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

生产用于靶向递送的腺相关病毒 (AAV) 血清型的先进技术

• 批准号: RGPIN-2015-05132
• 负责人: Kamen, Amine
• 金额: $ 2.55万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=685449
• 关键词: Advanced; Technology; Produce; Adeno; Associated

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. **

基于我在细胞培养工程和病毒生物制剂方面的 NSERC 发现资助所取得的进展，拟议的研究计划旨在阐明腺相关病毒 (AAV) 血清型生产中涉及的基本机制并量化表达动力学，使用因此，该计划旨在从生物加工的角度解决表达系统生物学和产物形成方面的知识空白，并解决限制系统运行的工程挑战； ***我们建议：i) 研究杆状病毒在昆虫细胞中诱导 AAV 血清型表达的机制，重点关注 AAV 空衣壳的表达和详细表征。指导努力研究转基因的包装并控制生产动力学，以使用不同的昆虫细胞系和杆状病毒构建体以及RNA干扰技术提高AAV载体的产量和完整性；基于细胞生理状态的在线测量，使用分批补料生产和闭环控制循环优化高细胞密度培养的生产和纯化，然后验证先进工艺； iii) 开发和实施工艺分析技术以记录工业化工艺；并评估 AAV 载体的特性、功能和纯度；***总体研究和开发策略将结合分子设计和代谢组学的不同方法；细胞/病毒相互作用的机制分析；生物过程工程，包括实验设计（DoE）、过程中的分析技术以及使用系统方法进行建模；利用新产生的知识和最新的技术进步，我们将合理设计、优化和开发集成的 AAV 生产/纯化工艺，重点关注 AAV 输送系统的高产量、质量和安全属性，以满足许多生物医学应用中未满足的需求.***这的结果当需要大量基于 AAV 的纳米粒子或临床质量的载体时，该计划对于基因和细胞治疗以及疫苗接种（包括传染病的暴露后治疗）的转化研究至关重要。该计划将在生物制造行业高需求领域培训高素质人才，并将有助于建设加拿大的专业知识和能力，以促进研究转化**。