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No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII<\/jats:styled-content> domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full\u2010length CTB<\/jats:styled-content>\u2010FVIII<\/jats:styled-content> sequence to cover the entire patient population, regardless of individual CD<\/jats:styled-content>4+<\/jats:sup> T\u2010cell epitope responses. Codon optimization of FVIII<\/jats:styled-content> heavy chain (HC<\/jats:styled-content>) and light chain (LC<\/jats:styled-content>) increased expression 15\u2010 to 42\u2010fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB<\/jats:styled-content> fusion proteins of FVIII<\/jats:styled-content>\u2010HC<\/jats:styled-content> (99.3\u00a0kDa), LC<\/jats:styled-content> (91.8\u00a0kDa), C2 (31\u00a0kDa) or single chain (SC<\/jats:styled-content>, 178.2\u00a0kDa) up to 3622, 263, 3321 and 852\u00a0\u03bcg\/g in lyophilized plant cells, when grown in a cGMP<\/jats:styled-content> hydroponic facility (Fraunhofer). CTB<\/jats:styled-content>\u2010FVIII<\/jats:styled-content>\u2010SC<\/jats:styled-content> is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891\u00a0kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long\u2010term storage as revealed by GM<\/jats:styled-content>1\u2010ganglioside receptor binding assays. Repeated oral gavages (twice\/week for 2\u00a0months) of CTB<\/jats:styled-content>\u2010FVIII<\/jats:styled-content>\u2010HC<\/jats:styled-content>\/CTB<\/jats:styled-content>\u2010FVIII<\/jats:styled-content>\u2010LC<\/jats:styled-content> reduced inhibitor titres ~10\u2010fold (average 44\u00a0BU\/mL to 4.7\u00a0BU\/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP<\/jats:styled-content>\u2010expressing CD<\/jats:styled-content>4+<\/jats:sup>CD<\/jats:styled-content>25+<\/jats:sup>FoxP3+<\/jats:sup> Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant\u2010based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII<\/jats:styled-content> injections.<\/jats:p>","DOI":"10.1111\/pbi.12859","type":"journal-article","created":{"date-parts":[[2017,11,6]],"date-time":"2017-11-06T15:42:37Z","timestamp":1509982957000},"page":"1148-1160","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":55,"title":["Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice"],"prefix":"10.1111","volume":"16","author":[{"given":"Kwang\u2010Chul","family":"Kwon","sequence":"first","affiliation":[{"name":"Department of Biochemistry School of Dental Medicine University of Pennsylvania Philadelphia PA 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