding disorder affecting 1 in 30,0000 men and results from the deficiency of FIX clotting activity. The current treatment involves infusion of either plasma derived or recombinant FIX. We have recently described a novel naturally occurring gain-of-function mutation in the FIX gene found associated with hyperfunctional activity of the protein (N Engl J Med, 2009). The proband presented with 776% FIX clotting activity (FIX:C), but normal FIX antigen levels (FIX:Ag of 92% of normal), and after detailed analysis the causative mutation was found to be a transversion of R338L in FIX (FIX-Padua) that segregates in a classical X-linked fashion. Furthermore, in vitro expression of FIX-Padua confirmed that the protein had a specific activity that was 8 fold higher than FIX-wild type. Arginine at 338 is highly conserved in FIX from mammals but is unique among other human vitamin K-dependent proteins. We sought to determine whether FIX-Padua would be an attractive strategy for novel therapy of hemophilia B by introducing this mutation in the canine FIX gene (cFIX) and testing in HB dog models. The HB colony at Chapel Hill is an outbred, immunocompetent large animal model that have a missense mutation in the cFIX gene and have no detectable circulating levels of cFIX antigen or activity, and its disease phenotype closely correlates with that seen in humans. Adeno-associated viral (AAV) vector encoding the cFIX-Padua and cFIX-wt under control of the CMV promoter was generated and administered to HB dogs. AAV6 was chosen as the serotype to overcome the high prevalence of neutralizing antibodies to AAV2 in the general population. Delivery of AAV6-cFIX wild type by intravascular delivery to the skeletal muscle via anterograde limb perfusion (ALP) coupled with transient immunosuppression (duration of 5 weeks) resulted in FIX:Ag and FIX:C of 2-5% of normal with partial shortening the whole blood clotting time. Post AAV6-cFIX Padua injection we observed a remarkable correction of the WBCT to the normal range (8-10 min) within the first 7 days that has continued for the duration of the study. One dog's (M55) FIX:Ag levels were ∼250 ng/mL (5% of normal) at the peak and 180 ng/mL (3.6%) at the plateau. Notably, his FIX:C levels were 49% at peak and 35% at plateau, indicating a specific activity ∼10 fold higher than wild type cFIX. Treatment of a second dog (M59) resulted in FIX:Ag levels of 175 ng/mL (3.5%) at the peak and 60 ng/mL (1.2%) at plateau. This is in contrast with his FIX:C levels that were 35% at peak and 12% at plateau levels, respectively. Like M55, the specific activity was 10 fold higher than wild type cFIX. An important consideration is the potential for an immune response against the cFIX-Padua neo-antigen. Notably, there is no evidence of formation of inhibitor to cFIX in these dogs for a cumulative observation period of 6 months post-immunosuppression. We have demonstrated that the cFIX-Padua mutant protein has 10-fold higher specific activity in a canine model of HB. This is significant as it allows for lower doses of AAV vectors to be used for the treatment of HB without compromising efficacy, thereby increasing the safety profile of clinical gene therapy for HB. In addition, FIX Padua provides an excellent alternative to overcome the limited ability of skeletal muscle to synthesize fully posttranslational modified functional FIX (Blood, 2001). Thus, FIX-Padua improves both the efficacy and safety of skeletal muscle-mediated FIX resulting in FIX:C levels sufficient to convert the severe HB phenotype to mild hemophilia (10-35% of normal) at vector doses already tested clinical trials. This strategy has the potential of improving other gene and/or cell therapy for HB targeting ectopic targets as well as the liver. Disclosures: High: Genzyme: Patent licensed to Genzyme, but Dr. High has waived all financial interest, Patents & Royalties.