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

Formation of pathogenic antibodies is a major problem in replacement therapies for inherited protein deficiencies. For example, antibodies to coagulation factors (‘inhibitors’) seriously complicate treatment of haemophilia. While immune tolerance induction (ITI) protocols have been developed, inhibitors against factor IX (FIX) are difficult to eradicate due to anaphylactic reactions and nephrotic syndrome and thus substantially elevate risks for morbidity and mortality. However, hepatic gene transfer with an adeno-associated virus (AAV) serotype 8 vector expressing FIX (at levels of ≥4% of normal) rapidly reversed pre-existing high-titre inhibitors in haemophilia B mice, eliminated antibody production by B cells, desensitized from anaphylaxis (even if protein therapy was resumed) and provided long-term correction. High levels of FIX protein suppressed memory B cells and increased Treg induction, indicating direct and indirect mechanisms of suppression of inhibitor formation. Persistent presence of Treg was required to prevent relapse of antibodies. Together, these data suggest that hepatic gene transfer-based ITI provides a safe and effective alternative to eradicate inhibitors. This strategy may be broadly applicable to reversal of antibodies in different genetic diseases.

The liver is a preferred target organ for gene therapy not only for liver-specific diseases but also for disorders that require systemic delivery of a protein. Diseases that could benefit from hepatic gene transfer include hemophilia, metabolic disorders, lysosomal storage disorders, and others. For a successful delivery of the transgene and sustained expression, the protocol must avoid immune responses in order to be efficacious. A growing number of studies have demonstrated that liverdirected transfer can induce transgene product-specific immune tolerance. Tolerance obtained via this route requires optimal engineering of the vector to eliminate transgene expression in antigen presenting cells while restricting high levels of therapeutic expression to hepatocytes. Innate immune responses may prevent tolerance induction, cause toxicity, and have to be minimized. Discussed in our review is the crucial role of CD4 + CD25 + regulatory T cells in tolerance to the hepatocyte-derived gene product, the immunobiology of the liver and our current understanding of its tolerogenic properties, current and proposed research as to the mechanisms behind the liver's unique cellular environment, as well as development of the tools for tolerance induction such as advanced vector systems.

Summary. The immune response to coagulation factors VIII or IX, in particular formation of inhibitory antibodies, complicates treatment of hemophilia. Therefore, a number of recent studies in animal models have explored novel approaches toward induction of immune tolerance in protein or gene replacement therapy. Strong evidence has emerged that regulatory T cells (Treg) are an important component of the mechanism by which tolerance is maintained and inhibitor formation, a T help dependent response, is prevented. Limited data in patients also support this concept. In particular, CD4 + CD25 + FoxP3 + Treg, whether naturally occurring or induced, have been invoked in suppression of antibody and of cytotoxic T lymphocyte responses to the therapeutic clotting factor. This review summarizes the data on this emerging concept of Treg-mediated regulation of the immune response in treatment of hemophilia, strategies and mechanisms of Treg induction and function, and the implications for development of immune tolerance protocols.

222 While the incidence of inhibitor formation against the infused coagulation factor is overall relatively low in factor IX-deficiency (1–4% of patients), 9–23% of patients with severe hemophilia B form inhibitors, which seriously complicates treatment. Factor IX (F.IX) inhibitors tend to be high titer and not effectively eliminated by immune tolerance induction protocols, and are often associated with serious anaphylactic reactions. Inhibitor formation and anaphylaxis is more typical for patients with F9 gene deletions or early stop codons. We sought to develop a novel approach to induction of antigen-specific immune tolerance that is safe, not based on immune suppression, and does not require knowledge of specific T cell epitopes. Chloroplast transgenic plants expressing human F.IX were generated. Because the plant cell provides bioencapsulation, the recombinant polypeptide is protected from acids and enzymes in the stomach and released in the gut. The hF.IX coding sequences were fused with the transmucosal carrier CTB. The fusion gene cassettes were expressed from the light-regulated psbA 5′ untranslated region/promoter in order to achieve hyper-expression. To prevent steric hindrance, a GPGP hinge was introduced between CTB and FIX. A furin cleavage site was also introduced in one of the constructs. Plasmid DNA was bombarded on nicotine-free tobacco leaves via a particle-mediated method using the gene gun, resulting in transgene integration into the chloroplast genome by homologous recombination. Transformed tissues were repeatedly cultured on selective media, and transplastomic plants were regenerated. Chloroplast transplastomic lines were examined by Southern blot analysis in order to confirm site-specific integration and to test for homoplasmic lines. Transplastomic lines of CTB-GPGP-hFIX (F1) and CTB-GPGP-Furin-hFIX (F2) had expression levels of up to 3.8% and 0.28% fusion protein, respectively, in the total soluble protein. Control and transplastomic plant materials (250 mg) were fed twice per week for 8 weeks by oral gavage to Hemophilia B mice, known to develop strong antibody responses against hF.IX (C3H/HeJ F9−/− mice with targeted F9 gene deletion). For the F2 material, this was equivalent to 2 μg of hF.IX per dose. After 4 weeks, animals received weekly infusions of hF.IX (Benefix) at 1 IU per dose for 4 weeks (1 IP followed by 3 IV doses). As expected, ≥80% of mice control groups had developed significant inhibitors at titers of 4–10 BU (average titers were 5.5 BU for wild-type material fed and 7 BU for unfed controls). In contrast, all F1 and F2 fed mice (n=15 and n=14, respectively) had undetectable or at most very low titer inhibitors (≤2 BU) and showed normal clearance of hF.IX. Some F2 fed mice (n=9) was subjected to 1 more month of weekly IV infusions of hF.IX. At the end of this experiment, inhibitor titers remained undetectable to low (< 3 BU) in 6/9 mice, while 3 mice had formed titers of 4–12 BU, in contrast to on average 80 BU in control mice. Importantly, a rise in inhibitor titers was not seen in another cohort, in which feeding was continued during subsequent treatment. Most importantly, 30–50% of control mice developed allergic reactions, went into respiratory arrest, and died instantly or shortly after the 4th and subsequent injections of hF.IX. This severe anaphylactic response was not observed for up to 12 hF.X injections in any F1/2-fed animals regardless of whether feeding was continued beyond 8 weeks. The underlying mechanism appears to involve a shift to non-inhibitory antibodies and shifts in immunoglobulin subclasses. In summary, repeated feeding of bioencapsulated hF.IX prevents inhibitor formation and life-threatening anaphylactic reactions. Disclosures: Daniell: Chlorogen Inc: Technical founder.