Advanced Technology to Produce Adeno-Associated Viruses (AAV) Serotypes for Targeted Delivery

点击翻译按钮获取中文摘要

Building on the progress made in my NSERC-Discovery Grant on cell culture engineering and viral-based biologics, the proposed research program aims to elucidate the basic mechanisms and quantify the expression kinetics involved in the production of Adeno-Associated Viruses (AAV) serotypes using the insect cell expression system. This program is therefore directed to address knowledge gaps in the biology of the expression systems and the product formation from a bioprocessing standpoint; and solving engineering challenges that limit the operation of the system at industrial scales for potential applications in the treatment and prevention of human diseases. We propose to: i) Study the mechanism of baculovirus induction of AAV serotypes expression in insect cells focusing on the expression and detailed characterization of AAV empty capsids, then directing efforts to study the packaging of the transgene and control the production kinetics to improve the yield and the integrity of AAV vector using different insect cell lines and baculovirus constructs as well as RNA interference technology;  ii) Optimize the production and purification at high cell density culture using fed-batch production and closed controlled loop based on on-line measurement of cell physiological states, then validate the advanced process; iii) Develop and implement process analytical technologies to document the process for industrialization and assess the identity, the functionality and the purity of AAV vectors. Generalization of the study will be extended to all 12 serotypes; The overall research and development strategy will combine different approaches of molecular design; cell engineering and metabolomics; kinetic and mechanistic analyses of the cell/viral interactions; bioprocess engineering including design of experiments (DoE), downstream processing, in-process analytical technologies; product characterization; process integration; data management as well as modeling using a system approach. By integrating the newly generated knowledge and the most recent technological advancements we will rationally design, optimize and develop integrated AAV production/purification processes with an emphasis on high yield, quality and safety attributes of the AAV delivery systems to address unmeet needs of a number of biomedical applications. The outcomes of this program are particularly significant for systemic delivery when large quantities of AAV-based nanoparticles or vectors of clinical quality are required. The achievements will be essential for translational research in gene and cell therapy and vaccination including post-exposure treatment of infectious diseases. Importantly, the program will train Highly Qualified Personnel in areas of high demand in the biomanufacturing industry and will contribute to building expertise and capacity in Canada to facilitate research translation.