Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-04, Vol.107 (15), p.7101-7106
• Main Authors: Verma, Dheeraj, Moghimi, Babak, LoDuca, Paul A, Singh, Harminder D, Hoffman, Brad E, Herzog, Roland W, Daniell, Henry
• Format: Article
• Language: English
• Subjects: Administration, Oral; Allergies; Anaphylaxis; Anaphylaxis - mortality; Anaphylaxis - prevention & control; Animals; Antibodies; Antigens; Biological Sciences; Chloroplasts; Chloroplasts - metabolism; Disease Models, Animal; Drug Delivery Systems; Factor IX - administration & dosage; Factor IX - antagonists & inhibitors; Factor IX - chemistry; Genetic disorders; Genetic Vectors; Hemophilia; Hemophilia A; Hemophilia B; Hemophilia B - blood; Hemophilia B - complications; Immunoglobulin E - metabolism; Male; Medical treatment; Mice; Mice, Inbred C3H; Models, Genetic; Nicotiana - genetics; Plant antigens; Plant cells; Rodents; Studies; Transgenic plants
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0912181107

To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin β-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20–25% of control animals survived after six to eight F.IX doses, 90–93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyer's patches in the ileum. Within 2–5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5–80 μg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (∼40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.