Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice

Abstract

Summary Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation.

Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells

“…Our strategy of oral tolerance induction to autoantigens and to therapeutic proteins used in replacement therapy of genetic disorder (such as hemophilia and lysosomal storage disorders) has in part relied on efficient targeting of gut epithelial cells with CTB followed by transmucosal delivery and proteolytic cleavage, resulting in the release of the antigen from the CTB tag and uptake by DCs [7, 9, 10, 11, 33, ]. However, DCs and macrophages also directly sample antigen in the gut lumen, and M cells may shuttle intact antigen across the epithelium to areas rich in DCs.…”

“…While infection of lymphocytes is a critical step of the HIV life cycle, insulin expression needs to be tightly regulated and responsive to environmental stimuli [64], which may in part account for the selectivity of PTD from PDX-1. At the same time, PTD-GFP was superior in vivo for systemic protein delivery, which can be exploited for therapies that require certain protein levels in the blood, such as in our published examples of treatment of hypertension and hormone or cytokine therapies [9, 11, 14, 15]. Interestingly, despite marked differences in systemic delivery, CTB-, DCpep-, and PTD-tags all resulted in very similar GFP antigen levels in the liver, as evidenced by immunohistochemistry and more quantitatively by ELISA.…”

“…Fusion of the (nontoxic) Cholera toxin B subunit (CTB) to green fluorescent protein (GFP) expressed in chloroplasts and bioencapsulated in plant cells was delivered across the gut epithelium through GM1 receptors and GFP was released into the circulatory system [6]. Fusion of CTB to therapeutic proteins facilitates their effective oral delivery for induction of oral tolerance [7–11] or functional proteins to sera [12–14] or even across blood brain or retinal barriers [15, 16]. …”

Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce

“…Unfortunately, no immune tolerance protocols are currently available for patients to prevent ADA formation. General immune suppression can be applied, as for example in treatment of Pompe disease, but is not generally acceptable 1, 2. In the case of the X-linked bleeding disorder hemophilia, immune tolerance induction (ITI) protocols are attempted only after neutralizing antibodies (termed “inhibitors”) have formed.…”

Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts

“…Oral administration of lyophilized transplastomic plant cells expressing domains of blood coagulation factor VIII or full length FIX in chloroplasts successfully eliminated inhibitor formation in mice and dog models [4••,35•,36]. Besides hemophilia, the oral administration of the acid alpha glucosidase (GAA) epitopes expressed in plant chloroplasts [37] also eliminated antibody response in Pompe disease mouse model. Therefore, the concept of immune modulation using antigens expressed in chloroplasts has been demonstrated in several disease models.…”