There are several major costs associated with the production of vaccines and biopharmaceuticals. Production of therapeutic proteins in plants should eliminate the use of cost-prohibitive fermenters. For example, we have shown that 1 acre of chloroplast transgenic plants can produce up to 360 million doses of clean, safe, and fully functional anthrax vaccine antigen. In our first NIH funded project, chloroplasts have been successfully engineered to produce various vaccine antigens and human blood proteins in the soluble stromal compartment. Transgenic plants expressing vaccine antigens (e.g.-anthrax plague) and human blood proteins (e.g.- interferon alpha 2b, human serum albumin) have been grown in the field and their functionality has been determined by in vitro assays and/or animal studies. However, many viral antigens and human blood proteins are anchored to membranes and require glycosylation for their stability and functionality. While 60% of all human drug targets are membrane proteins, very few have been studied due to their low abundance and limited availability. Therefore, new concepts in chloroplast genetic engineering to express membrane proteins, glycoproteins, and other completely folded peptides requiring unique post-translational modifications (e.g.-cyclization after formation of disulfide bonds) are proposed here. Oral delivery of therapeutic proteins expressed in plant cells offers several advantages including protection in the digestive system by bioencapsulation, followed by slow release in the gut;elimination of expensive purification steps, the cold chain (low temperature storage and transportation), medical personnel, and sterile injections for their delivery;and generation of both systemic and mucosal immunity from vaccine antigens. Therefore, the proposed objectives are: a)Express and analyze the anti-HIV-1 efficacy of retrocyclins produced in chloroplasts;b) Express multivalent vaccine antigens for subcutaneous or oral delivery (malaria);c) Investigate the oral delivery of therapeutic proteins (insulin, interferon) to the circulatory system and characterize their folding, assembly, and functionality using in vitro cell culture systems and suitable animal models;d) Express therapeutic membrane proteins via the chloroplast genome by targeting them to the chloroplast genome by targeting them to the thylakoid or inner membrane, and perform purification, functional, and structural studies;e) Create a chloroplast capable of glycosylation of a foreign protein using the pgl operon. Successful completion of these studies would make transgenic chloroplasts ideal bioreactors for the production of safe and less expensive therapeutic proteins and open the door for in depth studies of membrane proteins.
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