BACKGROUND:

FVIII neutralizing antibodies is 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 since 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 FVIIIindependent activity for gene and proteinbased therapy of 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:

1600 clones were screened and 3 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, 6 FIX mutants with the desired bypassing activity were designed. Candidate mutants FIXIDAV 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 5fold increase in catalytic efficiency. Expression of FIX-IDAV in FVIIIknockout 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 to improve HA treatment, especially for patients with inhibitors. This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.