Abstract 2048


Hemophilia B, a bleeding disorder caused by deficiency of coagulation factor IX (FIX), is well suited for a gene therapy approach since continuous plasma levels above 1% already prevent major long-term complications. Although viral vectors are more efficient, non-viral vectors become more and more attractive as gene delivery systems because of their excellent safety profiles. The transgene expression from those systems is relatively low but a repeated vector administration is possible to obtain durable transgene in a therapeutic range. In this study we explore oral gene delivery based on the cationic biopolymer chitosan and FIX variants with improved clotting factor function for gene delivery. The oral administration route is considered privileged not only because of the ease administration, but also because of the immunoprivileged presentation of antigens and the resulting potential to induce immune tolerance. Chitosan nanoparticles protect the encapsulated DNA from digestion in the gastrointestinal tract and enhance the uptake in the gut by improving intestinal trans- and paracellular permeability and its mucoadhesive nature.


Nucleotide substitutions for protein modification were introduced by site-directed mutagenesis into the pcDNA3.1 vector and a minicircle system, which differ from standard plasmid vectors by the lack of plasmid bacterial sequences. All vectors consist of a FIX expression cassette driven by a CMV promoter. Vectors were formulated as chitosan nanoparticles for oral delivery to treat FIX-deficient mice. Protection of vector DNA in nanoparticle constructs was confirmed by DNase I digest and subsequent gel electrophoresis.


First, HEK293 cells were transfected with chitosan nanoparticles containing the reporter gene eGFP. At 24h post-transfection, cells yielded in eGFP expression visualized with fluorescence microscope. Transfection with nanoparticles containing wild-type FIX resulted in secretion of FIX protein up to 100 ng/ml (2% of normal plasma concentration). Encouraged by the in vitro findings, FIX-deficient mice were administerd eGFP formulated chitosan nanoparticles to confirm gene transfer in vivo. Immunofluorescent staining revealed eGFP expression in small intestine. Next, we treated groups of mice (n=4–5/group) with naked DNA or nanoparticles complexes containing FIX or mock DNA. Circulating plasma levels in all groups remained below the therapeutic range (<1%). However, RT-PCR confirmed a 30-fold higher FIX expression in the small intestine compared to naked DNA treated mice. To improve therapeutic efficacy, different amino acid substitutions known to increase FIX clotting activity were combined. One protein variant was identified with 1500% FIX specific clotting activity compared to the wild-type protein. Following oral gene transfer with variant FIX, FIX antigen expression levels remained under the detection limit but the clotting times of mice were shortened due to a FIX activity of 1–4%. Immune histochemical staining confirmed FIX accumulation in small intestine caused by FIX binding to its physiological binding partner collagen IV in the extracellular matrix of the gut. To overcome the physiological affinity and improve protein release into the circulation a second variant was generated with impaired binding to collagen type IV and with 2000% FIX specific clotting activity. FIX antigen levels still remained low, but FIX activity in the therapeutic range of 3–4% was measured in the plasma. Immunofluorescent staining indicated remaining extracellular collagen IV binding in the gut. Further, to improve the low in vivo transfection efficiency a minicircle system was used to produce smaller chitosan nanoparticles by reducing vector size. Treatment of FIX-deficient mice provided haemostasis with FIX activity of 2–14% of individual mice while antigen levels still remained undetected. No inhibitory antibodies against FIX were detected in any treated animal. Experiments with repeated FIX challenges prior and post gene transfer are ongoing.


Oral gene transfer based on chitosan nanoparticles seems feasible but only results in low levels of FIX expression. Protein engineering for FIX and optimized expression vectors might provide a possibility to overcome this shortcoming and therefore help to develop a gene transfer strategy for prophylaxis of bleedings and/or inhibitory antibodies.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.