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

基于我在细胞培养工程和基于病毒的生物制剂方面取得的进展，拟议的研究计划旨在阐明使用昆虫细胞表达系统生产腺相关病毒（AAV）血清型的基本机制和量化表达动力学。因此，该项目旨在从生物加工的角度解决表达系统和产品形成生物学方面的知识空白；并解决限制该系统在工业规模上运行的工程挑战，以用于治疗和预防人类疾病的潜在应用。***我们建议：i)研究杆状病毒诱导AAV在昆虫细胞中血清型表达的机制，重点研究AAV空衣壳的表达和详细表征，然后利用不同的昆虫细胞系和杆状病毒构建体以及RNA干扰技术，研究转基因的包装和生产动力学控制，以提高AAV载体的产量和完整性；ii)基于在线测量细胞生理状态，采用补料批量生产和闭环控制优化高密度培养的生产和纯化，并对先进工艺进行验证；iii)开发和实施过程分析技术，以记录工业化过程，并评估AAV载体的特性、功能和纯度。该研究的推广将扩展到所有12种血清型；***整体研发策略将结合不同的分子设计方法；细胞工程与代谢组学；细胞/病毒相互作用的动力学和机理分析；生物过程工程，包括实验设计（DoE），下游处理，过程中分析技术；产品描述;流程集成;使用系统方法进行数据管理和建模。***通过整合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，重点是AAV输送系统的高产率，质量和安全属性，以解决许多生物医学应用的未满足需求。***当需要大量基于aav的纳米颗粒或临床质量载体时，该计划的结果对于全身递送尤为重要。这些成果将对基因和细胞治疗以及疫苗接种的转化研究，包括传染病的暴露后治疗至关重要。重要的是，该项目将培养生物制造行业高需求领域的高素质人才，并将有助于在加拿大建立专业知识和能力，以促进研究翻译。**

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. **