Low-cost production of rApoA-IM for the treatment of cardiovascular disease

描述（由申请人提供）：血管疾病如动脉粥样硬化是全球发病率和死亡率的主要原因。高密度脂蛋白（HDL）颗粒被认为是通过从血管壁去除多余的胆固醇和其他脂质并将其输送到肝脏进行消除来保护动脉免受动脉粥样硬化。HDL的关键蛋白质组分是载脂蛋白A-I（ApoA-I）。一种天然存在的遗传变异体，名为ApoA-I Milano（ApoA-IM），在减少动脉粥样硬化性血管疾病方面非常有效。ApoA-IM和HDL颗粒的这种特异性功能已成为基于HDL的治疗中的重要治疗焦点。然而，ApoA-IM的生产极其困难且昂贵，因此限制了其治疗潜力。Ventria Bioscience开发了一种在水稻谷粒中的植物蛋白表达系统，其产生的重组蛋白通常比其他植物蛋白表达系统高25至1000倍。我们假设，我们可以使用这种技术，以大规模和低成本生产功能性ApoA-IM。本建议有四个目标：1。产生具有三种不同构建体的转基因水稻植物，用于在水稻谷粒中表达ApoA-IM以实现重组ApoA-IM（rApoA-IM）的高水平表达; 2.转基因水稻种子的表达分析及rApoA-IM的纯化; 3.水稻rApoA-IM的生化分析及与大肠杆菌rApoA-IM的比较由我们的合作者The Medicines Company制备的大肠杆菌;以及4.水稻rApoA-IM的功能分析及其与大肠杆菌rApoA-IM的比较由我们的合作者The Medicines Company制备。首先将测试水稻衍生的rApoA-IM与脂质形成复合物的能力。然后，将在基于细胞的试验中检测复合物，该试验旨在测量反向胆固醇流出水平，这是The Medicines Company开发的复合物的作用机制。根据我们过去的经验，我们预期我们将能够在拟议的时间框架内实现我们的目标。

DESCRIPTION (provided by applicant): Vascular diseases such as athersclerosis are the major causes of morbidity and mortality worldwide. The high density lipoprotein (HDL) particle is thought to protect the arteries from atherosclerosis by removing excess cholesterol and other lipids from the vessel wall and delivering them to the liver for elimination. The key protein component of HDL is apolipoprotein A-I (ApoA-I). A naturally occurring genetic variant, named ApoA-I Milano (ApoA-IM) is highly potent in reducing atherosclerotic vascular disease. This specific function of ApoA-IM and the HDL particle has become an important therapeutic focus in the HDL-based therapies. However, production of ApoA-IM is extremely difficult and costly thus limiting its therapeutic potential. Ventria Bioscience has developed a plant-based protein expression system in rice grain that produces recombinant protein generally 25 to 1000 fold higher than other plant-based protein expression systems. We hypothesize that we can use this technology to produce functional ApoA-IM in large scale and at low cost. There are four aims in present proposal: 1. production of transgenic rice plants with three different constructs for expressing ApoA-IM in rice grain to achieve a high level expression of recombinant ApoA-IM (rApoA-IM); 2. Expression analysis of transgenic rice seeds and purification of rApoA-IM from transgenic rice seeds; 3. Biochemical analysis of rApoA-IM derived from rice grain and comparison of rice-derived rApoA-IM to that derived from E. coli prepared by our collaborator, The Medicines Company; and 4. Functional analysis of rApoA-IM derived from rice grain and comparison of rice-derived rApoA-IM to that derived from E. coli prepared by our collaborator, The Medicines Company. Rice-derived rApoA-IM will be first tested for its ability to form complex with lipid. Then the complex will be tested in a cell-based assay designed to measure the level of Reverse Cholesterol Efflux which is the mechanism of action of complex developed by The Medicines Company. Based on our past experience, we expect that we will be able to achieve our goals within the proposed time- frame.