Next generation FIX muteins with FVIII-independent activity for alternative treatment of hemophilia A.

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.

Oral gene therapy for hemophilia B using chitosan-formulated FIX mutants.

BACKGROUND: Oral gene delivery of non-viral vectors is an attractive strategy to achieve transgene expression. Although expected efficacy from non-viral delivery systems is relatively low, repeated vector administration is possible and may help to obtain durable transgene expression in a therapeutic range. OBJECTIVES: To test the principle feasibility of using factor (F) IX variants with improved function combined with an optimized oral delivery system in hemophilia B (HB) mice. METHODS: FIX modifications were introduced by site-directed mutagenesis into plasmid- or minicircle-based expression cassettes. Vectors were formulated as chitosan nanoparticles for oral delivery to HB mice. Protection of vector DNA in nanoparticle constructs and transfection efficiency were characterized. HB mice received eGFP-formulated chitosan nanoparticles to confirm gene transfer in vivo. FIX expression, phenotype correction and the potential of nanoparticles to induce immunotolerance (ITI) against exogenous FIX were evaluated after repeated oral administration. RESULTS: Transfection of HEK 293T cells or livers of FIX-knockout mice with nanoparticles resulted in GFP or functional FIX expression. Oral administration of FIX mutants resulted in exclusive FIX expression in the small intestine, as confirmed by RT-PCR and fluorescence staining. HB mice demonstrated transient FIX expression reaching > 14% of normal activity and partial phenotype correction after oral delivery of FIX mutants with high specific activity and improved tissue release. CONCLUSION: The feasibility of oral, non-viral delivery of FIX was established and improved by bioengineered FIX proteins and optimized vectors. Thus, these data might point the way for development of a clinically applicable oral gene transfer strategy for hemophilia B.