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.
ApoA-IM protein and its lipid (POPC) complex have become an important therapeutic focus in the HDL-based therapies for atherosclerotic vascular disease. Given that the production of ApoA-IM is extremely difficult and expensive, we propose to produce ApoA-IM with our rice grain-based expression system that can produce recombinant proteins in a large scale and at low cost.