Expression of Human Therapeutic Proteins in Transgenic Tobacco Chloroplasts

描述（由申请人提供）：与疫苗和生物药品的生产有关的几个主要费用。在植物中生产治疗性蛋白质应该消除使用成本过高的发酵罐。例如，我们已经证明，1英亩的叶绿体转基因植物可以生产多达3.6亿剂清洁、安全、功能齐全的炭疽疫苗抗原。在我们第一个NIH资助的项目中，叶绿体已经成功地在可溶性间质室中产生各种疫苗抗原和人类血液蛋白。表达疫苗抗原（如炭疽鼠疫）和人血液蛋白（如干扰素α 2b、人血清白蛋白）的转基因植物已在野外生长，其功能已通过体外试验和/或动物研究确定。然而，许多病毒抗原和人类血液蛋白被锚定在膜上，需要糖基化来维持它们的稳定性和功能。虽然60%的人类药物靶点是膜蛋白，但由于其丰度低且可用性有限，研究很少。因此，本文提出了叶绿体基因工程的新概念，以表达膜蛋白、糖蛋白和其他完全折叠的肽，这些肽需要独特的翻译后修饰（例如在形成二硫键后进行环化）。口服植物细胞表达的治疗性蛋白具有以下优点：通过生物包封保护消化系统，随后在肠道中缓慢释放；消除了昂贵的净化步骤、冷链（低温储存和运输）、医务人员和无菌注射剂；以及由疫苗抗原产生的全身和粘膜免疫。因此，我们提出的目标是：a)表达和分析叶绿体中产生的反转录细胞周期素的抗hiv -1功效；b)表达用于皮下或口服的多价疫苗抗原（疟疾）；c)利用体外细胞培养系统和合适的动物模型，研究治疗性蛋白（胰岛素、干扰素）口服给药到循环系统，并表征其折叠、组装和功能；d)通过叶绿体基因组靶向类囊体或内膜表达治疗性膜蛋白，并进行纯化、功能和结构研究；e)利用pgl操纵子制造能够将外源蛋白糖基化的叶绿体。这些研究的成功完成将使转基因叶绿体成为生产安全、廉价的治疗性蛋白的理想生物反应器，并为膜蛋白的深入研究打开大门。

DESCRIPTION (provided by applicant): 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.