Oral gene therapy for hemophilia B using chitosan‐formulated FIX mutants

Summary 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.