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

低成本生产 rApoA-IM 用于治疗心血管疾病

• 批准号: 8709796
• 负责人: NING HUANG
• 金额: $ 26.09万
• 依托单位: VENTRIA BIOSCIENCE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8709796
• 关键词: Animal Model; Apolipoprotein A-I; Apolipoproteins A; Arteries; Atherosclerosis; Biochemical; Biological Assay; Cardiovascular Diseases; Cells; Cereals; Cholesterol; Complex; Coronary heart disease; Development; Effectiveness; Escherichia coli; Feasibility Studies; Funding; Goals; High Density Lipoprotein Cholesterol; High Density Lipoproteins; In Vitro; Life; Lipids; Liver; Measures; Medicine; Morbidity - disease rate; Names; Phase; Plants; Process; Production; Proteins; Recombinant Proteins; Recombinants; Request for Proposals; Rice; Risk Factors; Seeds; Small Business Innovation Research Grant; System; Technology; Testing; Therapeutic; Time; Toxicology; Transgenic Organisms; Transgenic Plants; Vascular Diseases; apolipoprotein A-I Milano; base; cost; design; experience; genetic variant; in vivo; mortality; particle; protein expression; public health relevance; research clinical testing; reverse cholesterol transport; safety study; scale up

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.

描述(申请人提供)：动脉粥样硬化等血管疾病是全球发病率和死亡率的主要原因。高密度脂蛋白(高密度脂蛋白)颗粒被认为可以通过从血管壁中清除多余的胆固醇和其他脂质，并将它们输送到肝脏进行清除，从而保护动脉免受动脉粥样硬化的影响。高密度脂蛋白的关键蛋白质成分是载脂蛋白A-I(ApoA-I)。一种自然产生的基因变体，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。载脂蛋白A-IM和高密度脂蛋白颗粒的这种特殊功能已成为基于高密度脂蛋白的治疗中的一个重要治疗焦点。然而，ApoA-IM的生产极其困难和昂贵，从而限制了其治疗潜力。Ventria Bioscience已经开发出一种基于植物的稻米蛋白质表达系统，该系统产生的重组蛋白质通常比其他植物蛋白质表达系统高25到1000倍。我们假设可以利用这项技术来大规模、低成本地生产功能性ApoA-IM。本研究的目的有四个：1.在水稻籽粒中建立表达ApoA-IM的三种不同结构的转基因植株，以实现重组ApoA-IM(rApoA-IM)的高效表达：2.转基因水稻种子的表达分析和从转基因水稻种子中分离纯化rApoA-IM：3.对稻米来源的rApoA-IM进行生化分析，并将水稻来源的rApoA-IM与我们的合作者Medicines公司的大肠杆菌来源的rApoA-IM进行比较；4.米粒来源的rApoA-IM的功能分析，并与我们的合作者Medicines公司制备的来自大肠杆菌的rApoA-IM进行比较。从大米中提取的rApoA-IM将首先测试其与脂质形成复合体的能力。然后，该复合体将在基于细胞的测试中进行测试，该测试旨在测量反向胆固醇外流的水平，这是由Medicines公司开发的复合体的作用机制。根据我们过去的经验，我们预计我们将能够在拟议的时间框架内实现我们的目标。