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

Abstract

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.allergy | chloroplast | genetic disorders | oral tolerance | plant-made therapeutics

Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells

“…17,18 Oral delivery of transplastomic plant cells has been effective in prevention of insulitis in nonobese diabetic mice and of inhibitor formation in mice with hemophilia B. 19,20 For FIX inhibitors, immune tolerance induction is often not sustainable because of anaphylactic reactions and the development of nephrotic syndrome. In mice with hemophilia B, we demonstrated that repeated oral delivery of bioencapsulated FIX prevented inhibitor formation and fatal anaphylaxis in subsequent replacement therapy.…”

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Therefore, transgenes inserted into chloroplast genomes are expressed at high levels, up to 70% of total leaf protein (De Cosa et al, 2001;Ruhlman et al, 2010). A wide range of proteins, from very small antimicrobial peptides (Lee et al, 2011) or hormones (Boyhan and Daniell, 2011;Kwon et al, 2013) to very large proteins encoded by bacterial, viral, fungal, animal, and human genes, have been expressed successfully in plant chloroplasts (DeGray et al, 2001;Daniell et al, 2009;Verma et al, 2010;Shenoy et al, 2014;Sherman et al, 2014;Shil et al, 2014). Most importantly, expressed proteins are highly stable when lyophilized plant cells are stored at ambient temperature (Kwon et al, 2013;Lakshmi et al, 2013;Kohli et al, 2014;Jin and Daniell, 2015).…”

Immunology: Oral solutions

“…In 2010, they showed that oral delivery of factor IX expressed in chloroplasts in this way led to almost un detectable inhibitor levels in mice, and no sign of anaphylactic shock 1 . "The mice are healthy, they show no allergic responses and they don't form the inhibitors, " Herzog says.…”

“…The researchers behind the bio engineered lettuce have shown that inhibitor formation and severe allergic reactions can be prevented in mice by feeding the animals with a product based on these plants 1,2 . If the strategy also works in Anita and her kennel mates -and ultimately in humans -it could form the basis of the first product to protect against the immune responses associated with haemophilia treatment.…”