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 Pennsylvania
Schools of Dentistry
United States
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Kwon, Kwang-Chul; Sherman, Alexandra; Chang, Wan-Jung et al. (2018) 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. Plant Biotechnol J 16:1148-1160
Xiao, Yuhong; Daniell, Henry (2017) Long-term evaluation of mucosal and systemic immunity and protection conferred by different polio booster vaccines. Vaccine 35:5418-5425
Posgai, Amanda L; Wasserfall, Clive H; Kwon, Kwang-Chul et al. (2017) Plant-based vaccines for oral delivery of type 1 diabetes-related autoantigens: Evaluating oral tolerance mechanisms and disease prevention in NOD mice. Sci Rep 7:42372
Herzog, Roland W; Nichols, Timothy C; Su, Jin et al. (2017) Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce. Mol Ther 25:512-522
Zhang, Bei; Shanmugaraj, Balamurugan; Daniell, Henry (2017) Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts. Curr Opin Chem Biol 38:17-23
Kwon, Kwang-Chul; Chan, Hui-Ting; León, Ileana R et al. (2016) Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation. Plant Physiol 172:62-77
Pasoreck, Elise K; Su, Jin; Silverman, Ian M et al. (2016) Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling. Plant Biotechnol J 14:1862-75
Chan, Hui-Ting; Xiao, Yuhong; Weldon, William C et al. (2016) Cold chain and virus-free chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes. Plant Biotechnol J 14:2190-2200
Kwon, Kwang-Chul; Daniell, Henry (2016) Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells. Mol Ther 24:1342-50
Daniell, Henry; Lin, Choun-Sea; Yu, Ming et al. (2016) Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol 17:134

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