Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM 1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins. Keywords transmucosal carrier; furin; ganglioside receptors; genetically modified crops One of the most challenging problems of human health management is the high cost of prescription drugs in developed countries and their lack of availability in developing countries. For example, interferon (IFN) alpha 2b is used for the treatment of viral diseases such as hepatitis C, as well as for certain cancers. However, IFN treatment for four months costs $26,000 in the United States, where more than forty-five million Americans do not have health insurance (1). Several hundred million people in developing countries are infected with hepatitis, but the daily income of one-third of the world population is less than $2 per day (1). The high cost of prescription drugs is due to a number reasons, including fermentation-based production (each fermenter costs several hundred million dollars to build), expensive purification and in vitro processing methods (such as column chromatography, disulfide bond formation) (2), the need for storage and transportation at low temperature and delivery via sterile injections requiring the involvement of hospitals
NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript and highly qualified health professionals (1). Therefore, new approaches to minimize or eliminate most of these expenses are urgently needed. Transgenic plants offer many advantages, including the feasibility of the oral delivery of foreign proteins, low cost of production, storage and transportation, heat stability and protection through bioencapsulation, elimination of the need for expensive purification, in ...