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Current clinical immune tolerance protocols are often not effective due to complications such as anaphylactic reactions against FIX. Plant\u2010based 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\u2010FIX gene and homoplasmy (transformation of \u02dc10\u00a0000 copies in each cell) were maintained in both T1 and T2 generation marker\u2010free plants. CTB\u2010FIX expression in lyophilized leaves of T1 and T2 marker\u2010free plants was 1.0\u20131.5\u00a0mg\/g dry weight, confirming that the marker excision did not affect antigen levels. Oral administration of CTB\u2010FIX to Sprague Dawley rats at 0.25, 1 or 2.5\u00a0mg\/kg did not produce overt adverse effects or toxicity. The no\u2010observed\u2010adverse\u2010effect level (NOAEL) is at least 2.5\u00a0mg\/kg for a single oral administration in rats. Oral administration of CTB\u2010FIX at 0.3 or 1.47\u00a0mg\/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.<\/jats:p>","DOI":"10.1111\/pbi.13608","type":"journal-article","created":{"date-parts":[[2021,5,5]],"date-time":"2021-05-05T09:04:34Z","timestamp":1620205474000},"page":"1952-1966","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["Preclinical development of plant\u2010based oral immune modulatory therapy for haemophilia B"],"prefix":"10.1111","volume":"19","author":[{"given":"Aparajitha","family":"Srinivasan","sequence":"first","affiliation":[{"name":"Department of Basic and Translational Sciences School of Dental Medicine University of Pennsylvania Philadelphia PA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roland W.","family":"Herzog","sequence":"additional","affiliation":[{"name":"Department of Pediatrics Herman B Wells Center for Pediatric Research Indiana 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