Current treatment for many inherited protein deficiencies is based on intravenous infusion of recombinant proteins. The infused protein is expensive and is often encountered by antibody responses that neutralize therapy, thereby complicating treatment and further increasing costs. They also cause severe allergic or even life-threatening anaphylactic reactions. Oral immune modulatory therapy represents an ideal antigen-specific approach to prevent such responses because it is non-invasive and does not require immune suppression or genetic manipulation of the patient's cells. This proposal takes advantage of a unique concept based on the use of plant chloroplasts as highly efficient bioreactors for production and oral delivery of therapeutic protein antigens without a need for fermentation, purification, cold chain, or sterile injections. Bioencapsulated antigens are protected in the stomach from acids or enzymes, but are released in the gut when plant cell walls are digested by commensal bacteria. We propose to further advance this method for effective delivery of antigen-ligand fusions to the gut-associated lymphoid tissue. We have also made substantial progress towards development of an oral delivery system (based on the edible crop lettuce) suitable for clinical translation. In treatment of the X-linked bleeding disorder hemophilia, formation of inhibitory antibodies against the therapeutic clotting factor represents a serious complication that substantially increases morbidity and mortality. For example, patients with hemophilia B due to deletion or early stop codon in the factor IX (F.IX) gene are at increased risk for inhibitor formation. Not only are most F.IX inhibitors high-titer and difficult to eliminate, but these antibodies are often associated with potentially life-threatening anaphylactic reactions. Recently, we demonstrated that oral delivery of F.IX, produced by tobacco chloroplasts and bioencapsulated in plant cells, prevents formation of inhibitors and anaphylaxis in protein replacement therapy for hemophilia B (featured in PNAS 107:7101). These results illustrate the effectiveness of our approach towards immune modulatory therapy for inherited protein deficiencies. The goals of this proposal are to develop an oral immune modulatory protocol for hemophilia B that prevents or mitigates pathogenic antibody responses (including anaphylaxis);to dissect the underlying mechanisms of antigen presentation and immune regulation;to find alternative transmucosal carriers for effective antigen delivery;to test the approach for a different disease with similar antibody/anaphylactic response (the lysosomal storage disorder Pompe disease);to develop transplastomic lettuce to express F.IX for future oral delivery in humans;to investigate stability and microbial contamination of the freeze-dried transgenic material;and to perform translational studies in a large animal model of hemophilia B. We hypothesize that our low cost approach can be utilized in a prophylactic fashion in several diseases, such as hemophilia and lysosomal storage disorders, thereby eliminating the need for immune suppression.

Public Health Relevance

The immune system of patients with genetic diseases (such as hemophilia and Pompe disease) often rejects the protein drug that is given for therapy. This proposal seeks to develop a novel and effective method to achieve tolerance by using oral delivery of genetically modified plant cells.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BST-E (02))
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Link, Rebecca P
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University of Central Florida
Schools of Medicine
United States
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Nayak, Sushrusha; Doerfler, Phillip A; Porvasnik, Stacy L et al. (2014) Immune responses and hypercoagulation in ERT for Pompe disease are mutation and rhGAA dose dependent. PLoS One 9:e98336
Kohli, Neha; Westerveld, Donevan R; Ayache, Alexandra C et al. (2014) Oral delivery of bioencapsulated proteins across blood-brain and blood-retinal barriers. Mol Ther 22:535-46
Jin, Shuangxia; Daniell, Henry (2014) Expression of ?-tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal-induced oxidative stresses by reducing reactive oxygen species. Plant Biotechnol J 12:1274-85
Sherman, Alexandra; Su, Jin; Lin, Shina et al. (2014) Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells. Blood 124:1659-68
Clarke, Jihong Liu; Waheed, Mohammad Tahir; Lossl, Andreas G et al. (2013) How can plant genetic engineering contribute to cost-effective fish vaccine development for promoting sustainable aquaculture? Plant Mol Biol 83:33-40
Lakshmi, Priya Saikumar; Verma, Dheeraj; Yang, Xiangdong et al. (2013) Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro. PLoS One 8:e54708
Kwon, Kwang-Chul; Verma, Dheeraj; Jin, Shuangxia et al. (2013) Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress. PLoS One 8:e67106
Wang, Xiaomei; Sherman, Alexandra; Liao, Gongxian et al. (2013) Mechanism of oral tolerance induction to therapeutic proteins. Adv Drug Deliv Rev 65:759-73
Kwon, Kwang-Chul; Verma, Dheeraj; Singh, Nameirakpam D et al. (2013) Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev 65:782-99
Kwon, Kwang-Chul; Nityanandam, Ramya; New, James S et al. (2013) 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 11:77-86