Preclinical development of plant‐based oral immune modulatory therapy for haemophilia B

Anti‐drug antibody (ADA) formation is a major complication in treatment of the X‐linked bleeding disorder haemophilia B (deficiency in coagulation factor IX, FIX). Current clinical immune tolerance protocols are often not effective due to complications such as anaphylactic reactions against FIX. Plant‐based oral tolerance induction may address this problem, as illustrated by the recent first regulatory approval of orally delivered plant cells to treat peanut allergy. Our previous studies showed that oral delivery of plant cells expressing FIX fused to the transmucosal carrier CTB (cholera toxin subunit B) in chloroplasts suppressed ADA in animals with haemophilia B. We report here creation of the first lettuce transplastomic lines expressing a coagulation factor, in the absence of antibiotic resistance gene. Stable integration of the CTB‐FIX gene and homoplasmy (transformation of ˜10 000 copies in each cell) were maintained in both T1 and T2 generation marker‐free plants. CTB‐FIX expression in lyophilized leaves of T1 and T2 marker‐free plants was 1.0–1.5 mg/g dry weight, confirming that the marker excision did not affect antigen levels. Oral administration of CTB‐FIX to Sprague Dawley rats at 0.25, 1 or 2.5 mg/kg did not produce overt adverse effects or toxicity. The no‐observed‐adverse‐effect level (NOAEL) is at least 2.5 mg/kg for a single oral administration in rats. Oral administration of CTB‐FIX at 0.3 or 1.47 mg/kg either mixed in food or as an oral suspension to Beagle dogs did not produce any observable toxicity. These toxicology studies should facilitate filing of regulatory approval documents and evaluation in haemophilia B patients.