title: Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B.
abstract: Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ∼2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP(+) regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ∼870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft(2) per annum yielding 24,000-36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs.
authors: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Jin
authors: J
authors: Su
authors: Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA.
authors: Liqing
authors: L
authors: Zhu
authors: Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA.
authors: Alexandra
authors: A
authors: Sherman
authors: Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA.
authors: Xiaomei
authors: X
authors: Wang
authors: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Shina
authors: S
authors: Lin
authors: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Aditya
authors: A
authors: Kamesh
authors: Fraunhofer USA, Center for Molecular Biotechnology, Newark, DE, USA.
authors: Joey H
authors: JH
authors: Norikane
authors: Fraunhofer USA, Center for Molecular Biotechnology, Newark, DE, USA.
authors: Stephen J
authors: SJ
authors: Streatfield
authors: Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA. Electronic address: rherzog@ufl.edu.
authors: Roland W
authors: RW
authors: Herzog
authors: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address: hdaniell@upenn.edu.
authors: Henry
authors: H
authors: Daniell
mesh_terms: D000284:Administration, Oral; D000818:Animals; D000941:Antigens / Q000378:metabolism; D018533:Biomass; D002214:Capsules; D002736:Chloroplasts / Q000378:metabolism; D003365:Costs and Cost Analysis; D005164:Factor IX / Q000008:administration & dosage / Q000191:economics* / Q000627:therapeutic use*; D005612:Freeze Drying; D005822:Genetic Vectors / Q000378:metabolism; D002836:Hemophilia B / Q000188:drug therapy*; D007108:Immune Tolerance; D007073:Immunoglobulin E / Q000378:metabolism; D007221:Industry; D007421:Intestine, Small / Q000378:metabolism; D018545:Lactuca / Q000166:cytology*; D051379:Mice; D018515:Plant Leaves / Q000378:metabolism; D017510:Protein Folding; D055550:Protein Stability
chemical_list: D000941:Antigens; D002214:Capsules; D007073:Immunoglobulin E; D005164:Factor IX
keywords: Chloroplast; Hemophilia; Lettuce; Molecular pharming; Oral tolerance; cGMP plant production