Comparison of hemostatic dosing of factor IX wild-type and padua (R338L) in multiple hemophilia B mouse models Free

Introduction The hyperactive factor IX (FIX) variant Padua (R338L) has turbocharged gene therapy for hemophilia B (HB). FIX-Padua is the transgene for all three approved gene therapies for HB worldwide. FIX-Padua's 5-15-fold increased specific activity compared to wild-type (WT) FIX after liver-directed gene therapy (Samelson-Jones and Arruda, MTM&CD 2019) has allowed for higher FIX activity levels at lower vector doses. However, the quantification of the relative hemostatic potency of FIX-Padua compared to FIX-WT is complicated by the well-characterized 3-fold assay discrepancy of FIX-Padua but not FIX-WT (Foley et al. Blood Advances 2023); thus, the relative in vitro potency of FIX-Padua over FIX-WT depends on the assay.

Previous studies have demonstrated that rFIX-Padua and FIX-Padua after liver-directed gene therapy exhibit the same assay discrepancy (Robinson et al. JTH 2021). The bleeding phenotype of FIX-Padua gene therapy recipients has to-date not been able to inform on this question. Previous mouse studies demonstrated improved hemostasis of FIX-Padua compared to even higher levels of FIX-WT after gene therapy but did not quantify the difference (Cantore et al. Blood 2012). To address this question, we compared the dose-dependent hemostatic effect of recombinant (r) FIX-Padua and FIX-WT protein in HB mice subjected to 2 distinct injury models.

Methods rFIX WT and variant proteins were expressed in mammalian cell culture and then purified to homogeneity by affinity chromatography. rFIX protein was administered intravenously 5 minutes prior to injury. In the tail-clip (TC) assay, the tails of HB and wild-type (WT) BALB/c mice were cut at a diameter of 3mm completely transecting both veins and the artery. Blood was collected for 10 minutes and quantified spectrophotometrically. In the tail vein transection (TVT) assay, only the lateral tail vein was lacerated in C57bl6 mice. The initial clot was removed every 10 minutes and blood was collected for 40 minutes.

Results In the TC assay (n≥6 mice/cohort), HB mice that received only buffer exhibit excessive hemorrhage with blood loss (median (IQR)) of 17 (14-21) µL/g compared to 0.8 (0.2-4) µL/g in WT mice. Administration of rFIX dose-dependently decreases blood loss. Administration of rFIX-Padua at doses of 0.025 mg/kg and 0.05 mg/kg decreases blood loss to 13 (1-18) µL/g and 2 (1-9) µL/g (p<0.001) respectively. Administration of rFIX-WT at doses of 0.05 mg/kg, 0.225 mg/kg, and 0.4 mg/kg decreases blood loss to 22 (10-25) µL/g, 5 (3-10) µL/g, and 1(0.5-9) µL/g (p<0.001) respectively. 0.05 mg/kg rFIX-Padua significantly reduces blood loss compared to the equivalent dose of rFIX-WT (p<0.01). The rFIX dose-dependence for blood loss was quantified by empirically fitting the results to logistic curves (R²>0.94). Based on this fitting, there is a 6-fold decrease in EC50 and EC80 values for rFIX-Padua (0.031±0.0001 and 0.040±0.0001 mg/kg) compared to rFIX-WT (0.19±0.05 and 0.25±0.04 mg/kg).

In the TVT assay (n≥3 mice/cohort), HB mice that received only buffer exhibit excessive hemorrhage with blood loss and prolonged bleeding time (mean±SEM) 860±76 µL and 31±1 minutes, respectively versus 68±17 µL and 6±1 minutes in WT mice. Administration of rFIX-Padua at doses of 0.0625 mg/kg and 0.125 mg/kg decreases blood loss to 503±110 µL and 85±23 µL, respectively, and bleeding time to 12±3 minutes and 7±0.5 minutes, respectively. Administration of rFIX-WT at doses of 0.25 mg/kg, 0.5 mg/kg, and 1 mg/kg, reduced blood loss to 397±147 µL, 182±89 µL, and 35±13 µL, respectively, and bleeding time to 16 ± 2 minutes, 8 ± 0.2 minutes, and 4 ± 0.4 minutes. The rFIX dose-dependence was quantified by empirically fitting the results logistic curves (R²>0.91). Based on this fitting, there is a 4-fold decrease in EC50 and EC80 values for rFIX-Padua (0.065±0.0001 and 0.083±0.0001 mg/kg) compared to rFIX-WT (0.23± 0.09 and 0.3±0.2 mg/kg).Conclusion Preliminary characterization of the hyperactivity of rFIX-Padua compared to rFIX-WT in two hemostatic tail injury models demonstrate a 4-6-fold increased potency in vivo. Improved quantification of this potency difference between rFIX-Padua and rFIX-WT is expected with expanded experimental cohorts. These initial results support the continued therapeutic use of FIX-Padua and inform on its in vivo hemostatic effect.