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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL107904-03
Application #
8476263
Study Section
Special Emphasis Panel (ZRG1-BST-E (02))
Program Officer
Link, Rebecca P
Project Start
2011-07-15
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
3
Fiscal Year
2013
Total Cost
$433,499
Indirect Cost
$92,462
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Dentistry
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Wang, Xiaomei; Terhorst, Cox; Herzog, Roland W (2016) In vivo induction of regulatory T cells for immune tolerance in hemophilia. Cell Immunol 301:18-29
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
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
Singh, Nameirakpam Dolendro; Kumar, Shashi; Daniell, Henry (2016) Expression of β-glucosidase increases trichome density and artemisinin content in transgenic Artemisia annua plants. Plant Biotechnol J 14:1034-45
Daniell, Henry; Lin, Choun-Sea; Yu, Ming et al. (2016) Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol 17:134
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
Liu, Yuan; Kamesh, Aditya C; Xiao, Yuhong et al. (2016) Topical delivery of low-cost protein drug candidates made in chloroplasts for biofilm disruption and uptake by oral epithelial cells. Biomaterials 105:156-66
Kwon, Kwang-Chul; Daniell, Henry (2016) Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells. Mol Ther 24:1342-50
Shahid, Naila; Daniell, Henry (2016) Plant-based oral vaccines against zoonotic and non-zoonotic diseases. Plant Biotechnol J 14:2079-2099
Xiao, Yuhong; Kwon, Kwang-Chul; Hoffman, Brad E et al. (2016) Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells. Biomaterials 80:68-79

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