Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells
Authors: Kwang-Chul Kwon, Henry Daniell
Journal: Molecular Therapy (2016)

Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood–brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer's disease, diabetes, hypertension, Gaucher's or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery. Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood–brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer's disease, diabetes, hypertension, Gaucher's or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery. Peptides or proteins, including hormones, enzymes, ligands, or inhibitors regulate various cellular functions. Therefore, they are useful in the clinic to treat or prevent human disorders by modulating physiological or pathological processes. The use of proteins or peptides for therapeutic purposes will continue to increase in the treatment of cancer, metabolic disorders and neurodegenerative or infectious diseases. In contrast to small-molecule drugs, the high selectivity of peptides or proteins to their targets may reduce side effects and toxicity to host cells.1Craik DJ Fairlie DP Liras S Price D The future of peptide-based drugs.Chem Biol Drug Des. 2013; 81: 136-147Crossref PubMed Scopus (378) Google Scholar In 2013, cumulative sales of biopharmaceuticals reached $140 billion, and this value is expected to increase steadily in clinics.2Walsh G Biopharmaceutical benchmarks.Nat Biotechnol. 2014; 32: 992-1000Crossref PubMed Scopus (0) Google Scholar Currently, protein drugs are largely manufactured using mammalian, yeast or bacterial cell culture systems. These expressed proteins must be extracted and purified, which requires expensive and complex processes and cold storage and transportation, but they have short shelf-lives.3Daniell H Singh ND Mason H Streatfield SJ Plant-made vaccine antigens and biopharmaceuticals.Trends Plant Sci. 2009; 14: 669-679Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar,4Kwon KC Verma D Singh ND Herzog R Daniell H Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells.Adv Drug Deliv Rev. 2013; 65: 782-799Crossref PubMed Scopus (63) Google Scholar Moreover, there is a risk of facility and/or product contamination with toxins or human pathogens.5Ma JK Drake PM Christou P The production of recombinant pharmaceutical proteins in plants.Nat Rev Genet. 2003; 4: 794-805Crossref PubMed Scopus (0) Google Scholar Injectable forms of PDs often require health care personnel for administration, resulting in hospital visits and decreased patient compliance. In addition to issues of high cost, current facilities have limited production capacity. It is estimated that it would require ~77–500 million € and up to 5 years to build a mammalian cell culture production unit,6Spök A Karner S Plant Molecular Farming: Opportunities and Challenges.in: AJ European Commission: The Institute for Prospective Technological Studies. Seville, 2008http://ftp.jrc.es/EURdoc/Google Scholar excluding additional years for regulatory approval. Proteins drugs produced in current manufacturing systems are mostly delivered by injection; oral delivery is not possible due to drug degradation by stomach acids, proteases in the digestive system and the inability to cross intestinal membrane barriers.7Renukuntla J Vadlapudi AD Patel A Boddu SH Mitra AK Approaches for enhancing oral bioavailability of peptides and proteins.Int J Pharm. 2013; 447: 75-93Crossref PubMed Scopus (0) Google Scholar Attaching molecules like polyethylene glycol,8Diago M Crespo J Olveira A Pérez R Bárcena R Sánchez-Tapias JM et al.Clinical trial: pharmacodynamics and pharmacokinetics of re-treatment with fixed-dose induction of peginterferon alpha-2a in hepatitis C virus genotype 1 true non-responder patients.Aliment Pharmacol Ther. 2007; 26: 1131-1138Crossref PubMed Scopus (0) Google Scholar an antibody Fc domain9Kumar TR Soppimath K Nachaegari SK Novel delivery technologies for protein and peptide therapeutics.Curr Pharm Biotechnol. 2006; 7: 261-276Crossref PubMed Scopus (97) Google Scholar or human serum albumin10Baggio LL Huang Q Brown TJ Drucker DJ A recombinant human glucagon-like peptide (GLP)-1-albumin protein (albugon) mimics peptidergic activation of GLP-1 receptor-dependent pathways coupled with satiety, gastrointestinal motility, and glucose homeostasis.Diabetes. 