Cell-Free Protein Synthesis (CFPS) for improved production strategies of adeno-associated virus vectors

研究背景：最广泛使用的基因治疗产品之一是基于重组腺相关病毒（AAV）的载体，用于基因治疗适应症的递送。重组AAV的生产非常复杂且耗时，需要哺乳动物细胞系、强大的下游纯化和去除空的无功能衣壳。AAV载体的开发主要集中在优化基因传递上，然而，越来越需要开发基于平台的制造工艺和新型载体的筛选工具。无细胞蛋白合成（Cell-free protein synthesis， CFPS）是一种强大的经济高效的筛选技术平台，可用于从微尺度到生产规模的各种复杂蛋白产品。CFPS在解决AAV载体制造挑战方面的可行性尚未得到探索，这也是该项目的重点。目的和目标：本项目旨在通过应用合成生物学和无细胞蛋白合成技术，为AAV的快速筛选和翻译后修饰的控制开发新的解决方案。为实现这些目标，将追求下列目标：AAV衣壳蛋白的无细胞蛋白合成（CFPS）和组装成空的AAV颗粒。衣壳蛋白文库的生成与筛选。2. 用遗传物质装载空AAV颗粒，首先编码模型蛋白（即荧光蛋白），然后是感兴趣的基因。3. 细胞培养中AAV颗粒的摄取。4. 翻译后修改和CFPS可扩展性的范围需求。应用和好处：该项目将成为CFPS作为筛选和/或制造技术的试验台，重点是AAV载体的生产。CFPS可以在受控条件下快速体外生产和分析衣壳组装体。与细胞培养系统相比，像CFPS这样的开放式系统的一个优势是快速筛选能力（即衣壳蛋白工程，提高稳定性和不同血清型）。CFPS还提供翻译后修饰控制（糖基化，泛素化，磷酸化，sumo酰化，乙酰化，肉豆蔻酰化）。由于颗粒不需要从细胞培养中纯化，因此在制造、分离、纯度和分析方面可以得到改进。合成AAV颗粒的应用包括基因治疗的遗传有效载荷、小分子药物的细胞递送和疫苗开发。研究方法：候选人将发展生物信息学，微生物学，分子生物学，CFPS，蛋白质纯化，生化/生物物理方法，分析和生物处理方面的技能。UCL的专业知识和培训将与Pall在AAV病毒载体生产和纯化方面的生物工艺专业知识以及无细胞衍生AAV颗粒性能的测试能力形成高度互补。研究结果将在监督小组的定期会议上、会议上发表，并发表在同行评议的期刊上。与EPSRC的战略和研究领域保持一致：该项目与EPSRC的主题“制造未来”和“医疗保健技术”保持一致。它位于生物化学工程系，生物化学工程系在生物工艺研究方面处于世界领先地位，为未来的生物制造工艺创造了新颖的工程解决方案。该部门通过中心提供研究，中心由生化和系统工程师、合成生物学家和疫苗学家组成，他们一起工作。该项目与未来目标医疗保健制造中心和未来疫苗制造研究中心（Vax-Hub）保持一致，后者的愿景是通过整合发现和生物过程来制造下一代疫苗。该项目是UCL-Pall生物技术卓越中心（CoE）的一部分。

Context of research: One of the most widely used gene therapy products in development are recombinant Adeno-Associated Virus (AAV)-based vectors, used for delivery in gene therapy indications. Recombinant AAV production is complex and extremely time consuming, requiring mammalian cell lines, robust downstream purification and removal of empty non-functional capsids. The AAV vector development has largely focused on optimising gene delivery, however there is an increasing need to develop platform-based manufacturing processes and screening tools of novel vectors. Cell-free protein synthesis (CFPS) is a powerful cost-effective screening technology platform for a variety of complex protein products from microscale to manufacturing scale. CFPS has yet to be explored for its feasibility to address AAV vector manufacturing challenges and is the focus of this project.Aims and Objectives: The aim of this project is to develop novel solutions for rapid AAV bioprocesses by application of synthetic biology and cell-free protein synthesis technologies for rapid screening capability and control over posttranslational modifications.To meet these goals the following objectives will be pursued: 1. Cell-free protein synthesis (CFPS) of AAV capsid proteins and assembly into empty AAV particles. Generating and screening of a library of capsid proteins. 2. Loading empty AAV particles with genetic material, initially coding for a model protein (i.e. fluorescent protein) followed by genes of interest. 3. Uptake of AAV particles in cell culture. 4. Scoping requirements for posttranslational modifications and scalability of the CFPS. Applications and Benefits:This project will be a test bed for the use of CFPS as screening and/or manufacturing technology with focus on AAV vector production. CFPS could enable quick in vitro production and analysis of capsid assemblies under controlled conditions. One advantage of an open system like CFPS over a cell-culture system is the rapid screening capability (i.e. for capsid protein engineering, improved stability and different serotypes). CFPS also offers control over post translational modifications (glycosylation, ubiquitination, phosphorylation, SUMOylation, acetylation, myristoylation). With regards to manufacturing, separation, purity and analytics may be improved as particles will not need to be purified from cell culture. Applications of synthetic AAV particles include genetic payloads for gene therapy, cellular delivery of small-molecule drugs and vaccine development. The research methodology: The candidate will develop skills in bioinformatics, microbiology, molecular biology, CFPS, protein purification, biochemical/biophysical methods, analytics and bioprocessing. UCL's expertise and training will be highly complementary with Pall's bioprocesses expertise in AAV viral vector production and purification as well as testing capabilities for the performance of cell-free derived AAV particles. Results will be presented at regular meetings with the supervisory team, at conferences and published in peer-reviewed journals. Alignment to EPSRC's strategies and research areas: This project is aligned with the EPSRC themes 'manufacturing the future' and 'healthcare technologies'. It is based in the Department of Biochemical Engineering, a world leader in bioprocess research creating novel engineering solutions to underpin future biomanufacturing processes. The Department delivers research through hubs which are teams of biochemical and system engineers, synthetic biologists and vaccinologists, that work together. The project is aligned with the Future Targeted Healthcare Manufacturing Hub and the The Future Vaccine Manufacturing Research Hub (Vax-Hub) whose vision it is to manufacture the next-generation vaccines by integrating discovery and bioprocess. The project is part of the UCL-Pall Biotech Centre of Excellence (CoE).