Factor IX Expressed in Lettuce Chloroplasts Induces Oral Tolerance in Hemophilia B Mice

Abstract

Inhibitor formation represents a major complication of factor replacement therapy for hemophilia. There are currently no prophylactic protocols against this antibody response. To address this problem, we sought to develop an antigen-specific immune tolerance protocol that does not rely on immune suppressive drugs and that would be feasible in pediatric patients. Oral tolerance fits these requirements but is hampered by vulnerability of the protein antigen to degradation in the stomach, cost of production, and inadequate delivery across the gut epithelium to the immune system (the gut-associated lymphoid tissue or GALT). We had hypothesized that transgenic expression of coagulation factors fused to a transmucosal carrier in the chloroplasts of plant cells would provide high level and cost-effective antigen production, bioencapsulation, and transmucosal delivery. Although we demonstrated this concept by of expression cassettes for factor VIII (FVIII) and factor IX (FIX) antigens fused to cholera toxin B (CTB) subunit in tobacco, further advancement of this system to the clinic is not feasible. Translation of this approach would require generation of transgenic edible plant cells expressing these fusion proteins. Therefore, in this study, we expressed CTB-FIX using species-specific chloroplast vectors regulated by endogenous psbA sequences, a requirement for optimal transgene expression (Plant Physiology 152: 2088-2104). Transplastomic plants were homoplasmic (inserted into all copies of chloroplast genomes in each plant cell) as evidenced by Southern blots. CTB-FIX antigen levels reached ~1mg antigen/g of lyophilized leaf cells and was stable with proper folding, disulfide bond formation, and CTB pentamer assembly when stored at ambient temperature for up to 2 years. Oral gavage of these cells (twice per week for 2 months) to hemophilia B mice (FIX gene deletion, C3H/HeJ background, n=7-11/experimental group) delivered CTB-FIX efficiently to the gut epithelium and immune system (as shown by immunohistochemistry), and induced LAP+ regulatory T cells in response to intravenous challenge with recombinant FIX protein. Moreover, FIX-CTB antigen doses of 1.5 mg, 5 mg, or 15 mg all effectively prevented inhibitor/IgG formation against intravenous FIX and averted anaphylaxis and associated fatal reactions by suppression of FIX-specific IgE production. Average inhibitor titers were 15-fold lower for CTB-FIX fed compared to control mice. Of orally treated mice, >80% had inhibitor titers that were undetectable to <2 BU, while controls formed high-titer inhibitors that were >5BU, with 9/11 showing >10 BU. Induction of tolerance in such a broad dose range should greatly facilitate translation of the approach. Our results support initiation of clinical studies on oral tolerance for hemophilia B using transplastomic lettuce. Disclosures Herzog: Spark Therapeutics: Patents & Royalties: Patent licenses; Novo Nordisk: Research Funding. Daniell:Novo Nordisk: Research Funding.