2004; 53: 2492-2500Crossref PubMed Scopus (0) Google Scholar,11Subramanian GM Fiscella M Lamousé-Smith A Zeuzem S McHutchison JG Albinterferon alpha-2b: a genetic fusion protein for the treatment of chronic hepatitis C.Nat Biotechnol. 2007; 25: 1411-1419Crossref PubMed Scopus (0) Google Scholar increases peptide stability in serum during circulation. In addition, peptide drugs can be modified to protect from serum proteases and peptidases; such modifications include N-terminal acetylation, C-terminal amidation, the use of non-natural amino acids, and cyclization via disulfide bonds.12Werle M Bernkop-Schnürch A Strategies to improve plasma half life time of peptide and protein drugs.Amino Acids. 2006; 30: 351-367Crossref PubMed Scopus (0) Google Scholar However, there are still no clinically approved oral peptide drugs. Plants offer an ideal alternative to conventional manufacturing systems and invasive methods of PD administration. Plants are not hosts for human pathogens. The plant cell wall—heavily packed with lignin and cellulose—provides natural protection for PDs because human enzymes are incapable of breaking down glycosidic bonds of plant cell wall carbohydrates. However, gut bacteria digest the plant cell wall and release bioencapsulated PDs into the gut lumen.13Martens EC Lowe EC Chiang H Pudlo NA Wu M McNulty NP et al.Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts.PLoS Biol. 2011; 9: e1001221Crossref PubMed Scopus (159) Google Scholar,14Flint HJ Scott KP Duncan SH Louis P Forano E Microbial degradation of complex carbohydrates in the gut.Gut Microbes. 2012; 3: 289-306Crossref PubMed Scopus (42) Google Scholar Plant cells have similar capacity as mammalian cells to produce protein drugs.15Stoger E Fischer R Moloney M Ma JK Plant molecular pharming for the treatment of chronic and infectious diseases.Annu Rev Plant Biol. 2014; 65: 743-768Crossref PubMed Scopus (0) Google Scholar Antibodies against Ebola virus (ZMapp)16Gao J Yin L Drug development for controlling Ebola epidemic - a race against time.Drug Discov Ther. 2014; 8: 229-231Crossref PubMed Google Scholar or glucocerebrosidase, an enzyme replacement therapy for Gaucher's disease have been produced in tobacco plants16Gao J Yin L Drug development for controlling Ebola epidemic - a race against time.Drug Discov Ther. 2014; 8: 229-231Crossref PubMed Google Scholar or carrot cell suspension cultures.17Shaaltiel Y Bartfeld D Hashmueli S Baum G Brill-Almon E Galili G et al.Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system.Plant Biotechnol J. 2007; 5: 579-590Crossref PubMed Scopus (0) Google Scholar Long shelf life and stability of PDs are critical for successful clinical applications of PDs. Protein drugs made in chloroplasts are stable in lyophilized plant cells when stored at ambient temperature, maintaining their folding and functional efficacy for several years,18Su J Zhu L Sherman A Wang X Lin S Kamesh A et al.Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B.Biomaterials. 2015; 70: 84-93Crossref PubMed Scopus (21) Google Scholar thereby eliminating expensive cold chain. In addition, the freeze-drying process increases the concentration of PDs and eliminates bacterial contamination.4Kwon KC Verma D Singh ND Herzog R Daniell H Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells.Adv Drug Deliv Rev. 2013; 65: 782-799Crossref PubMed Scopus (63) Google Scholar Advantages of plant systems as bioreactors for production of biopharmaceuticals as well as their delivery, and remaining clinical challenges are discussed in this review. Similar to mammalian cells, plant cells facilitate formation of disulfide bonds, glycosylation, folding, and assembly of PDs.15Stoger E Fischer R Moloney M Ma JK Plant molecular pharming for the treatment of chronic and infectious diseases.Annu Rev Plant Biol. 2014; 65: 743-768Crossref PubMed Scopus (0) Google Scholar,19Sanz-Barrio R Fernández-San Millán A Carballeda J Corral-Martínez P Seguí-Simarro JM Farran I Chaperone-like properties of tobacco plastid thioredoxins f and m.J Exp Bot. 2012; 63: 365-379Crossref PubMed Scopus (0) Google Scholar The first plant-made pharmaceutical protein, human growth hormone, was made in tobacco and sunflower callus tissue via nuclear transformation and was reported in 1986.20Barta A Sommergruber K Thompson D Hartmuth K Matzke MA Matzke AJ The