Expression of Human Therapeutic Proteins in Chloroplasts

点击翻译按钮获取中文摘要

DESCRIPTION (provided by the applicant): Interferons (IFN) are cytokines with antiviral, antigrowth and immuno-modulatory properties. IFNalpha 2 is employed as a therapy for leukemia, metastasizing carcinoma, kaposi sarcoma and viral hepatitis. The annual cost of IFNalpha 2 therapy is $4000, making it unavailable for most patients worldwide. Among 120 million people infected with hepatitis C virus, 70 percent have abnormal liver function and 33 percent have severe cirrhosis. IFNalpha 5 expressed in liver has the most potent antiviral activity, making it an appealing therapeutic candidate. Insulin-like Growth Factor I (IGF-I) is a potent multifunctional anabolic hormone produced by the liver, and its deficiency results in several systematic complications occurring in liver cirrhosis. The annual requirement of IGF-l per cirrhotic patient is 600 mg, and the cost per mg is $30,000. Transgenic chloroplast technology provides a novel solution to recombinant protein production because of hyper-expression capabilities and ability to fold and process eukaryotic proteins with disulfide bridges (thereby eliminating the need for expensive post-purification processing). Tobacco is an ideal choice because of its large biomass, ease of scale-up (million seeds pen plant), genetic manipulation and an impending need to explore alternative uses for this hazardous crop. Oral delivery of functional biopharmaceuticals reduces 90 percent of the production cost, eliminating the need for expensive purification. Furthermore, bio-encapsulation within plant cells offers protection against proteolytic degradation during digestion. Interferon given orally has biological activity in human and animals. Therefore, IFNalpha2, IFNalpha5 and IGF-l will be expressed as follows: a) Develop recombinant DNA vectors for enhanced expression or oral delivery b) Generate transgenic tobacco and tomato plants c) Characterize transgenic expression of proteins or fusion proteins using molecular and biochemical methods d) Purify therapeutic proteins from transgenic chloroplasts e) Characterize and compare therapeutic proteins (yield, purity, functionality) produced in yeast, E.coli or transgenic plants f) Study in vitro and in vivo (pre-clinical trials) protein bio-functionality, parenteral and oral delivery. Large scale and low cost production via transgenic chloroplasts should provide treatment to patients at an affordable cost and tobacco farmers alternate uses for this hazardous crop.