Expression of Human Therapeutic Proteins in Chloroplasts

说明(申请人提供)：干扰素(干扰素)是一种细胞因子 抗病毒、抗生长和免疫调节特性。使用IFNAlpha 2 作为白血病、转移癌、卡波西肉瘤和病毒的治疗 肝炎。IFNAlpha 2疗法每年的费用为4000美元，使其 不适用于世界各地的大多数患者。在1.2亿感染了艾滋病的人中 丙型肝炎病毒，70%有肝功能异常，33%有 严重的肝硬变。在肝脏中表达的IFNα5具有最强的抗病毒作用 活动，使其成为一个有吸引力的治疗候选者。胰岛素样生长 胰岛素样生长因子-I(IGF-I)是一种有效的多功能合成激素，由 肝脏，其不足会导致几种系统并发症的发生 在肝硬变中。每位肝硬变患者对胰岛素样生长因子-L的年需要量为 600毫克，每毫克的成本是3万美元。 转基因叶绿体技术提供了一种新的重组解决方案 由于具有超表达能力和折叠能力而产生蛋白质 并用二硫键处理真核蛋白(从而消除了 需要昂贵的净化后处理)。烟草是一个理想的选择 因为它的生物量大，易于放大(百万种子围栏植物)， 基因操作以及探索其替代用途的迫切需要 危险的作物。口服功能性生物药物可减少90% %的生产成本，不再需要昂贵的 净化。此外，植物细胞内的生物封装提供了 防止蛋白质在消化过程中降解。给予干扰素 口服在人和动物体内具有生物活性。 因此，IFNalpha2、IFNalpha5和IGF-L将表达如下：a) 开发用于增强表达或口服的重组DNA载体 产生转基因烟草和番茄植株c)鉴定转基因 利用分子和生化技术表达蛋白质或融合蛋白 方法d)从转基因叶绿体中提纯治疗蛋白 鉴定和比较治疗性蛋白质(产量、纯度、功能性) 在酵母、大肠杆菌或转基因植物中产生f)体外和体内研究 (临床前试验)蛋白质生物功能性、非肠道和口服给药。 通过转基因叶绿体进行大规模和低成本的生产应该提供 以负担得起的成本为患者提供治疗，烟农可以替代使用 为了这种危险的作物。

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.