expression of a nopaline synthase —human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue.Plant Mol Biol. 1986; 6: 347-357Crossref PubMed Scopus (0) Google Scholar First chloroplast-made therapeutic proteins, cholera toxin B subunit (CTB), human serum albumin, and somatotropin were reported in the early 2000s.21Daniell H Lee SB Panchal T Wiebe PO Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts.J Mol Biol. 2001; 311: 1001-1009Crossref PubMed Scopus (0) Google Scholar,22Fernández-San Millán A Mingo-Castel A Miller M Daniell H A chloroplast transgenic approach to hyper-express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradation.Plant Biotechnol J. 2003; 1: 71-79Crossref PubMed Google Scholar,23Staub JM Garcia B Graves J Hajdukiewicz PT Hunter P Nehra N et al.High-yield production of a human therapeutic protein in tobacco chloroplasts.Nat Biotechnol. 2000; 18: 333-338Crossref PubMed Scopus (0) Google Scholar Plants stably transformed with transgenes can be easily propagated from seeds. Agrobacterium tumefaciens is used to deliver transgenes to the nucleus; whereas a particle delivery system is used to transform plants that are recalcitrant to A. mediated transformation.24Basaran P Rodríguez-Cerezo E Plant molecular farming: opportunities and challenges.Crit Rev Biotechnol. 2008; 28: 153-172Crossref PubMed Scopus (0) Google Scholar Recombinant proteins that require glycosylation for their functionality are expressed via the nuclear genome and targeted to the endoplasmic reticulum (ER). Targeting recombinant proteins to the ER, apoplast or other subcellular compartments improves yield.15Stoger E Fischer R Moloney M Ma JK Plant molecular pharming for the treatment of chronic and infectious diseases.Annu Rev Plant Biol. 2014; 65: 743-768Crossref PubMed Scopus (0) Google Scholar Biopharmaceuticals are also produced in plant cell suspension cultures to minimize regulatory concerns and expedite FDA approval.25Xu J Dolan MC Medrano G Cramer CL Weathers PJ Green factory: plants as bioproduction platforms for recombinant proteins.Biotechnol Adv. 2012; 30: 1171-1184Crossref PubMed Scopus (0) Google Scholar However, despite several decades of research on nuclear transformation, low expression levels have hampered production of industrial-level expression of biopharmaceuticals in plants.26Fahad S Khan FA Pandupuspitasari NS Ahmed MM Liao YC Waheed MT et al.Recent developments in therapeutic protein expression technologies in plants.Biotechnol Lett. 2015; 37: 265-279Crossref PubMed Scopus (0) Google Scholar There are also risks of dissemination of engineered genes to the environment via pollen and of contamination of food or feed chains by transgenic seeds.24Basaran P Rodríguez-Cerezo E Plant molecular farming: opportunities and challenges.Crit Rev Biotechnol. 2008; 28: 153-172Crossref PubMed Scopus (0) Google Scholar Chloroplast genomes have also been utilized for stable transformation of numerous heterologous genes since the early 1990s.27Daniell H Khan MS Allison L Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology.Trends Plant Sci. 2002; 7: 84-91Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,28Chebolu S Daniell H Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality.Curr Top Microbiol Immunol. 2009; 332: 33-54PubMed Google Scholar,29Jin S Daniell H The engineered chloroplast genome just got smarter.Trends Plant Sci. 2015; 20: 622-640Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,30Waheed MT Ismail H Gottschamel J Mirza B Lössl AG Plastids: The green frontiers for vaccine production.Front Plant Sci. 2015; 6: 1005Crossref PubMed Scopus (0) Google Scholar The most striking feature of the chloroplast genome over the nuclear genome is its high copy number (>10,000 per cell), enabling transgenes to be expressed at up to 70% of total leaf protein.31Ruhlman T Verma D Samson N Daniell H The role of heterologous chloroplast sequence elements in transgene integration and expression.Plant Physiol. 2010; 152: 2088-2104Crossref PubMed Scopus (106) Google Scholar Double homologous recombination and transgene integration at target sites eliminate positional effects. Gene silencing of transgenes has not yet been reported in chloroplasts. In addition, engineering multiple genes into the chloroplast genome is achieved with a single transformation event,32De Cosa B Moar W Lee SB Miller M Daniell H Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals.Nat Biotechnol. 