Current standard treatment of the X-linked bleeding disorder hemophilia is based on intravenous administration of recombinant protein. The infused protein is expensive, has a short half-life (thereby limiting duration of the therapeutic effect), is often targeted by antibody responses (thereby complicating/neutralizing therapy, creating immunotoxicities, and further increasing costs);and repeated intravenous access is required, which is painful and inconvenient. Upon treatment, 20-30% of patients with hemophilia A (factor VIII deficiency) and 1.5-3% of hemophilia B patients (factor IX deficiency) form inhibitory antibodies (""""""""inhibitors"""""""") against the infused factor. Inhibitor formation represents a serious complication of treatment and increases morbidity and mortality of the disease. Bypass reagents are available, but are expensive and cannot be routinely given because they pose a thrombotic risk. Clinical immune tolerance induction (ITI) protocols consist of frequent high-dose factor administrations for a long period of time and are very expensive. Bioengineering has created powerful reagents for non-invasive and cost-effective oral delivery of the protein (or a nucleic acid encoding the protein) to the gut. Within this Bioengineering Research Partnership application, we propose to advance this approach for treatment and for oral tolerance induction. Recently, the Daniell and Herzog labs found that oral delivery of bioencapsulated factor IX (in form of chloroplast transgenic plant leaf material) effectively prevented formation of inhibitory antibodies and anaphylactic reactions in subsequent protein replacement therapy in hemophilia B mice. The Leong lab developed an oral gene therapy for hemophilia based on plasmid vectors packaged into chitosan nanoparticles. Thus, oral delivery to the intestinal epithelium can provide both therapy that partially restores hemostasis in animals with hemophilia (thereby providing prophylaxis against spontaneous bleeding) and immune modulation (thereby preventing deleterious immune responses without use of immune suppressive drugs). In order to establish effective oral therapy for hemophilia A (which is the more prevalent form of the disease and has a much higher risk of inhibitor formation), we assembled an interdisciplinary team, encompassing plant biotechnology, bioengineering, immunology, and animal models. Specifically, we propose to i) determine the ability of nanoparticle-based oral gene therapy to correct coagulation and suppress antibody formation to F.VIII in murine models of hemophilia;ii) generate F.VIII chloroplast transgenic lettuce for oral delivery and prevention/reversal of inhibitor formation in protein replacement;and iii) scale up oral therapies to canine hemophilia A. Ultimately, we intend to integrate the approaches to provide an entirely oral-based treatment regimen that accomplished prophylaxis against bleeds and prevents inhibitor formation. This approach could readily be adapted to other inherited protein deficiencies or certain allergies.

Public Health Relevance

This project will develop novel methods for treatment of the inherited bleeding disorder hemophilia and for prevention of detrimental immune responses. The oral delivery technologies utilized here will improve quality of life of patients, reduce the risk of life-threatening bleeds, and reduce health care costs.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
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University of Florida
Schools of Medicine
United States
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Shahid, Naila; Daniell, Henry (2016) Plant-based oral vaccines against zoonotic and non-zoonotic diseases. Plant Biotechnol J 14:2079-2099
Chan, Hon Fai; Zhang, Ying; Leong, Kam W (2016) Efficient One-Step Production of Microencapsulated Hepatocyte Spheroids with Enhanced Functions. Small 12:2720-30
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
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
Kwon, Kwang-Chul; Daniell, Henry (2016) Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells. Mol Ther 24:1342-50

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