BACKGROUND: FVIII neutralizing antibodies are the main complication of substitution therapy in hemophilia A (HA); auto-antibodies against FVIII causing acquired HA can also occur. Treatment of inhibitor patients remains challenging because prophylactic treatment with existing FVIII bypassing agents, all based on constitutively active coagulation factors, is difficult due to their short half-life. OBJECTIVES: To generate zymogenic FIX variants with FVIII-independent activity for gene- and protein-based therapy for HA. METHODS: Modifications were introduced into FIX based on current knowledge of FIX structure and FVIII-independent function followed by random screening. Activity, thrombin generation and FX activation by FIX mutants were characterized in the presence and absence of FVIII. Phenotype correction of promising candidates was assessed by the tail-clip assay in FVIII-knockout mice. RESULTS: About 1600 clones were screened and three mutations (L6F, S102N and E185D) identified, which improved FVIII-independent activity in combination with our previously described variant FIX-ITV. By systematic combination of all mutations, six FIX mutants with the desired bypassing activity were designed. Candidate mutants FIX-IDAV and FIX-FIAV demonstrated the most efficient thrombin generation in FVIII-deficient plasma and had considerably increased activities towards FX in the absence of FVIII, in that they showed an up to 5-fold increase in catalytic efficiency. Expression of FIX-IDAV in FVIII knockout mice reduced blood loss after the tail-clip assay, even in the presence of neutralizing FVIII antibodies. CONCLUSION: Activatable bioengineered FIX molecules (as opposed to pre-activated coagulation factors) with FVIII-independent activity might be a promising tool for improving HA treatment, especially for patients with inhibitors.
BACKGROUND:FVIII neutralizing antibodies are the main complication of substitution therapy in hemophilia A (HA); auto-antibodies against FVIII causing acquired HA can also occur. Treatment of inhibitor patients remains challenging because prophylactic treatment with existing FVIII bypassing agents, all based on constitutively active coagulation factors, is difficult due to their short half-life. OBJECTIVES: To generate zymogenic FIX variants with FVIII-independent activity for gene- and protein-based therapy for HA. METHODS: Modifications were introduced into FIX based on current knowledge of FIX structure and FVIII-independent function followed by random screening. Activity, thrombin generation and FX activation by FIX mutants were characterized in the presence and absence of FVIII. Phenotype correction of promising candidates was assessed by the tail-clip assay in FVIII-knockout mice. RESULTS: About 1600 clones were screened and three mutations (L6F, S102N and E185D) identified, which improved FVIII-independent activity in combination with our previously described variant FIX-ITV. By systematic combination of all mutations, six FIX mutants with the desired bypassing activity were designed. Candidate mutants FIX-IDAV and FIX-FIAV demonstrated the most efficient thrombin generation in FVIII-deficient plasma and had considerably increased activities towards FX in the absence of FVIII, in that they showed an up to 5-fold increase in catalytic efficiency. Expression of FIX-IDAV in FVIII knockout mice reduced blood loss after the tail-clip assay, even in the presence of neutralizing FVIII antibodies. CONCLUSION: Activatable bioengineered FIX molecules (as opposed to pre-activated coagulation factors) with FVIII-independent activity might be a promising tool for improving HA treatment, especially for patients with inhibitors.