2001; 19: 71-74Crossref PubMed Scopus (0) Google Scholar,33Quesada-Vargas T Ruiz ON Daniell H Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation.Plant Physiol. 2005; 138: 1746-1762Crossref PubMed Scopus (0) Google Scholar,34Kumar S Hahn FM Baidoo E Kahlon TS Wood DF McMahan CM et al.Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts.Metab Eng. 2012; 14: 19-28Crossref PubMed Scopus (54) Google Scholar,35Saxena B Subramaniyan M Malhotra K Bhavesh NS Potlakayala SD Kumar S Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and impact on plant growth.J Biosci. 2014; 39: 33-41Crossref PubMed Scopus (11) Google Scholar facilitating expression of complex proteins. Chloroplasts also minimize the effect of toxic PDs, like CTB, by sequestering transgene products within this compartment.21Daniell H Lee SB Panchal T Wiebe PO Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts.J Mol Biol. 2001; 311: 1001-1009Crossref PubMed Scopus (0) Google Scholar,36Lee SB Kwon HB Kwon SJ Park SC Jeong MJ Han SE et al.Accumulation of trehalose within transgenic chloroplasts confers drought tolerance.Mol Breeding. 2003; 11: 1-13Crossref Scopus (0) Google Scholar Transgene confinement via maternal inheritance and harvesting leaves before flowering offers complete transgene containment.37Daniell H Transgene containment by maternal inheritance: effective or elusive?.Proc Natl Acad Sci USA. 2007; 104: 6879-6880Crossref PubMed Scopus (0) Google Scholar The large mucosal area of the human intestine ~ 1.8–2.7 m2,38Wilson JP Surface area of the small intestine in man.Gut. 1967; 8: 618-621Crossref PubMed Google Scholar offers an ideal surface for drug delivery. Upon oral delivery, the plant cell wall protects PDs from acids and enzymes in the stomach via bio-encapsulation.39Limaye A Koya V Samsam M Daniell H Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.FASEB J. 2006; 20: 959-961Crossref PubMed Scopus (10) Google Scholar,40Xiao Y Kwon KC Hoffman BE Kamesh A Jones NT Herzog RW et al.Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells.Biomaterials. 2016; 80: 68-79Crossref PubMed Google Scholar Human digestive enzymes are incapable of breaking down all glycosidic bonds in the plant cell wall. Figure 1b shows intact plant cells expressing GFP in the gut lumen that survived acids and enzymes secreted in the stomach, thereby providing direct evidence for protection of proteins via bioencapsulation. However, when intact plant cells containing PDs reach the gut, commensal microbes digest the plant cell wall and release PDs (Figure 1a).13Martens EC Lowe EC Chiang H Pudlo NA Wu M McNulty NP et al.Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts.PLoS Biol. 2011; 9: e1001221Crossref PubMed Scopus (159) Google Scholar,14Flint HJ Scott KP Duncan SH Louis P Forano E Microbial degradation of complex carbohydrates in the gut.Gut Microbes. 2012; 3: 289-306Crossref PubMed Scopus (42) Google Scholar Among gut microbes, only Bacteroidetes and Firmicutes can break down the recalcitrant, insoluble plant cell wall.41Flint HJ Bayer EA Rincon MT Lamed R White BA Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis.Nat Rev Microbiol. 2008; 6: 121-131Crossref PubMed Scopus (438) Google Scholar The cellulosome, which is found in anaerobic cellulolytic bacteria (e.g., Ruminococcus flavefaciens, a representative of the Firmicutes), is an extracellular enzyme complex that contains catalytic, structural, and cellulose-binding domains. Through the cellulosome, bacteria that retain highly concentrated catalytic activities necessary for cleaving plant cell wall glycosidic bonds on their surface make close contact with cell wall substrates and disrupt plant cells. In addition, specialized groups of bacteria colonize the gut mucus layer. For example, a fluorescence in situ hybridization study showed enrichment of Bacteroidetes in the mucous layer. Moreover, mucopolysaccharides in the mucous layer are substrates for gut bacteria. Bacteroides fragilis is known to degrade mucin glycoproteins to allow it to penetrate the mucous layer.42Koropatkin NM Cameron EA Martens EC How glycan metabolism shapes the human gut microbiota.Nat Rev Microbiol. 2012; 10: 323-335Crossref PubMed Scopus (225) Google Scholar As seen in Figure 1b, intact plant cells captured by cellulosomes or pilli of bacteria that colonize the mucosal layer undergo cell wall degradation to release GFP. By these mechanisms, bioencapsulated therapeutic proteins are orally delivered. The presence of plant cells expressing GFP in between villi of the ileum offers visible proof of the protection of plant cells from the digestive system and the uptake of proteins by epithelial cells in the upper gut (Figure 1b).40Xiao Y Kwon KC Hoffman BE Kamesh A Jones NT Herzog RW et al.Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells.Biomaterials. 2016; 80: 68-79Crossref PubMed Google Scholar When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. In our recent study, systemic distribution of GFP was observed in sera and organs including liver, lung, brain, retina, and the tibialis anterior muscle when plant cells expressing CTB-, protein transduction domain (PTD)-, or dendritic cell peptide (DCpep)-fused GFP were orally delivered.40Xiao Y Kwon KC Hoffman BE Kamesh A Jones NT Herzog RW et al.Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells.Biomaterials. 2016; 80: 68-79Crossref PubMed Google Scholar To investigate the route of delivery, an Alexa Fluor 488-labeled antibody against GFP was used for immunohistochemical studies on mice intestine that were fed with plant cells expressing GFP-tagged proteins. The intestines were rolled up to observe proximal and distal portions simultaneously. GFP tagged with PTD was observed in gut epithelial cells, whereas more widespread delivery of GFP to epithelial cells was seen with CTB tag. However, GFP was not detected in epithelial cells when DCpep-GFP was orally delivered. All three fusion tags delivered GFP to microfold cells (M cells). Therefore, in addition to the gut-liver axis, systemic delivery of DCpep-GFP was achieved via M cells, whereas CTB- and PTD-GFP reached circulation through both epithelial and M cells. When tags are used to move proteins across epithelial cells, various endocytic subpathways are employed. CTB can use either clathrin-, caveolar- and nonclathrin/noncaveolar-mediated pathways followed by retrograde transport43Wernick NL Chinnapen DJ Cho JA Lencer WI Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum.Toxins (Basel). 2010; 2: 310-325Crossref PubMed Scopus (0) Google Scholar or transcytosis.44Saslowsky DE te Welscher YM Chinnapen DJ Wagner JS Wan J Kern E et al.Ganglioside GM1-mediated transcytosis of cholera toxin bypasses the retrograde pathway and depends on the structure of the ceramide domain.J Biol Chem. 2013; 288: 25804-25809Crossref PubMed Scopus (0) Google Scholar In the case of PTD, uptake occurs via electrostatic interactions with the plasma membrane, followed by endocytosis and retrograde transport.45Noguchi H Matsushita M Matsumoto S Lu YF Matsui H Bonner-Weir S Mechanism of PDX-1 protein transduction.Biochem Biophys Res Commun. 2005; 332: 68-74Crossref PubMed Scopus (0) Google Scholar The main mechanism for PTD penetrating epithelial cells is through endocytic pathways involving membrane invagination, rather than direct entry via pores, membrane thinning, inverted micelle formation or direct translocation.46Durzyńska J Przysiecka Ł Nawrot R Barylski J Nowicki G Warowicka A et al.Viral and other cell-penetrating peptides as vectors of therapeutic agents in medicine.J Pharmacol Exp Ther. 2015; 354: 32-42Crossref PubMed Scopus (0) Google Scholar DCpep-GFP is exclusively observed in M cells after oral delivery40Xiao Y Kwon KC Hoffman BE Kamesh A Jones NT Herzog RW et al.Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells.Biomaterials. 2016; 80: 68-79Crossref PubMed Google Scholar suggesting that the delivery of DCpep-GFP into circulation is possible by transcytosis through M cells, in addition to gut-liver axis.47Ohno H Intestinal M cells.J Biochem. 2016; 159: 151-160Crossref PubMed Scopus (114) Google Scholar In addition, DCpep fusion proteins can be directly captured in the intestinal lumen by extended dendrites of DCs, thereby facilitating delivery to the immune system.48Wang X Sherman A Liao G Leong KW Daniell H Terhorst C et al.Mechanism of oral tolerance induction to therapeutic proteins.Adv Drug Deliv Rev. 2013; 65: 759-773Crossref PubMed Scopus (24) Google Scholar Therefore, DCpep fusion proteins can cross the epithelial barrier through M cells to enter circulation or can be directly taken up by DCs in the intestinal lumen. In addition to facilitating PD delivery, fusion tags provide N-terminal protection for fused proteins, increasing their stability in sera for several hours over the same proteins after injection.49Kwon KC Nityanandam R New JS Daniell H Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells.Plant Biotechnol J. 2013; 11: 77-86Crossref PubMed Scopus (41) Google Scholar,50Shenoy V Kwon KC Rathinasabapathy A Lin S Jin G Song C et al.Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension.Hypertension. 2014; 64: 1248-1259Crossref PubMed Scopus (0) Google Scholar After crossing the epithelium, proteases like furin, which is present in all cell types, cleave tags at engineered sites.51Thomas G Furin at the cutting edge: from protein traffic to embryogenesis and disease.Nat Rev Mol Cell Biol. 2002; 3: 753-766Crossref PubMed Scopus (617) Google Scholar In the section below, we discuss mechanistic aspects of PD delivery to the circulatory or immune system using different tags. Human body is connected to the environment by mucosal systems where nutrient uptake/waste excretion and gas exchange take place; however, these systems are also contact and entry points for pathogens or toxins. So, the mucosal-associated lymphoid tissue is loaded with a large number of immune cells. In particular, the gut-associated lymphoid tissue represents almost 70% of the entire immune system and contains ~80% of immunoglobulin A-bearing cells.52Vighi G Marcucci F Sensi L Di Cara G Frati F Allergy and the gastrointestinal system.Clin Exp Immunol. 2008; 153 Suppl 1: 3-6Crossref PubMed Scopus (0) Google Scholar Therefore, when therapeutic proteins are orally delivered to treat metabolic diseases, delivery to immune cells, which are heavily represented along the gastrointestinal tract, should be minimized to avoid complications (e.g. developing toxic antibodies). Differentiating immune cells from nonimmune cells is therefore important for oral drug delivery. PTD fusion proteins are not delivered to immune cells but are delivered efficiently to nonimmune cells in the pancreas and kidney.40Xiao Y Kwon KC Hoffman BE Kamesh A Jones NT Herzog RW et al.Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells.Biomaterials. 2016; 80: 68-79Crossref PubMed Google Scholar This unique selectivity of PTD can be used clinically to treat metabolic disorders such as diabetes or nephropathy by targeting PTD-fused drugs to the pancreas and kidney, respectively. Likewise, to treat autoimmune diseases or induce specific immune responses, it is important to deliver therapeutic proteins to immune cells. In contrast to splenic DCs, DCs from Peyer's patches induce tolerance to intestinal antigens transported by M cells, rather than prime T-cells as effectors.48Wang X Sherman A Liao G Leong KW Daniell H Terhorst C et al.Mechanism of oral tolerance induction to therapeutic proteins.Adv Drug Deliv Rev. 2013; 65: 759-773Crossref PubMed Scopus (24) Google Scholar Indeed, antigen uptake by M cells and transportation to APCs is also considered a prime target to enhance oral vaccine efficacy. Therefore, it is widely accepted that initial systemic priming followed by mucosal boosting with the use of adjuvants is likely to prevent mucosal tolerance induction.53Chan HT Daniell H Plant-made oral vaccines against human infectious diseases-Are we there yet?.Plant Biotechnol J. 2015; 13: 1056-1070Crossref PubMed Scopus (0) Google Scholar,54Chan HT Xiao Y Weldon WC Oberste SM Chumakov K Daniell H Cold chain and virus free chloroplast-made booster vaccine to confer immunity against different polio virus serotypes.Plant Biotechnol J. 2016; https://doi.org/10.1111/pbi.12575Crossref Google Scholar Therefore, proper targeting of drugs by distinguishing immune and nonimmune cells is very important. DCs deliver GFP only to the immune system and not to any nonimmune cell type. In addition, different strategies can assure efficient transmucosal delivery of the antigen, once it is released from plant cells. CTB is a highly efficient carrier molecule for transporting PDs across the gut epithelium.55Sun JB Holmgren J Czerkinsky C Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance.Proc Natl Acad Sci USA. 1994; 91: 10795-10799Crossref PubMed Scopus (0) Google Scholar,56Odumosu O Nicholas D Yano H Langridge W AB toxins: a paradigm swit